JP7432929B2 - RNA interference-inducing nucleic acids that suppress non-canonical targets of microRNAs and their uses - Google Patents

Description

The present invention relates to RNA interference-inducing nucleic acids that inhibit gene expression and their uses, and more particularly to interference-inducing nucleic acids that have useful effects exerted by selectively suppressing non-canonical targets of microRNAs and their uses.

This application is Korean Patent Application No. 10-2018-0063054 filed on May 31, 2018, Korean Patent Application No. 10-2018-0063055 filed on May 31, 2018, and May 2019. Korean Patent Application No. 10-2019-0064333 filed on May 31, Korean Patent Application No. 10-2019-0064334 filed on May 31, 2019, Korean Patent Application No. 10-2019-0064334 filed on May 31, 2019 Claims priority based on Application No. 10-2019-0064335 and Korean Patent Application No. 10-2019-0064386 filed on May 31, 2019, and everything disclosed in the specification and drawings of the application The contents of this application are incorporated by reference into this application.

RNA interference refers to a phenomenon that suppresses gene expression at a post-transcriptional stage. The naturally occurring RNA interference phenomenon is caused by microRNAs (microRNAs, miRNAs). MicroRNA is composed of 18 to 25 bases, most of which are small RNAs composed of about 21 bases, and are the messenger RNA of the target gene through the Argonaute protein. (mRNAs) has a complementary base sequence. In the case of animal microRNA, it binds to Argonaute protein and forms a partial base sequence with the target messenger RNA. If the defined seed region has a minimum of 6 or more consecutive base sequences, it is recognized as a target, and most importantly, the 6 base sequences from the 2nd to 7th bases of the minimum 5' end standard are recognized as a target. When the base sequence continuously binds to a target messenger RNA, it sufficiently suppresses the decomposition and translation of the target messenger RNA and inhibits its expression (Lewis BP, et. al. , 2003, Cell, 115(7), 787-98). In this way, microRNAs recognize the messenger RNA of a target gene through a partial base sequence, so one microRNA can normally affect the expression of several hundred to several thousand genes.

The inhibitory effect of microRNA on target gene expression is one of the important mechanisms of gene expression. Under normal circumstances, it is involved in cell differentiation and growth, and when its function is abnormal, it is involved in cancer and degenerative diseases. It is attracting attention as a key to life phenomena, as it induces diseases such as diabetes. Therefore, in order to artificially induce the gene expression inhibitory effect of microRNA, an RNA interference material (siRNA or shRNA) containing the seed region of microRNA is designed and introduced into cells, thereby artificially inhibiting cells. They are differentiated or their functions are changed, and depending on the situation, they are used as therapeutic agents for diseases. Therefore, the complementary base sequence in the microRNA seed region that recognizes the target messenger RNA mediated by Argonaute is important for the RNA interference material including the microRNA to exert its function. In particular, in order to utilize such RNA interference materials, it is necessary to understand the function of each microRNA, and in this case, the function of a microRNA is determined by which target gene is suppressed. , it has become necessary to analyze the entire target gene (target body) of microRNA.

Research on microRNA targets from the transcript level was first conducted by the present inventor through the Ago HITS-CLIP (or CLIP-Seq) experiment. The Ago HITS CLIP experimental method involves irradiating cells and tissue samples with UV to form a complex between RNA and Argonaute protein (Ago) within the cell through covalent bonds. This is a method in which the RNA-Argonaute complex is separated by immunoprecipitation using an Argonaute-specific recognition antibody, and then the separated RNA is analyzed by next-generation sequencing. Through this, it is possible to accurately analyze the microRNA bound to Argonaute, target messenger RNA groups with complementary base sequences, and their positions (Chi S et al, Nature, 2009, 460(7254): 479-86) .

As a result, the present inventors revealed that when a microRNA binds to a target messenger RNA, it may bind even if the microRNA seed region is not in an exact complementary relationship. More specifically, the microRNA seed region defined by the 1st to 8th base sequence of the 5' end standard of the microRNA is a messenger with a minimum of 6 or more continuous and perfect base sequences. Binding to RNA, most importantly binding at the 2nd to 7th base sequence of the 5' end standard, is called canonical target recognition. Recognizing and binding to a microRNA target even if there is no continuous and exact complementary sequence relationship with the RNA seed region is called non-canonical target recognition. According to the Ago HITS-CLIP analysis results, it can be seen that the frequency of microRNA non-canonical recognition of the target through the seed region is approximately 50% of the normal recognition frequency.

Therefore, RNA interference materials (e.g., siRNA or shRNA) designed to contain the sequence of the seed region of traditional microRNAs can inhibit biology by suppressing not only canonical target genes but also various non-canonical target genes. It can be seen that a certain function appears.

Therefore, the problem to be solved by the present invention is to solve the limitations of conventional RNA interference materials designed to include the sequence of the seed region of microRNA as they are, and to increase the efficiency.

More specifically, RNA interference materials designed to contain the seed region sequence of conventional microRNAs suppress both canonical and non-canonical target genes, but strongly suppress the canonical target genes. In comparison, non-canonical target genes are suppressed very weakly. Therefore, the problem to be solved by the present invention is to efficiently increase the biological functions exhibited by conventional microRNAs by suppressing non-canonical target genes, or to increase one of the functions exhibited by conventional microRNAs. The object of the present invention is to provide an RNA interference-inducing nucleic acid that has the effect of selectively exhibiting only the biological function exhibited by suppressing a non-canonical target gene.

In order to solve the above problems, the present invention provides an RNA interference-inducing nucleic acid in which a partial sequence of a specific microRNA is modified to suppress a noncanonical target gene of the microRNA.

More specifically,
The present invention provides that a partial sequence of a specific microRNA is modified in one or more single strands of the double strands of a nucleic acid that induces RNA interference, thereby generating a noncanonical target gene of the microRNA. An RNA interference-inducing nucleic acid that suppresses a gene),
The RNA interference-inducing nucleic acid is based on the base sequence from the 2nd to the 7th base from the 5' end, which is the most important site for binding to the target messenger RNA in the specific microRNA. The 6th and 7th bases are the same, and the 6th and 7th bases are complementary base sequences to bases that can be aligned with the 6th base of the specific microRNA. The bases that can be aligned with the 6th base of the specific microRNA include all complementary bases including G:A, G:U wobble sequences, and the 5th base of the microRNA is The RNA interference is characterized in that a bulge is generated in the target gene between the 6th and 6th nucleotide sequences, and the bulge disappears at the non-canonical target nucleotide sequence to be bound, so that the bulge is bound to a continuous nucleotide sequence, or In the derived nucleic acid, among the base sequences between the 5' end and the 9th base of the specific microRNA, preferably, the guanine base and the uracil base in the 2nd to 7th base of the 5' end are or adenine (Adenine), so that the G:A or G:U wobble sequence at the relevant site becomes the normal base sequence of U:A or A:U. An RNA interference-inducing nucleic acid is provided.

Preferably, the specific microRNA is selected from the group consisting of miR-124, miR-155, miR-122, miR-1, let-7, miR-133, miR-302 and miR-372, and is derived from the same seed. Provided is an RNA interference-inducing nucleic acid, which has a sequence and is characterized by being composed of 18 to 24 bases.

Preferably, the RNA interference-inducing nucleic acid is characterized in that the second to seventh base sequences at the 5' end are any one or more of the following:
An RNA interference-inducing nucleic acid that suppresses a non-canonical target gene of miR-124, the RNA interference-inducing nucleic acid (miR-124-BS) having a base sequence of 5'-AA GGC C-3' (SEQ ID NO: 1);
An RNA interference-inducing nucleic acid that suppresses a non-canonical target gene of miR-122, the RNA interference-inducing nucleic acid (miR-122-BS) having a base sequence of 5'-GG AGU U-3' (SEQ ID NO: 2);
An RNA interference-inducing nucleic acid that suppresses a non-canonical target gene of miR-155, the RNA interference-inducing nucleic acid (miR-155-BS) having a base sequence of 5'-UA AUG G-3' (SEQ ID NO: 3); or an RNA interference-inducing nucleic acid that suppresses a non-canonical target gene of miR-1, the RNA interference-inducing nucleic acid (miR-1-BS) having a base sequence of 5'-GG AAU U-3' (SEQ ID NO: 4) .

Preferably, an RNA interference-inducing nucleic acid is provided, characterized in that the base sequence of the RNA interference-inducing nucleic acid is one or more of the following:
An RNA interference-inducing nucleic acid (miR-124-BS) (sequence Number 5);
RNA interference-inducing nucleic acid (miR-122-BS) (sequence Number 6);
RNA interference-inducing nucleic acid (miR-155-BS) (sequence No. 7); or an RNA interference-inducing nucleic acid that suppresses a non-canonical target gene of miR-1, the RNA interference-inducing nucleic acid having a base sequence of 5'-UGG AAU UGU AAA GAA GUA UGU-3' (miR-1 -BS) (SEQ ID NO: 8).

Preferably, the RNA interference-inducing nucleic acid is characterized in that the base sequence between the 9th base from the 5' end is one or more of the following:
An RNA interference-inducing nucleic acid that suppresses a non-canonical target gene of miR-124, preferably in which at least one guanine base is replaced by Uracil in the second to seventh sequences of the 5'-end reference. 5'-UAA UGC ACG-3' (miR-124-G4U) (SEQ ID NO: 9) and 5'-UAA GUC ACG-3' (miR-124-G5U) with substituted modified base sequences. (SEQ ID NO: 10) or an RNA interference-inducing nucleic acid comprising the base sequence of 5'-UAA UUC ACG-3' (miR-124-G4,5U) (SEQ ID NO: 11);
An RNA interference-inducing nucleic acid that suppresses a non-canonical target gene of miR-1, preferably in which at least one guanine base is replaced by Uracil in the second to seventh sequences of the 5'-end reference. 5'-UUG AAU GUA-3' (miR-1-G2U) (SEQ ID NO: 12), 5'-UUG AAU GUA-3' (miR-1-G3U) with a substituted modified base sequence. (SEQ ID NO: 13), 5'-UGG AAU UUA-3' (miR-1-G7U) (SEQ ID NO: 14), 5'-UUU AAU GUA-3' (miR-1-G2,3U) (SEQ ID NO: 15 ), 5'-UUG AAU UUA-3' (miR-1-G3,7U) (SEQ ID NO: 16), 5'-UUG AAU UUA-3' (miR-1-G2,7U) (SEQ ID NO: 17), or , 5'-UUU AAU UUA-3' (miR-1-G2,3,7U) (SEQ ID NO: 18);
An RNA interference-inducing nucleic acid that suppresses a non-canonical target gene of miR-122, comprising:
5'-UUG AGU GUG-3' (miR-122-G2U) (SEQ ID NO: 19), 5'-UGU AGU GUG-3' (miR-122-G3U) (SEQ ID NO: 20), 5'-UGG AUU GUG -3' (miR-122-G5U) (SEQ ID NO: 21), 5'-UGG AGU UUG-3' (miR-122-G7U) (SEQ ID NO: 22), 5'-UGG AGU GUU-3' (miR- 122-G9U) (SEQ ID NO: 23), 5'-UUU AGU GUG-3' (miR-122-G2,3U) (SEQ ID NO: 24), 5'-UUG AUU GUG-3' (miR-122-G2, 5U) (SEQ ID NO: 25), 5'-UUG AGU UUG-3' (miR-122-G2, 7U) (SEQ ID NO: 26), 5'-UUG AGU GUU-3' (miR-122-G2, 9U) (SEQ ID NO: 27), 5'-UGU AUU GUG-3' (miR-122-G3,5U) (SEQ ID NO: 28), 5'-UGU AGU UUG-3' (miR-122-G3,7U) (SEQ ID NO: No. 29), 5'-UGU AGU GUU-3' (miR-122-G3,9U) (SEQ ID NO: 30), 5'-UGG AUU UUG-3' (miR-122-G5,7U) (SEQ ID NO: 31 ), 5'-UGG AUU GUU-3' (miR-122-G5,9U) (SEQ ID NO: 32), or 5'-UGG AGU UUU-3 (miR-122-G7,9U) (SEQ ID NO: 33) An RNA interference-inducing nucleic acid comprising a nucleotide sequence, preferably a modified nucleotide sequence in which at least one guanine base in the second to seventh 5' end reference sequences is replaced with uracil. 5'-UUG AGU GUG-3' (miR-122-G2U) (SEQ ID NO: 19), 5'-UUG AGU GUG-3' (miR-122-G3U) (SEQ ID NO: 20), 5'-UGG AUU GUG-3' (miR-122-G5U) (SEQ ID NO: 21), 5'-UGG AGU UUG-3' (miR-122-G7U) (SEQ ID NO: 22), 5'-UUU AGU GUG -3' (miR-122-G2,3U) (SEQ ID NO: 24), 5'-UUG AUU GUG-3' (miR-122-G2,5U) (SEQ ID NO: 25), 5'-UUG AGU UUG-3 '(miR-122-G2,7U) (SEQ ID NO: 26), 5'-UGU AUU GUG-3' (miR-122-G3,5U) (SEQ ID NO: 28), 5'-UGU AGU UUG-3' ( RNA interference-inducing nucleic acid comprising the base sequence of miR-122-G3,7U) (SEQ ID NO: 29), 5'-UGG AUU UUG-3' (miR-122-G5,7U) (SEQ ID NO: 31);
An RNA interference-inducing nucleic acid that suppresses a non-canonical target gene of miR-133, preferably in which at least one guanine base is replaced by uracil in the second to seventh sequences of the 5'-end reference. 5'-UUU UGU CCC-3' (miR-133-G4U) (SEQ ID NO: 34), 5'-UUU GUU CCC-3' (miR-133-G5U) with substituted modified base sequences. (SEQ ID NO: 35) or an RNA interference-inducing nucleic acid comprising the base sequence of 5'-UUU UUU CCC-3' (miR-124-G4,5U) (SEQ ID NO: 36);
An RNA interference-inducing nucleic acid that suppresses a non-canonical target gene of let-7, comprising:
5'-UUA GGU AGU-3' (let-7-G2U) (SEQ ID NO: 37), 5'-UGA UGU AGU-3' (let-7-G4U) (SEQ ID NO: 38), 5'-UGA GUU AGU -3' (let-7-G5U) (SEQ ID NO: 39), 5'-UGA GGU AUU-3' (let-7-G8U) (SEQ ID NO: 40), 5'-UUA UGU AGU-3' (let- 7-G2,4U) (SEQ ID NO: 41), 5'-UUA GUU AGU-3' (let-7-G2,5U) (SEQ ID NO: 42), 5'-UUA GGU AUU-3' (let-7- G2,8U) (SEQ ID NO: 43), 5'-UGA UUU AGU-3' (let-7-G4,5U) (SEQ ID NO: 44), 5'-UGA UGU AUU-3' (let-7-G4, 8U) (SEQ ID NO: 45), or 5'-UGA GUU AUU-3' (let-7-G5,8U) (SEQ ID NO: 46), preferably 5'5'-UUA GGU AGU-3' (let- 7-G2U) (SEQ ID NO: 37), 5'-UGA UGU AGU-3' (let-7-G4U) (SEQ ID NO: 38), 5'-UGA GUU AGU-3' (let-7-G5U) (Sequence No. 39), 5'-UUA UGU AGU-3' (let-7-G2, 4U) (SEQ ID NO: 41), 5'-UUA GUU AGU-3' (let-7-G2, 5U) (SEQ ID NO: 42) ), an RNA interference-inducing nucleic acid comprising the base sequence of 5'-UGA UUU AGU-3' (let-7-G4,5U) (SEQ ID NO: 44);
An RNA interference-inducing nucleic acid that suppresses a non-canonical target gene of miR-302a, preferably in which at least one guanine base is replaced by Uracil in the second to seventh sequences of the 5'-end reference. 5'-UAA UUG CUU-3' (miR-302a-G4U) (SEQ ID NO: 47), 5'-UAA GUU CUU-3' (miR-302a-G6U) with a substituted modified base sequence. (SEQ ID NO: 48), or an RNA interference-inducing nucleic acid comprising the base sequence of 5'-UAA UUU CUU-3' (miR-302a-G4,6U) (SEQ ID NO: 49); or a non-canonical target gene of miR-372. The RNA interference-inducing nucleic acid to be suppressed preferably has a modified base sequence in which at least one guanine base in the second to seventh sequences of the 5' end standard is replaced with uracil. and
5'-AAA UUG CUG-3' (miR-372-G4U) (SEQ ID NO: 50), 5'-AAA GUU CUG-3' (miR-372-G6U) (SEQ ID NO: 51), 5'-AAA GUG CUU -3' (miR-372-G9U) (SEQ ID NO: 52), 5'-AAA UUU CUG-3' (miR-372-G4,6U) (SEQ ID NO: 53), 5'-AAA UUG CUU-3' ( An RNA interference-inducing nucleic acid comprising the base sequence of miR-372-G4,9U) (SEQ ID NO: 54) or 5'-AAA GUU CUU-3' (miR-372-G6,9U) (SEQ ID NO: 55).

Preferably, an RNA interference-inducing nucleic acid is provided, characterized in that the base sequence of the RNA interference-inducing nucleic acid is one or more of the following:
An RNA interference-inducing nucleic acid that suppresses a non-canonical target gene of miR-124, comprising:
5'-UAA UGC ACG CGG UGA AUG CCA A-3' (miR-124-G4U) (SEQ ID NO: 56), 5'-UAA GUC ACG CGG UGA AUG CCA A-3' (miR-124-G5U) (SEQ ID NO: 56) No. 57) or an RNA interference-inducing nucleic acid showing the base sequence of 5'-UAA UUC ACG CGG UGA AUG CCA A-3' (miR-124-G4,5U) (SEQ ID No. 58);
An RNA interference-inducing nucleic acid that suppresses a non-canonical target gene of miR-1, comprising:
5'-UUG AAU GUA AAG AAG UAU GUA U-3' (miR-1-G2U) (SEQ ID NO: 59), 5'-UUG AAU GUA AAG AAG UAU GUA U-3' (miR-1-G3U) (SEQ ID NO: 59) No. 60), 5'-UGG AAU UUA AAG AAG UAU GUA U-3' (miR-1-G7U) (SEQ ID NO: 61), 5'-UUU AAU GUA AAG AAG UAU GUA U-3' (miR-1- G2,3U) (SEQ ID NO: 62), 5'-UGU AAU UUA AAG AAG UAU GUA U-3' (miR-1-G3,7U) (SEQ ID NO: 63), 5'-UUG AAU UUA AAG AAG UAU GUA U -3' (miR-1-G2,7U) (SEQ ID NO: 64) or 5'-UUU AAU UUA AAG AAG UAU GUA U-3' (miR-1-G2,3,7U) (SEQ ID NO: 65) base RNA interference-inducing nucleic acids showing sequences;
An RNA interference-inducing nucleic acid that suppresses a non-canonical target gene of miR-122, comprising: 5'-UUG AGU GUG ACA AUG GUG UUU G-3' (miR-122-G2U) (SEQ ID NO: 66), 5'-UGU AGU GUG ACA AUG GUG UUU G-3 (miR-122-G3U) (SEQ ID NO: 67), 5'-UGG AUU GUG ACA AUG GUG UUU G-3' (miR-122-G5U) (SEQ ID NO: 68), 5 '-UGG AGU UUG ACA AUG GUG UUU G-3' (miR-122-G7U) (SEQ ID NO: 69), 5'-UGG AGU GUU ACA AUG GUG UUU G-3' (miR-122-G9U) (SEQ ID NO: 70), 5'-UUU AGU GUG ACA AUG GUG UUU G-3' (miR-122-G2,3U) (SEQ ID NO: 71), 5'-UUG AUU GUG ACA AUG GUG UUU G-3' (miR-122 -G2,5U) (SEQ ID NO: 72), 5'-UUG AGU UUG ACA AUG GUG UUU G-3' (miR-122-G2,7U) (SEQ ID NO: 73), 5'-UUG AGU GUU ACA AUG GUG UUU G-3' (miR-122-G2,9U) (SEQ ID NO: 74), 5'-UGU AUU GUG ACA AUG GUG UUU G-3 (miR-122-G3,5U) (SEQ ID NO: 75), 5'- UGU AGU UUG ACA AUG GUG UUU G-3 (miR-122-G3,7U) (SEQ ID NO. 76), 5'-UGU AGU GUU ACA AUG GUG UUU G-3 (miR-122-G3,9U) (SEQ ID NO. 77), 5'-UGG AUU UUG ACA AUG GUG UUU G-3' (miR-122-G5,7U) (SEQ ID NO: 78), 5'-UGG AUU GUU ACA AUG GUG UUU G-3' (miR-122 -G5,9U) (SEQ ID NO: 79) or 5'-UGG AGU UUU ACA AUG GUG UUU G-3' (miR-122-G7,9U) (SEQ ID NO: 80);
An RNA interference-inducing nucleic acid that suppresses a non-canonical target gene of miR-133, comprising: 5'-UUU UGU CCC CUU CAA CCA GCU G-3' (miR-133-G4U) (SEQ ID NO: 81), 5'-UUU GUU CCC CUU CAA CCA GCU G-3' (miR-133-G5U) (SEQ ID NO: 82) or 5'-UUU UUU CCC CUU CAA CCA GCU G-3' (miR-133-G4,5U) (SEQ ID NO: 83 ) RNA interference-inducing nucleic acid comprising the base sequence;
An RNA interference-inducing nucleic acid that suppresses a non-canonical target gene of let-7, comprising: 5'-UUA GGU AGU AGG UUG UAU AGU U-3' (let-7-G2U) (SEQ ID NO: 84), 5'-UGA UGU AGU AGG UUG UAU AGU U-3' (let-7-G4U) (SEQ ID NO: 85), 5'-UGA GUU AGU AGG UUG UAU AGU U-3' (let-7-G5U) (SEQ ID NO: 86), 5'-UGA GGU AUU AGG UUG UAU AGU U-3' (let-7-G8U) (SEQ ID NO: 87), 5'-UUA UGU AGU AGG UUG UAU AGU U-3' (let-7-G2, 4U) (SEQ ID NO: 88), 5'-UUA GUU AGU AGG UUG UAU AGU U-3' (let-7-G2,5U) (SEQ ID NO: 89), 5'-UUA GGU AUU AGG UUG UAU AGU U-3' ( let-7-G2,8U) (SEQ ID NO: 90), 5'-UGA UUU AGU AGG UUG UAU AGU U-3' (let-7-G4, 5U) (SEQ ID NO: 91), 5'-UGA UGU AUU AGG UUG UAU AGU U-3' (let-7-G4, 8U) (SEQ ID NO: 92) or 5'-UGA GUU AUU AGG UUG UAU AGU U-3' (let-7-G5, 8U) (SEQ ID NO: 93) An RNA interference-inducing nucleic acid having a base sequence of;
An RNA interference-inducing nucleic acid that suppresses a non-canonical target gene of miR-302a, comprising: 5'-UAA UUG CUU CCA UGU UUU GGU GA-3' (miR-302a-G4U) (SEQ ID NO: 94), 5'-UAA GUU CUU CCA UGU UUU GGU GA-3' (miR-302a-G6U) (SEQ ID NO: 95), or 5'-UAA UUU CUU CCA UGU UUU GGU GA-3' (miR-302a-G4,6U) (SEQ ID NO: 96); or an RNA interference-inducing nucleic acid that suppresses a non-canonical target gene of miR-372, which is 5'-AAA UUG CUG CGA CAU UUG AGC GU-3' (miR-372 -G4U) (SEQ ID NO: 97), 5'-AAA GUU CUG CGA CAU UUG AGC GU-3' (miR-372-G6U) (SEQ ID NO: 98), 5'-AAA GUG CUU CGA CAU UUG AGC GU-3' (miR-372-G9U) (SEQ ID NO: 99), 5'-AAA UUU CUG CGA CAU UUG AGC GU-3' (miR-372-G4,6U) (SEQ ID NO: 100), 5'-AAA UUG CUU CGA CAU UUG AGC GU-3' (miR-372-G4,9U) (SEQ ID NO: 101), or 5'-AAA GUU CUU CGA CAU UUG AGC GU-3' (miR-372-G6,9U) (SEQ ID NO: 102) An RNA interference-inducing nucleic acid having the base sequence of

A composition for suppressing the expression of a non-canonical target gene of microRNA, which includes the RNA interference-inducing nucleic acid of the present invention, is provided.

Provided is a method for suppressing the expression of non-canonical target genes of microRNAs, which includes the step of administering to an individual a composition containing the RNA interference-inducing nucleic acid of the present invention.

Provided is a use of the RNA interference-inducing nucleic acid of the present invention to suppress the expression of a non-canonical target gene of microRNA.

Provided are compositions for regulating cell cycle, differentiation, reverse differentiation, morphology, migration, reverse differentiation, division, proliferation, or death, which contain the RNA interference-inducing nucleic acid of the present invention.

A method of regulating cell cycle, differentiation, reverse differentiation, morphology, migration, division, proliferation, or death is provided, which comprises administering to an individual a composition comprising an RNA interference-inducing nucleic acid of the invention.

Uses of the RNA interference-inducing nucleic acids of the present invention for regulating cell cycle, differentiation, reverse differentiation, morphology, migration, division, proliferation, or death are provided.

Preferably, the composition provides a composition characterized in that it is any one or more of the following:
A composition for inducing cancer cell death, comprising an RNA interference inducing nucleic acid that suppresses a non-canonical target gene of miR-124;
A composition for inducing neurite branching differentiation, comprising an RNA interference inducing nucleic acid that suppresses a non-canonical target gene of miR-124;
A composition for inducing cell cycle arrest in hepatoma cells, comprising an RNA interference-inducing nucleic acid that suppresses a non-canonical target gene of miR-122;
A composition for promoting muscle cell differentiation or muscle fibrosis, comprising an RNA interference-inducing nucleic acid that suppresses a non-canonical target gene of miR-1;
A composition for inducing muscle cell death, comprising an RNA interference inducing nucleic acid that suppresses a non-canonical target gene of miR-155;
A composition for promoting cell death of neuroblastoma, comprising an RNA interference-inducing nucleic acid that suppresses a non-canonical target gene of miR-124;
A composition for promoting cell division and proliferation of neuroblastoma, comprising an RNA interference-inducing nucleic acid that suppresses a non-canonical target gene of miR-124;
A composition for inducing myocardial hypertrophy, comprising an RNA interference-inducing nucleic acid that suppresses a non-canonical target gene of miR-1;
A composition for inducing myocardial hypertrophy comprising an RNA interference inducing nucleic acid that suppresses a non-canonical target gene of miR-133;
A composition for inducing cell cycle arrest in cancer cells, comprising an RNA interference-inducing nucleic acid that suppresses a non-canonical target gene of let-7;
A composition for inducing cell cycle progression activity of hepatocytes, comprising an RNA interference-inducing nucleic acid that suppresses a non-canonical target gene of let-7;
A composition for promoting reverse differentiation comprising an RNA interference-inducing nucleic acid that suppresses a non-canonical target gene of miR-302a;
A composition for promoting reverse differentiation comprising an RNA interference-inducing nucleic acid that suppresses a non-canonical target gene of miR-372; or a composition for inhibiting cell migration of hepatoma cells comprising an RNA interference-inducing nucleic acid that suppresses a non-canonical target gene of miR-122 thing.

The present invention involves the steps below, in which a partial sequence of a specific microRNA is modified in one or more single strands of the double strands of a nucleic acid that induces RNA interference, resulting in non-specific microRNAs. Provided is a method for producing an RNA interference-inducing nucleic acid that suppresses the expression of a noncanonical target gene:
Contains the 4 base sequence from the 2nd to 5th base from the 5' end of the specific microRNA, the 6th and 7th bases are the same, and the 6th and 7th bases are the 6th base sequence of the specific microRNA. All complementary bases that have a complementary base sequence to a base that can be aligned with the base and can be aligned with the 6th base of the specific microRNA include G:A, G:U wobble sequences. A bulge is created in the target gene between the 5th and 6th positions of the microRNA, and the bulge disappears at the non-canonical target base sequence that binds, so that it binds with a continuous base sequence. or a modified base in which at least one guanine base is substituted with uracil in the base sequence between the 9th base from the 5' end of the specific microRNA. A step of producing an RNA interference-inducing nucleic acid so that the G:A or G:U wobble sequence at the site becomes a regular base sequence of U:A or A:U.

The present invention also provides a method for screening test substances for modulating cell cycle, differentiation, retrodifferentiation, morphology, migration, division, proliferation, retrodifferentiation or death, including the following stages:
transfecting a target cell with an RNA interference-inducing nucleic acid;
A step of treating the target cells with a test substance; and a step of confirming the amount or presence or absence of expression of the non-canonical target gene of the microRNA suppressed by the RNA interference-inducing nucleic acid in the target cells.

Furthermore, the present invention provides the following 1 to 3 for RNA interference-inducing nucleic acids:

1.2'OMe-modified RNA interference-inducing nucleic acid The present invention provides a method for detecting a part of a specific microRNA in one or more single strands of the double strands of a nucleic acid that induces RNA interference. An RNA interference-inducing nucleic acid whose sequence is modified to suppress a noncanonical target gene of a microRNA, the nucleic acid comprising:
The RNA interference-inducing nucleic acid includes a sequence of four bases from the second to the fifth from the 5' end of the specific microRNA, the sixth and seventh bases are the same, and the sixth and seventh bases are , has a complementary base sequence to a base that can be aligned with the 6th base of the specific microRNA, and the base that can be aligned with the 6th base of the specific microRNA includes G:A, G:U wobble. A bulge is created in the target gene between the 5th and 6th positions of the microRNA so that all complementary bases containing the sequence are included, and the bulge disappears at the non-canonical target base sequence that binds, creating a continuous sequence. It is characterized by binding with a specific base sequence,
The specific microRNA provides an RNA interference-inducing nucleic acid characterized in that a methyl group (2'OMe) is added to the 2' position of the ribose ring of the 6th nucleotide from the 5' end.

Preferably, the RNA interference-inducing nucleic acid suppresses only the expression of a regular seed target gene of the RNA interference-inducing nucleic acid.

Preferably, the RNA interference-inducing nucleic acid is characterized in that it specifically suppresses only non-canonical nuclear protrusion sites of specific microRNAs and removes non-canonical nuclear protrusion sites that may newly occur. provide.

The present invention also provides a composition for suppressing the expression of a non-canonical target gene of microRNA, which includes an RNA interference-inducing nucleic acid.

The present invention also provides a method for suppressing the expression of a non-canonical target gene of microRNA, which includes the step of administering to an individual a composition containing an RNA interference-inducing nucleic acid.

The present invention also provides uses of RNA interference-inducing nucleic acids to suppress the expression of non-canonical target genes of microRNAs.

In addition, the present invention includes the following steps, in which a part of the sequence of a specific microRNA is modified in one or more single strands of the double strands of a nucleic acid that induces RNA interference, and the microRNA is transformed into a microRNA. A method for producing an RNA interference-inducing nucleic acid that suppresses the expression of a noncanonical target gene, the method comprising:
Contains the four base sequences from the second to the fifth from the 5' end of the specific microRNA, the 6th and 7th bases are the same, and the 6th and 7th bases are the 6th base sequence of the specific microRNA. The bases that can be aligned with the 6th base of the specific microRNA include all complementary bases including G:A, G:U wobble sequences. A bulge is created in the target gene between the 5th and 6th nucleotides of the microRNA, and the bulge disappears at the non-canonical target nucleotide sequence, and the bulge is combined with a continuous nucleotide sequence. and the step of adding a methyl group (2'OMe) to the 2' position of the ribose ring of the 6th nucleotide from the 5' end of the specific microRNA. A method for producing an RNA interference-inducing nucleic acid is provided.

2. Regarding RNA interference-inducing nucleic acids (SEQ ID NOs: 103-528) that suppress non-canonical nuclear protuberance target sites of microRNAs An RNA interference-inducing nucleic acid that suppresses a noncanonical target gene of a microRNA by modifying a partial sequence of a specific microRNA in a single strand,
The RNA interference-inducing nucleic acid includes a sequence of four bases from the second to the fifth from the 5' end of the specific microRNA, the sixth and seventh bases are the same, and the sixth and seventh bases are , has a complementary base sequence to a base that can be aligned with the 6th base of the specific microRNA, and the base that can be aligned with the 6th base of the specific microRNA includes G:A, G:U wobble. A bulge is created in the target gene between the 5th and 6th positions of the microRNA so that all complementary bases containing the sequence are included, and the bulge disappears at the non-canonical target base sequence that binds, creating a continuous sequence. Provided is an RNA interference-inducing nucleic acid, which is characterized in that it binds with a unique base sequence.

Preferably, the RNA interference-inducing nucleic acid is characterized in that it selectively suppresses only non-canonical nuclear protuberance target sites and does not suppress normal target genes of microRNA.

Preferably, the specific microRNA is let-7/98/4458/4500, miR-125a-5p/125b-5p/351/670/4319, miR-124/124ab/506, miR-9/9ab, miR -29abcd, miR-103a/107/107ab, miR-221/222/222ab/1928, miR-26ab/1297/4465, miR-15abc/16/16abc/195/322/424/497/1907, miR-126 -3p, miR-30abcdef/30abe-5p/384-5p, miR-33ab/33-5p, miR-34ac/34bc-5p/449abc/449c-5p, miR-19ab, miR-99ab/100, miR-17 /17-5p/20ab/20b-5p/93/106ab/427/518a-3p/519d, miR-27abc/27a-3p, miR-218/218a, miR-22/22-3p, miR-185/882 /3473/4306/4644, miR-181abcd/4262, miR-338/338-3p, miR-127/127-3p, miR-101/101ab, miR-149, miR-324-5p, miR-24/24ab /24-3p, miR-33a-3p/365/365-3p, miR-139-5p, miR-138/138ab, miR-143/1721/4770, miR-25/32/92abc/363/363-3p /367, miR-574-5p, miR-7/7ab, miR-145, miR-135ab/135a-5p, miR-148ab-3p/152, miR-28-5p/708/1407/1653/3139, miR -130ac/301ab/301b/301b-3p/454/721/4295/3666, miR-3132, miR-155, miR-485-3p, miR-132/212/212-3p, hsa-miR-9-3p , miR-374ab, miR-129-3p/129ab-3p/129-1-3p/129-2-3p, hsa-miR-126-5p, miR-425/425-5p/489, miR-423-3p , miR-21/590-5p, miR-31, hsa-miR-20b-3p, hsa-let-7d-3p, miR-191, miR-18ab/4735-3p, miR-369-3p, hsa-miR -5187-5p, miR-382, miR-485-5p/1698/1703/1962, hsa-miR-136-3p, miR-576-3p, miR-204/204b/211, miR-769-5p, miR -342-5p/4664-5p, miR-361-5p, miR-199ab-3p/3129-5p, miR-142-3p, miR-299-5p/3563-5p, miR-193/193b/193a-3p , hsa-miR-1277-5p, miR-140/140-5p/876-3p/1244, hsa-miR-30a/d/e-3p, hsa-let-7i-3p, miR-409-5p/409a , miR-379/1193-5p/3529, miR-136, miR-154/872, miR-4684-3p, miR-361-3p, miR-335/335-5p, miR-423a/423-5p/3184 /3573-5p, miR-371/373/371b-5p, miR-1185/3679-5p, miR-3613-3p, miR-93/93a/105/106a/291a-3p/294/295/302abcde/372 /373/428/519a/520be/520acd-3p/1378/1420ac, miR-876-5p/3167, miR-329/329ab/362-3p, miR-582-5p, miR-146ac/146b-5p, miR -380/380-3p, miR-499-3p/499a-3p, miR-551a, miR-142-5p, hsa-miR-17-3p, miR-199ab-5p, miR-542-3p, miR-1277 , hsa-miR-29c-5p, miR-3145-3p, hsa-miR-106b-3p, hsa-miR-22-5p, miR-744/1716, hsa-miR-132-5p, miR-488, miR -501-3p/502-3p/500/502a, miR-486-5p/3107, miR-450a/451a, hsa-miR-30c-3p, miR-499-5p, miR-421, miR-197, miR -296-5p, miR-326/330/330-5p, miR-214/761/3619-5p, miR-612/1285/3187-5p, miR-409-3p, miR-378/422a/378bcdefhi, miR -342-3p, miR-338-5p, miR-625, miR-200bc/429/548a, hsa-miR-376a-5p, miR-584, miR-411, miR-573/3533/3616-5p/3647 -5p, miR-885-5p, hsa-miR-99-3p, miR-876-3p, miR-654-3p, hsa-miR-340-3p, miR-3614-5p, hsa-miR-124-5p , miR-491-5p, miR-96/507/1271, miR-548a-3p/548ef/2285a, hsa-miR-32-3p, miR-3942-5p/4703-5p, miR-34b/449c/1360 /2682, hsa-miR-23a/b-5p, miR-362-5p/500b, miR-677/4420, miR-577, miR-3613-5p, miR-369-5p, miR-150/5127, miR -544/544ab/544-3p, hsa-miR-29a-5p, miR-873, miR-3614-3p, miR-186, miR-483-3p, hsa-miR-374a-3p, miR-196abc, hsa -miR-145-3p, hsa-miR-29b-2-5p, hsa-miR-221-5p, miR-323b-3p, miR-616, miR-330-3p, hsa-miR-7-3p, miR -187, hsa-miR-26a-3p, miR-452/4676-3p, miR-129-5p/129ab-5p, miR-223, miR-4755-3p, miR-1247, miR-3129-3p, hsa -miR-335-3p, miR-542-5p, hsa-miR-181a-3p, hsa-miR-186-3p, hsa-miR-27b-5p, miR-491-3p, miR-4687-3p, hsa -miR-101-5p, miR-4772-5p, miR-337-3p, hsa-miR-223-5p, hsa-miR-16/195-3p, miR-3677-3p, hsa-miR-766-5p , miR-299/299-3p/3563-3p, miR-3140-3p, miR-532-5p/511, hsa-miR-24-5p, miR-4778-5p, miR-642b, miR-483-5p , miR-767-5p, hsa-miR-31-3p, miR-574-3p, miR-3173-3p, miR-2127/4728-5p, hsa-miR-103a-2-5p, miR-3591-3p , hsa-miR-625-3p, hsa-miR-15b-3p, miR-522/518e/1422p, miR548d-3p/548acbz, hsa-miR-452-3p, miR-192/215, miR-1551/4524 , hsa-miR-425-3p, miR-3126-3p, hsa-miR-125b-2-3p, miR-324-3p/1913, hsa-miR-141-5p, hsa-miR-365a/b-5p , hsa-miR-29b-1-5p, miR-563/380-5p, miR-1304, miR-216c/1461/4684-5p, hsa-miR-2681-5p, miR-194, miR-296-3p , hsa-miR-205-3p, miR-888, miR-4802-3p, hsa-let-7a/g-3p, miR-762/4492/4498, hsa-miR-744-3p, hsa-miR-148b -5p, miR-514/514b-3p, miR-28-3p, miR-550a, hsa-miR-125b-1-3p, hsa-miR-506-5p, hsa-miR-1306-5p, miR-3189 -3p, miR-675-5p/4466, hsa-miR-34a-3p, hsa-miR-454-5p, miR-509-5p/509-3-5p/4418, hsa-miR-19a/b-5p , miR-4755-5p, hsa-miR-93-3p, miR-3130-5p/4482, hsa-miR-488-5p, hsa-miR-378a-5p, miR-575/4676-5p, miR-1307 , miR-3942-3p, miR-4677-5p, miR-339-3p, miR-548b-3p, hsa-miR-642b-5p, miR-188-5p, hsa-miR-652-5p, miR-2114 , miR-3688-5p, hsa-miR-15a-3p, hsa-miR-181c-3p, miR-122/122a/1352, miR-556-3p, hsa-miR-218-2-3p, miR-643 , miR-140-3p, miR-1245, hsa-miR-2115-3p, miR-518bcf/518a-3p/518d-3p, miR-3200-3p, miR-545/3065/3065-5p, miR-1903 /4778-3p, hsa-miR-302a-5p, hsa-miR-183-3p, miR-3144-5p, miR-582-3p, miR-4662a-3p, miR-3140-5p, hsa-miR-106a -3p, hsa-miR-135a-3p, miR-345/345-5p, miR-125a-3p/1554, miR-3145-5p, miR-676, miR-3173-5p, hsa-miR-5586-3p , miR-615-3p, miR-3688-3p, miR-4662a-5p, miR-4659ab-5p, hsa-miR-5586-5p, hsa-miR-514a-5p, miR-10abc/10a-5p, hsa -miR-888-3p, miR-3127-5p, miR-508-3p, hsa-miR-185-3p, hsa-miR-200c-5p, hsa-miR-550a-3p, miR-513c/514b-5p , miR-490-3p, hsa-miR-5187-3p, miR-3664-3p, miR-3189-5p, miR-4670-3p, miR-105/105ab, hsa-miR-135b-3p, hsa-miR -5010-3p, miR-493/493b, miR-3605-3p, miR-188-3p, hsa-miR-449c-3p, miR-4761-5p, miR-224, miR-4796-5p, hsa-miR -551b-5p, miR-556-5p, hsa-miR-122-3p, miR-4677-3p, miR-877, miR-576-5p, miR-490-5p, hsa-miR-589-3p, miR -4786-3p, hsa-miR-374b-3p, hsa-miR-26b-3p, miR-3158-3p, miR-4423-3p, miR-518d-5p/519bc-5p520c-5p/523b/526a, miR -4707-3p, hsa-miR-10a-3p, miR-526b, hsa-miR-676-5p, hsa-miR-660-3p, hsa-miR-5004-3p, miR-193a-5p, hsa-miR -222-5p, miR-4661-3p, hsa-miR-25-5p, miR-4670-5p, miR-659, miR-1745/3194-3p, hsa-miR-182-3p, miR-298/2347 /2467-3p, hsa-miR-130b-5p, miR-4746-3p, miR-1




893/2277-5p, miR-3619-3p, hsa-miR-138-1-3p, miR-4728-3p, miR-3127-3p, miR-671-3p, hsa-miR-211-3p, hsa- miR-2114-3p, hsa-miR-877-3p, miR-3157-5p, miR-502-5p, miR-500a, miR-548g, miR-523, hsa-miR-584-3p, miR-205/ 205ab, miR-4793-5p, hsa-miR-363-5p, hsa-miR-214-5p, miR-3180-5p, miR-1404/2110, miR-3157-3p, hsa-miR-191-3p, miR-1346/3940-5p/4507, miR-4746-5p, miR-3939, hsa-miR-181a-2-3p, hsa-miR-500a-3p, hsa-miR-196b-3p, hsa-miR- 675-3p, hsa-miR-548aj/g/x-5p, miR-4659ab-3p, hsa-miR-5001-3p, hsa-miR-1247-3p, miR-2890/4707-5p, hsa-miR- 150-3p, hsa-miR-629-3p, miR-2277-3p, miR-3547/3663-3p, miR-34bc-3p, miR-518ef, miR-3187-3p, miR-1306/1306-3p, miR-3177-3p, miR-1ab/206/613, miR-128/128ab, miR-1296, miR-598/598-3p, miR-887, miR-1-5p, miR-376c/741-5p, miR-374c/655, miR-494, miR-651, miR-1301/5047, miR-381-5p, miR-216a, miR-300/381/539-3p, miR-1249, miR-579, miR- 656, miR-433, miR-1180, miR-597/1970, miR-190a-3p, miR-1537, miR-874-5p, miR-410/344de/344b-1-3p, miR-370, miR- 219-2-3p/219-3p, miR-3620, miR-504/4725-5p, miR-2964/2964a-5p, miR-450a-2-3p, miR-511, miR-6505-3p, miR- 433-5p, miR-6741-3p, miR-370-5p, miR-579-5p, miR-376c-5p, miR-376b-5p, miR-552/3097-5p, miR-1910, miR-758, 4 elements selected from the group consisting of miR-6735-3p, miR-376a-2-5p, miR-585miR-451, and miR-137/137ab, from the second to the fifth from the 5' end The 6th and 7th bases are the same, and the 6th and 7th bases have a complementary base sequence to a base that can be aligned with the 6th base of the specific microRNA. Provided is an RNA interference-inducing nucleic acid characterized by being composed of 6 to 24 bases.

Preferably, the RNA interference-inducing nucleic acid is characterized in that it contains a base sequence represented by any one or more of SEQ ID NOs: 103 to 528 (see Table 3).

The present invention also provides a composition for suppressing the expression of a non-canonical target gene of microRNA, which includes an RNA interference-inducing nucleic acid.

The present invention also provides a method for suppressing the expression of a non-canonical target gene of microRNA, which includes the step of administering to an individual a composition containing an RNA interference-inducing nucleic acid.

The present invention also provides uses of RNA interference-inducing nucleic acids to suppress the expression of non-canonical target genes of microRNAs.

In addition, the present invention includes the following steps, in which a part of the sequence of a specific microRNA is modified in one or more single strands of the double strands of a nucleic acid that induces RNA interference, resulting in microRNA A method for producing an RNA interference-inducing nucleic acid that suppresses the expression of a noncanonical target gene, the method comprising:
Contains the four base sequences from the second to the fifth from the 5' end of the specific microRNA, the 6th and 7th bases are the same, and the 6th and 7th bases are the 6th base sequence of the specific microRNA. The bases that can be aligned with the 6th base of the specific microRNA include all complementary bases including G:A, G:U wobble sequences. A bulge is created in the target gene between the 5th and 6th nucleotides of the microRNA, and the bulge disappears at the non-canonical target nucleotide sequence, and the bulge is combined with a continuous nucleotide sequence. Provided is a method for producing an RNA interference-inducing nucleic acid, the method comprising the step of producing an RNA interference-inducing nucleic acid as described above.

3. Regarding RNA interference-inducing nucleic acids (SEQ ID NOs: 529-863) that suppress the non-canonical G:A wobble target site of microRNAs, the present invention is directed to one of the double strands of nucleic acids that induce RNA interference (RNA interference). An RNA interference-inducing nucleic acid that suppresses a noncanonical target gene of a microRNA by modifying a partial sequence of a specific microRNA in the above single strand,
The RNA interference-inducing nucleic acid is a modified base in which at least one guanine base is substituted with a uracil base in the base sequence between the second to ninth bases from the 5' end of a specific microRNA. The present invention provides an RNA interference-inducing nucleic acid, which has a sequence in which the G:A wobble at the site becomes a regular base sequence of U:A.

Preferably, the RNA interference-inducing nucleic acid includes a sequence of 6 to 8 consecutive bases starting from the second base from the 5' end of the specific microRNA,
The base sequence provides an RNA interference-inducing nucleic acid characterized in that at least one guanine base is replaced with a uracil base.

Preferably, the RNA interference-inducing nucleic acid selectively suppresses only non-canonical target genes of microRNAs bound by a G:A wobble sequence, but does not suppress regular target genes of microRNAs. , provides RNA interference-inducing nucleic acids.

Preferably, the specific microRNA is hsa-miR-1-3p, hsa-miR-194-5p, hsa-miR-193a-5p, hsa-miR-15b-3p, hsa-miR-200c-5p, hsa -miR-214-5p, hsa-miR-134-5p, hsa-miR-145-3p, hsa-miR-22-5p, hsa-miR-423-3p, hsa-miR-873-3p, hsa-miR -122-5p, hsa-miR-143-3p, hsa-miR-485-5p, hsa-miR-409-5p, hsa-miR-24-3p, hsa-miR-223-3p, hsa-miR-144 -5p, hsa-miR-379-5p, hsa-miR-146b-5p/hsa-miR-146a-5p, hsa-miR-539-5p, hsa-miR-296-5p, hsa-miR-767-5p , hsa-miR-34a-5p/hsa-miR-34c-5p, hsa-let-7f-5p/hsa-let-7d-5p/hsa-let-7b-5p/hsa-let-7a-5p/hsa -let-7e-5p/hsa-miR-202-3p/hsa-let-7i-5p/hsa-miR-98-5p/hsa-let-7c-5p/hsa-let-7g-5p, hsa-miR -1271-3p, hsa-miR-138-5p, hsa-miR-19b-3p/hsa-miR-19a-3p, hsa-miR-27a-5p, hsa-miR-146b-3p, hsa-miR-7 -5p, hsa-miR-423-5p, hsa-miR-324-5p, hsa-miR-629-5p, hsa-miR-139-3p, hsa-miR-30d-5p/hsa-miR-30e-5p /hsa-miR-30a-5p/hsa-miR-30c-5p/hsa-miR-30b-5p, hsa-miR-221-3p/hsa-miR-222-3p, hsa-miR-509-3p, hsa -miR-769-5p, hsa-miR-142-3p, hsa-miR-185-5p, hsa-miR-508-3p/hsa-miR-219a-5p, hsa-miR-31-5p, hsa-miR -103a-3p/hsa-miR-107, hsa-miR-542-3p, hsa-miR-219a-2-3p, hsa-miR-29c-3p/hsa-miR-29a-3p/hsa-miR-29b -3p, hsa-miR-125b-1-3p, hsa-miR-411-5p, hsa-miR-196a-5p/hsa-miR-196b-5p, hsa-miR-3622a-5p, hsa-miR-127 -5p, hsa-miR-22-3p, hsa-miR-153-3p, hsa-miR-15b-5p/hsa-miR-16-5p/hsa-miR-424-5p, hsa-let-7g-3p /hsa-miR-493-5p/hsa-let-7c-3p, hsa-let-7i-3p, hsa-miR-218-5p, hsa-miR-1307-5p, hsa-miR-127-3p, hsa -miR-210-3p, hsa-miR-187-3p, hsa-miR-192-3p, hsa-miR-192-5p, hsa-miR-21-5p, hsa-miR-500a-3p, hsa-miR -203a-3p, hsa-miR-30c-2-3p, hsa-miR-488-3p, hsa-miR-301a-3p/hsa-miR-301b-3p, hsa-miR-126-3p, hsa-miR -26b-3p, hsa-miR-324-3p, hsa-miR-3065-3p, hsa-miR-124-5p, hsa-miR-345-5p, hsa-miR-615-3p, hsa-miR-889 -5p/hsa-miR-135a-5p/hsa-miR-135b-5p, hsa-miR-18a-5p, hsa-miR-708-5p/hsa-miR-28-5p, hsa-miR-224-5p , hsa-miR-100-3p, hsa-miR-873-5p, hsa-miR-4662a-5p, hsa-miR-99b-3p/hsa-miR-99a-3p, hsa-miR-433-5p, hsa -miR-3605-5p, hsa-miR-744-5p, hsa-miR-1296-5p, hsa-miR-133a-3p, hsa-miR-382-5p, hsa-miR-425-5p, hsa-miR -377-5p, hsa-miR-3180-3p, hsa-miR-758-3p, hsa-miR-93-3p, hsa-miR-154-5p, hsa-miR-124-3p, hsa-miR-194 -3p, hsa-miR-375, hsa-miR-148a-5p, hsa-miR-2277-5p, hsa-miR-17-3p, hsa-miR-4772-3p, hsa-miR-329-5p, hsa -miR-182-5p/hsa-miR-96-5p, hsa-miR-2467-5p/hsa-miR-485-5p, hsa-miR-149-5p, hsa-miR-29b-2-5p, hsa -miR-122-3p, hsa-miR-302a-3p/hsa-miR-520a-3p/hsa-miR-519b-3p/hsa-miR-520b/hsa-miR-519c-3p/hsa-miR-520c -3p/hsa-miR-519a-3p, hsa-miR-532-5p, hsa-miR-132-5p, hsa-miR-541-5p, hsa-miR-671-3p, hsa-miR-518e-3p , hsa-miR-487a-5p, hsa-miR-589-5p/hsa-miR-146b-5p/hsa-miR-146a-5p, hsa-miR-196b-5p/hsa-miR-196a-5p, hsa -miR-486-3p, hsa-miR-378a-3p, hsa-miR-27b-5p, hsa-miR-6720-3p, hsa-miR-574-3p, hsa-miR-29a-5p, hsa-miR -30c-2-3p/hsa-miR-30c-1-3p, hsa-miR-199b-3p, hsa-miR-574-5p, hsa-miR-4677-3p, hsa-miR-654-3p, hsa -miR-652-3p, hsa-miR-19a-3p/hsa-miR-19b-3p, hsa-let-7c-5p/hsa-miR-98-5p/hsa-let-7g-5p/hsa-let -7f-5p/hsa-miR-202-3p/hsa-let-7b-5p/hsa-let-7e-5p/hsa-let-7a-5p/hsa-let-7d-5p/hsa-let-7i -5p, hsa-miR-3663-3p, hsa-miR-152-3p/hsa-miR-148b-3p/hsa-miR-148a-3p, hsa-miR-193b-5p, hsa-miR-502-3p /hsa-miR-501-3p, hsa-miR-299-3p, hsa-miR-140-5p, hsa-miR-96-5p/hsa-miR-182-5p, hsa-miR-193b-3p, hsa -miR-365a-3p, hsa-miR-486-5p, hsa--493-3p, hsa-miR-548am-5p, hsa-miR-20b-5p/hsa-miR-20a-5p/hsa-miR- 93-5p/hsa-miR-17-5p/hsa-miR-106b-5p, hsa-miR-541-3p, hsa-miR-452-5p, hsa-miR-221-5p, hsa-miR-518f- 3p, hsa-miR-370-3p, hsa-miR-107/hsa-miR-103a-3p, hsa-miR-338-3p, hsa-miR-409-3p, hsa-let-7d-5p/hsa- let-7g-5p/hsa-let-7i-5p/hsa-let-7f-5p/hsa-let-7e-5p/hsa-let-7a-5p/hsa-let-7b-5p/hsa-let- 7c-5p, hsa-miR-130b-3p/hsa-miR-301a-3p/hsa-miR-130a-3p/hsa-miR-301b-3p, hsa-miR-512-3p, hsa-miR-191- 5p, hsa-miR-509-3-5p, hsa-miR-92a-3p/hsa-miR-92b-3p/hsa-miR-363-3p/hsa-miR-25-3p/hsa-miR-32- 5p, hsa-miR-183-5p, hsa-miR-1307-3p, hsa-miR-499a-5p/hsa-miR-208a-3p, hsa-miR-186-5p, hsa-miR-450b-5p, hsa-miR-450a-5p, hsa-miR-101-3p/hsa-miR-144-3p, hsa-miR-320a, hsa-miR-199b-5p/hsa-miR-199a-5p, hsa-miR- 135a-5p, hsa-miR-145-5p, hsa-miR-26a-5p, hsa-miR-34c-5p, hsa-miR-125b-5p, hsa-miR-526b-5p, hsa-miR-16- 5p/hsa-miR-15b-5p/hsa-miR-424-5p/hsa-miR-15a-5p, hsa-miR-9-3p, hsa-miR-363-5p, hsa-miR-1298-3p, hsa-miR-148a-3p, hsa-miR-302a-3p, hsa-miR-9-5p, hsa-miR-28-3p, hsa-miR-508-3p, hsa-miR-137, hsa-miR- 5010-5p, hsa-miR-523-5p, hsa-miR-128-3p, hsa-miR-199a-5p/hsa-miR-199b-5p, hsa-miR-181a-2-3p, hsa-miR- 27a-3p/hsa-miR-27b-3p, hsa-let-7d-3p, hsa-miR-129-5p, hsa-miR-424-3p, hsa-miR-181a-3p, hsa-miR-10a- 5p, hsa-miR-196b-5p, hsa-miR-92a-1-5p, hsa-miR-483-5p, hsa-miR-1537-3p, hsa-miR-106b-5p/hsa-miR-20a- 5p/hsa-miR-17-5p/hsa-miR-93-5p, hsa-miR-30a-3p/hsa-miR-30e-3p, hsa-miR-374a-3p, hsa-miR-675-5p, hsa-miR-503-5p, hsa-miR-340-5p, hsa-miR-208a-3p, hsa-miR-200b-3p/hsa-miR-200c-3p, hsa-miR-518f-5p/hsa- miR-523-5p, hsa-miR-625-3p, hsa-miR-194-5p, hsa-let-7g-3p, hsa-miR-514a-5p, hsa-miR-381-3p, hsa-miR- 513c-5p/hsa-miR-514b-5p, hsa-miR-520a-5p, hsa-miR-125b-5p/hsa-miR-125a-5p, hsa-miR-141-3p, hsa-miR-874- 3p, hsa-miR-202-5p, hsa-miR-140-3p, hsa-miR-361-3p, hsa-miR-513b-5p, hsa-miR-33a-5p, hsa-let-7a-5p/ hsa-let-7c-5p/hsa-let-7b-5p/hsa-let-7d-5p/hsa-let-7f-5p/hsa-let-7e-5p/hsa-let-7i-5p/hsa- let-7g-5p, hsa-miR-136-3p, hsa-miR-508-5p, hsa-miR-204-5p/hsa-miR-211-5p, hsa-miR-146a-5p/hsa-miR- 146b-5p, hsa-miR-23a-3p, hsa-miR-21-3p, hsa-miR-877-5p, hsa-miR-302a-5p, hsa-miR-139-5p, hsa-miR-99a- 5p/hsa-miR-100-5p/hsa-miR-99b-5p,




selected from the group consisting of hsa-miR-216a-5p and hsa-miR-3157-3p, preferably guanine ( 6, while having a modified base sequence in which at least one (Guanine) base is substituted with uracil (Uracil) so that the G:A wobble sequence at the site becomes a regular U:A base sequence. Provided is an RNA interference-inducing nucleic acid characterized by being composed of ~24 bases.

Preferably, the RNA interference-inducing nucleic acid has a sequence of 6 to 8 consecutive bases starting from the 2nd base from the 5' end to the 7th base, or from the 2nd base to the 9th base, as shown in SEQ ID NO: The present invention provides an RNA interference-inducing nucleic acid characterized by being represented by any one or more of 529 to 863 (see Table 4).

The present invention also provides a composition for suppressing the expression of a non-canonical target gene of microRNA, which includes the RNA interference-inducing nucleic acid.

The present invention also provides a method for suppressing the expression of a non-canonical target gene of microRNA, which includes the step of administering to an individual a composition containing an RNA interference-inducing nucleic acid.

The present invention also provides uses of RNA interference-inducing nucleic acids to suppress the expression of non-canonical target genes of microRNAs.

In addition, the present invention includes the following steps, in which a part of the sequence of a specific microRNA is modified in one or more single strands of the double strands of a nucleic acid that induces RNA interference, resulting in microRNA A method for producing an RNA interference-inducing nucleic acid that suppresses the expression of a noncanonical target gene, the method comprising:
A modified base sequence in which at least one guanine base is replaced with a uracil base among 6 to 8 consecutive base sequences starting from the second base from the 5' end of a specific microRNA. Provided is a method for producing an RNA interference-inducing nucleic acid, the method comprising the step of producing an RNA interference-inducing nucleic acid having the following properties.

The present invention will be explained below.

When a microRNA binds to a target messenger RNA, it may be recognized as a target of the microRNA and bind to the messenger RNA even if it is not in an exact complementary relationship with the microRNA seed region. This is called non-canonical target recognition. The present inventors focused on such recognition of non-canonical targets of microRNAs and developed RNA interference-inducing nucleic acids that selectively suppress non-canonical target genes of microRNAs by modifying a partial sequence of microRNAs. The present invention was completed by clarifying its efficacy.

The present invention provides that a partial sequence of a specific microRNA is modified in one or more single strands of the double strands of a nucleic acid that induces RNA interference, thereby generating a noncanonical target gene of the microRNA. An RNA interference-inducing nucleic acid that suppresses only the expression of gene),
The RNA interference-inducing nucleic acid includes a sequence of four bases from the second to the fifth from the 5' end of the specific microRNA, the sixth and seventh bases are the same, and the sixth and seventh bases are , has a complementary base sequence to a base that can be aligned with the 6th base of the specific microRNA, and the base that can be aligned with the 6th base of the specific microRNA includes G:A, G:U wobble. A bulge is created in the target gene between the 5th and 6th positions of the microRNA so that all complementary bases containing the sequence are included, and the bulge disappears at the non-canonical target base sequence that binds, creating a continuous sequence. The RNA interference-inducing nucleic acid preferably binds with a 5'-end reference base sequence of the 9th base from the 5'-end of the specific microRNA. G:A or G at the site by having a modified base sequence in which at least one guanine base in the second to seventh sequences is replaced with uracil or adenine. Provided is an RNA interference-inducing nucleic acid characterized in that the :U wobble sequence becomes a regular base sequence of U:A or A:U.

The RNA interference-inducing nucleic acid according to the present invention is a single strand of one or more of the double strands of a nucleic acid that induces RNA interference, preferably micro RNA, small hairpin RNA, shRNA) and small interfering RNA (siRNA), DsiRNA, lsiRNA, ss-siRNA, asiRNA, piRNA, or endo-siRNA.

The RNA interference-inducing nucleic acid according to the present invention is based on two non-canonical target recognition of microRNA.

More specifically, microRNAs recognize not only canonical target genes but also non-canonical target genes. Non-canonical target genes recognized by microRNAs do not have exact complementary relationships with the microRNAs.

One embodiment of a non-canonical target gene recognized by microRNA and an RNA interference-inducing nucleic acid that suppresses the non-canonical target gene is as follows.

One recognition type of the non-canonical target gene to which the microRNA binds has a continuous complementary relationship with all of the five bases from the 5' end of the microRNA. However, the gene recognized by the 6th base from the 5' end of the microRNA (aligned with the 6th base from the 5' end of the microRNA) is complementary to the 5th base from the 5' end of the microRNA. A base that is not immediately contiguous with a related base is pushed out into a raised shape, and then a base that has a complementary relationship with the microRNA is recognized by the 6th base from the 5' end of the microRNA. It becomes a base.

The RNA interference-inducing nucleic acid that suppresses the non-canonical target gene recognized by the microRNA of the above embodiment includes a four base sequence from the second to the fifth base from the 5' end of the specific microRNA. Furthermore, two consecutive bases in the four base sequences are the same, and the two base sequences have a base sequence complementary to a base that can be aligned with the sixth base of the specific microRNA. However, the bases that can be aligned with the 6th base of the specific microRNA include all complementary bases including G:A and G:U wobble sequences.

When compared with the base sequence of the microRNA, the RNA interference-inducing nucleic acid that suppresses the non-canonical target gene recognized by such a specific microRNA has at least 4 bases from the 2nd to the 5th 5' end of the specific microRNA. common base sequences.

In addition, the 6th base sequence and the 7th base sequence from the 5' end of the RNA interference-inducing nucleic acid can be the same, and these two base sequences are complementary to the base that can be aligned with the 6th base of the specific microRNA. Bases that have a typical base sequence and can be aligned with the 6th base of the specific microRNA include all complementary bases including G:A and G:U wobble sequences. For example, the 6th base of a specific microRNA: A base that can be aligned with the 6th base: A base that is complementary to the base that can be aligned with the 6th base is (A:U, G:A, C), ( G:A,U,C:U,A,G), (U:G,A:C,U) or (C:G:C). For example, when the 6th base of the specific microRNA is A, the 6th and 7th base sequences from the 5' end of the RNA interference-inducing nucleic acid may be AA and CA; When the 6th base is G, the 6th and 7th base sequences from the 5' end of the RNA interference-inducing nucleic acid can be UG, AG, or GG; , the 6th base sequence and the 7th base sequence from the 5' end of the RNA interference-inducing nucleic acid may be CU or UU; or when the 6th base of the specific microRNA is C, RNA interference The 6th base sequence and the 7th base sequence from the 5' end of the derived nucleic acid may be CC.

Preferably, the 6th base sequence from the 5' end of the RNA interference-inducing nucleic acid and the 6th base sequence from the 5' end of the specific microRNA may be the same, and preferably The seventh base sequence and the sixth base sequence from the 5' end of the specific microRNA may be the same.

Preferably, the RNA interference-inducing nucleic acid can include the seventh base sequence from the 5' end of the specific microRNA as the eighth base sequence from the 5' end.

Still another embodiment of a non-canonical target gene recognized by microRNA and an RNA interference-inducing nucleic acid that suppresses the non-canonical target gene is as follows.

The non-canonical target genes recognized by microRNAs have a complementary sequence of A:U, G:C, C:G, U:A as well as a G:A wobble in which the bases of the microRNA and the target gene recognized by the microRNA are complementary. Contains an array of relationships or an array of G:U wobble relationships.

When the microRNA and the target gene recognized by the microRNA have a G:A wobble relationship, the base of the specific microRNA: the base of the target gene recognized by the specific microRNA: the target gene recognized by the specific microRNA is suppressed. The base sequence of the RNA interference-inducing nucleic acid is G:A:U. Furthermore, when the microRNA and the target gene recognized by the microRNA have a G:U wobble relationship, the base of the specific microRNA: the base of the target gene recognized by the specific microRNA: the target gene recognized by the specific microRNA The base sequence of the RNA interference-inducing nucleic acid that inhibits is G:U:A.

The RNA interference-inducing nucleic acid according to the present invention has a modified base sequence in which one or more guanine (G) bases are substituted with uracil (U) in the base sequence between the 9th base from the 5' end of a specific microRNA. have Alternatively, the RNA interference-inducing nucleic acid according to the present invention includes a base sequence between the 9th base from the 5' end of a specific microRNA, preferably one or more guanines ( G) It has a modified base sequence in which the base is replaced with adenine (A). If one or more guanine (G) bases are included in the base sequence between the 9th base from the 5' end of a specific microRNA, all of the included guanine bases are converted to uracil (U) or adenine (A). Substitutions can also be made, with at least one guanine base being replaced by uracil (U) or adenine (A). Among the base sequences between the 5' end and the 9th base of the specific microRNA, preferably one or more guanine (G) bases are included in the 5' end reference 2nd to 7th base, Besides the bases that are replaced with uracil or adenine bases, the remaining bases can be the same as the bases of the particular microRNA.

The RNA interference-inducing nucleic acid according to the present invention selectively suppresses the non-canonical target genes of microRNAs, but does not suppress the canonical target genes of microRNAs.

According to specific embodiment 2 of the present invention (see FIG. 2), the expression regulation function of messenger RNA for the target gene of the RNA interference-inducing nucleic acid according to the present invention is specific for the non-canonical target gene, and is specific for the canonical target gene. No suppressive effect was shown on the gene.

Therefore, using the RNA interference-induced nucleic acid according to the present invention, it is possible to selectively suppress only non-canonical target gene expression, and therefore, selectively induce only the expected effect through suppression of RNA interference-induced nucleic acid expression. be able to.

In the present invention, the specific microRNA is one or more selected from the group consisting of miR-124, miR-155, miR-122, miR-1, miR-133, let-7, miR-302a, and miR-372. could be.

In the present invention, the nucleotide sequences of human miR-124, miR-155, miR-122, miR-1, miR-133, let-7, miR-302a and miR-372, respectively, are a microRNA sequence database. MIMAT0000422, MIMAT0000646, MIMAT0000421, MIMAT0000416, MIMAT0000427, MIMAT0000062, MIMAT0000684 with miRBase (http://www.mirbase.org/) , and as described in MIMAT0000724.

However, the specific microRNA according to the present invention is not limited to human microRNA, but includes animal microRNA including mammals.

An RNA interference-inducing nucleic acid that suppresses a non-canonical target gene recognized by a specific microRNA according to the present invention, which comprises four base sequences from the second to the fifth base from the 5' end of the specific microRNA, and the sixth and seventh base sequences from the 5' end of the specific microRNA. The 6th base may be the same, and the 6th and 7th bases have a complementary base sequence to a base that can be aligned with the 6th base of the specific microRNA, and the 6th base of the specific microRNA The length of the RNA interference-inducing nucleic acid is characterized in that the bases that can be sequenced include all complementary bases including G:A, G:U wobble sequences. The base sequence includes, but is not limited to, usually about 21 bases, and can have a base sequence of 24 or less.

The RNA interference-inducing nucleic acid according to the present invention is an RNA interference-inducing nucleic acid that suppresses a non-canonical target gene of miR-124 when the specific microRNA is miR-124, and is an RNA interference-inducing nucleic acid that suppresses a non-canonical target gene of miR-124. The 6th and 7th bases may be the same, and the 6th and 7th bases are complementary to bases that can be aligned with the 6th base of miR-124. It is an RNA interference-inducing nucleic acid that has a base sequence and includes all complementary bases including G:A and G:U wobble sequences as bases that can be aligned with the 6th base of miR-124. .

For example, the RNA interference-inducing nucleic acid may be an RNA interference-inducing nucleic acid (miR-124BS) in which the second to seventh base sequences at the 5' end are 5'-AA GGC C-3'. More preferably, the RNA interference-inducing nucleic acid may be an RNA interference-inducing nucleic acid (miR-124BS) having a base sequence of 5'-UAA GGC CAC GCG GUG AAU GCC-3'.

The RNA interference-inducing nucleic acid according to the present invention is an RNA interference-inducing nucleic acid that suppresses a non-canonical target gene of miR-122 when the specific microRNA is miR-122, and is an RNA interference-inducing nucleic acid that suppresses a non-canonical target gene of miR-122, starting from the second from the 5' end of miR-122. The 6th and 7th bases are the same, and the 6th and 7th bases are complementary bases that can be aligned with the 6th base of miR-122. It is an RNA interference-inducing nucleic acid that has a sequence and includes all complementary bases including G:A and G:U wobble sequences as bases that can be aligned with the 6th base of miR-122.

For example, the RNA interference-inducing nucleic acid may be an RNA interference-inducing nucleic acid (miR-122BS) in which the second to seventh base sequences at the 5' end have a base sequence of 5'-GG AGU U-3'. More preferably, the RNA interference-inducing nucleic acid may be an RNA interference-inducing nucleic acid (miR-122BS) having a base sequence of 5'-UGG AGU UGU GAC AAU GGU GUU-3'.

The RNA interference-inducing nucleic acid according to the present invention is an RNA interference-inducing nucleic acid that suppresses a non-canonical target gene of miR-155 when the specific microRNA is miR-155, and is an RNA interference-inducing nucleic acid that suppresses a non-canonical target gene of miR-155. The 6th and 7th bases are the same, and the 6th and 7th bases are complementary bases that can be aligned with the 6th base of miR-155. It is an RNA interference-inducing nucleic acid that has a sequence and includes all complementary bases including G:A and G:U wobble sequences as bases that can be aligned with the 6th base of miR-155.

For example, the RNA interference-inducing nucleic acid may be an RNA interference-inducing nucleic acid (miR-155BS) in which the second to seventh base sequences at the 5' end are 5'-UA AUG G-3'. More preferably, the RNA interference-inducing nucleic acid may be an RNA interference-inducing nucleic acid (miR-155BS) having a base sequence of 5'-UUA AUG GC UAA U CGU GAU AGG-3'.

The RNA interference-inducing nucleic acid according to the present invention is an RNA interference-inducing nucleic acid that suppresses a non-canonical target gene of miR-1 when the specific microRNA is miR-1, and is an RNA interference-inducing nucleic acid that suppresses a non-canonical target gene of miR-1, starting from the second from the 5' end of miR-1. The 6th and 7th bases are the same, and the 6th and 7th bases are complementary bases that can be aligned with the 6th base of miR-1. It is an RNA interference-inducing nucleic acid that has a sequence and includes all complementary bases including G:A and G:U wobble sequences as bases that can be aligned with the 6th base of miR-1.

For example, the RNA interference-inducing nucleic acid may be an RNA interference-inducing nucleic acid (miR-1BS) in which the second to seventh base sequences at the 5' end are 5'-GG AAU U-3'. More preferably, the RNA interference-inducing nucleic acid may be an RNA interference-inducing nucleic acid (miR-1BS) having a base sequence of 5'-UGG AAU UGU AAA GAA GUA UGU-3'.

The RNA interference-inducing nucleic acid that suppresses a non-canonical target gene recognized by a specific microRNA according to the present invention preferably contains a base sequence from the 5' end to the 9th base of the specific microRNA. Length of an RNA interference-inducing nucleic acid characterized by having a modified base sequence in which at least one guanine (G) base is substituted with uracil (U) or adenine (A) in the reference sequence 2nd to 7th. has a nucleotide sequence of 6 or more, but is not limited to this, usually about 21 nucleotides, and can have a nucleotide sequence of 24 or less.

The RNA interference-inducing nucleic acid according to the present invention is an RNA interference-inducing nucleic acid that suppresses a non-canonical target gene of miR-124 when the specific microRNA is miR-124, and is an RNA interference-inducing nucleic acid that suppresses a non-canonical target gene of miR-124. Among the base sequences between the bases, preferably a modification in which at least one guanine (G) base in the second to seventh 5' end reference sequences is replaced with uracil (U) or adenine (A) base. An RNA interference-inducing nucleic acid having a base sequence.

For example, the RNA interference-inducing nucleic acid has a base sequence between the 9th base from the 5' end of 5'-UAA UGC AC-3' (miR-124-G4U), 5'-UAA GUC AC-3' ( miR-124-G5U) or 5'-UAA UUC AC-3' (miR-124-G4,5U). More preferably, the RNA interference-inducing nucleic acid is 5'-UAA UGC ACG CGG UGA AUG CCA A-3' (miR-124-G4U), 5'-UAA GUC ACG CGG UGA AUG CCA A-3' (miR- 124-G5U) or 5'-UAA UUC ACG CGG UGA AUG CCA A-3' (miR-124-G4,5U).

The RNA interference-inducing nucleic acid according to the present invention is an RNA interference-inducing nucleic acid that suppresses a non-canonical target gene of miR-1 when the specific microRNA is miR-1, and is an RNA interference-inducing nucleic acid that suppresses a non-canonical target gene of miR-1, It is an RNA interference-inducing nucleic acid having a modified base sequence in which at least one guanine (G) base in the base sequence between the bases is replaced with a uracil (U) or adenine (A) base.

For example, the RNA interference-inducing nucleic acid has a base sequence between the 9th base from the 5' end of 5'-UUG AAU GUA-3' (miR-1-G2U), 5'-UUG AAU GUA-3' ( miR-1-G3U), 5'-UGG AAU UUA-3' (miR-1-G7U), 5'-UUU AAU GUA-3' (miR-1-G2,3U), 5'-UGU AAU UUA- 3' (miR-1-G3,7U), 5'-UUG AAU UUA-3' (miR-1-G2,7U) or 5'-UUU AAU UUA-3' (miR-1-G2,3,7U ) may be an RNA interference-inducing nucleic acid. More preferably, the RNA interference-inducing nucleic acid is 5'-UUG AAU GUA AAG AAG UAU GUA U-3' (miR-1-G2U), 5'-UGU AAU GUA AAG AAG UAU GUA U-3' (miR- 1-G3U), 5'-UGG AAU UUA AAG AAG UAU GUA U-3' (miR-1-G7U), 5'-UUU AAU GUA AAG AAG UAU GUA U-3' (miR-1-G2,3U) , 5'-UGU AAU UUA AAG AAG UAU GUA U-3' (miR-1-G3,7U), 5'-UUG AAU UUA AAG AAG UAU GUA U-3' (miR-1-G2,7U) or 5 It may be an RNA interference-inducing nucleic acid having a base sequence of '-UUU AAU UUA AAG AAG UAU GUA U-3' (miR-1-G2,3,7U).

The RNA interference-inducing nucleic acid according to the present invention is an RNA interference-inducing nucleic acid that suppresses a non-canonical target gene of miR-122 when the specific microRNA is miR-122, and is an RNA interference-inducing nucleic acid that suppresses a non-canonical target gene of miR-122. Among the base sequences between the bases, preferably a modification in which at least one guanine (G) base in the second to seventh 5' end reference sequences is replaced with uracil (U) or adenine (A) base. An RNA interference-inducing nucleic acid having a base sequence.

For example, the RNA interference-inducing nucleic acid has a base sequence between the 9th base from the 5' end of 5'-UUG AGU GUG-3' (miR-122-G2U), 5'-UUG AGU GUG-3' ( miR-122-G3U), 5'-UGG AUU GUG-3' (miR-122-G5U), 5'-UGG AGU UUG-3' (miR-122-G7U), 5'-UGG AGU GUU-3' (miR-122-G9U), 5'-UUU AGU GUG-3' (miR-122-G2, 3U), 5'-UUG AUU GUG-3' (miR-122-G2, 5U), 5'-UUG AGU UUG-3' (miR-122-G2, 7U), 5'-UUG AGU GUU-3' (miR-122-G2, 9U), 5'-UGU AUU GUG (miR-122-G3, 5U), 5'-UGU AGU UUG-3' (miR-122-G3,7U), 5'-UGU AGU GUU-3' (miR-122-G3,9U), 5'-UGG AUU UUG-3' (miR- 122-G5,7U), 5'-UGG AUU GUU-3' (miR-122-G5,9U), or 5'-UGG AGU UUU-3 (miR-122-G7,9U). It can be. More preferably, the RNA interference-inducing nucleic acid is 5'-UUG AGU GUG ACA AUG GUG UUU G-3' (miR-122-G2U), 5'-UUG AGU GUG ACA AUG GUG UUU G-3 (miR-122 -G3U), 5'-UGG AUU GUG ACA AUG GUG UUU G-3' (miR-122-G5U), 5'-UGG AGU UUG ACA AUG GUG UUU G-3' (miR-122-G7U), 5' -UGG AGU GUU ACA AUG GUG UUU G-3' (miR-122-G9U), 5'-UUU AGU GUG ACA AUG GUG UUU G-3' (miR-122-G2,3U), 5'-UUG AUU GUG ACA AUG GUG UUU G-3' (miR-122-G2, 5U), 5'-UUG AGU UUG ACA AUG GUG UUU G-3' (miR-122-G2, 7U), 5'-UUG AGU GUU ACA AUG GUG UUU G-3' (miR-122-G2, 9U), 5'-UGU AUU GUG ACA AUG GUG UUU G-3 (miR-122-G3, 5U), 5'-UGU AGU UUG ACA AUG GUG UUU G -3 (miR-122-G3, 7U), 5'-UGU AGU GUU ACA AUG GUG UUU G-3 (miR-122-G3, 9U), 5'-UGG AUU UUG ACA AUG GUG UUU G-3' ( miR-122-G5,7U), 5'-UGG AUU GUU ACA AUG GUG UUU G-3' (miR-122-G5,9U) or 5'-UGG AGU UUU ACA AUG GUG UUU G-3' (miR- 122-G7,9U) may be an RNA interference-inducing nucleic acid having the base sequence.

The RNA interference-inducing nucleic acid according to the present invention is an RNA interference-inducing nucleic acid that suppresses a non-canonical target gene of miR-133 when the specific microRNA is miR-133, and is an RNA interference-inducing nucleic acid that suppresses a non-canonical target gene of miR-133. Among the base sequences between the bases, preferably a modification in which at least one guanine (G) base in the second to seventh 5' end reference sequences is replaced with uracil (U) or adenine (A) base. An RNA interference-inducing nucleic acid having a base sequence.

For example, the RNA interference-inducing nucleic acid has a base sequence between the 9th base from the 5' end of 5'-UUU UGU CCC-3' (miR-133-G4U), 5'-UUU GUU CCC-3' ( miR-133-G5U) or 5'-UUU UUU CCC-3' (miR-124-G4,5U). More preferably, the RNA interference-inducing nucleic acid is 5'-UUU UGU CCC CUU CAA CCA GCU G-3' (miR-133-G4U), 5'-UUU GUU CCC CUU CAA CCA GCU G-3' (miR- 133-G5U) or 5'-UUU UUU CCC CUU CAA CCA GCU G-3' (miR-133-G4,5U).

The RNA interference-inducing nucleic acid according to the present invention is an RNA interference-inducing nucleic acid that suppresses a non-canonical target gene of let-7 when the specific microRNA is let-7, Among the base sequences between the bases, preferably a modification in which at least one guanine (G) base in the second to seventh 5' end reference sequences is replaced with uracil (U) or adenine (A) base. An RNA interference-inducing nucleic acid having a base sequence.

For example, the RNA interference-inducing nucleic acid has a base sequence between the 9th base from the 5' end such as 5'-UUA GGU AGU-3' (let-7-G2U), 5'-UGA UGU AGU-3' ( let-7-G4U), 5'-UGA GUU AGU-3' (let-7-G5U), 5'-UGA GGU AUU-3' (let-7-G8U), 5'-UUA UGU AGU-3' (let-7-G2, 4U), 5'-UUA GUU AGU-3' (let-7-G2, 5U), 5'-UUA GGU AUU-3' (let-7-G2, 8U), 5' -UGA UUU AGU-3' (let-7-G4, 5U), 5'-UGA UGU AUU-3' (let-7-G4, 8U), or 5'-UGA GUU AUU-3' (let-7 -G5,8U). More preferably, the RNA interference-inducing nucleic acid is 5'-UUA GGU AGU AGG UUG UAU AGU U-3' (let-7-G2U), 5'-UGA UGU AGU AGG UUG UAU AGU U-3' (let- 7-G4U), 5'-UGA GUU AGU AGG UUG UAU AGU U-3' (let-7-G5U), 5'-UGA GGU AUU AGG UUG UAU AGU U-3' (let-7-G8U), 5 '-UUA UGU AGU AGG UUG UAU AGU U-3' (let-7-G2, 4U), 5'-UUA GUU AGU AGG UUG UAU AGU U-3' (let-7-G2, 5U), 5'- UUA GGU AUU AGG UUG UAU AGU U-3' (let-7-G2, 8U), 5'-UGA UUU AGU AGG UUG UAU AGU U-3' (let-7-G4, 5U), 5'-UGA UGU RNA interference induction showing the base sequence of AUU AGG UUG UAU AGU U-3' (let-7-G4, 8U) or 5'-UGA GUU AUU AGG UUG UAU AGU U-3' (let-7-G5, 8U) Can be a nucleic acid.

The RNA interference-inducing nucleic acid according to the present invention is an RNA interference-inducing nucleic acid that suppresses a non-canonical target gene of miR-302a when the specific microRNA is miR-302a, and is an RNA interference-inducing nucleic acid that suppresses a non-canonical target gene of miR-302a. Among the base sequences between the bases, preferably a modification in which at least one guanine (G) base in the second to seventh 5' end reference sequences is replaced with uracil (U) or adenine (A) base. An RNA interference-inducing nucleic acid having a base sequence.

For example, the RNA interference-inducing nucleic acid has a base sequence between the 9th base from the 5' end of 5'-UAA UUG CUU-3' (miR-302a-G4U), 5'-UAA GUU CUU-3' ( miR-302a-G6U), or 5'-UAA UUU CUU-3' (miR-302a-G4,6U). More preferably, the RNA interference-inducing nucleic acid is 5'-UAA UUG CUU CCA UGU UUU GGU GA-3' (miR-302a-G4U), 5'-UAA GUU CUU CCA UGU UUU GGU GA-3' (miR- 302a-G6U), or 5'-UAA UUU CUU CCA UGU UUU GGU GA-3' (miR-302a-G4,6U).

The RNA interference-inducing nucleic acid according to the present invention is an RNA interference-inducing nucleic acid that suppresses a non-canonical target gene of miR-372 when the specific microRNA is miR-372, and is an RNA interference-inducing nucleic acid that suppresses a non-canonical target gene of miR-372. Among the base sequences between the bases, preferably a modification in which at least one guanine (G) base in the second to seventh 5' end reference sequences is replaced with uracil (U) or adenine (A) base. An RNA interference-inducing nucleic acid having a base sequence.

For example, the RNA interference-inducing nucleic acid has a base sequence between the 9th base from the 5' end of 5'-AAA UUG CUG-3' (miR-372-G4U), 5'-AAA GUU CUG-3' ( miR-372-G6U), 5'-AAA GUG CUU-3' (miR-372-G9U), 5'-AAA UUU CUG-3' (miR-372-G4,6U), 5'-AAA UUG CUU- 3' (miR-372-G4,9U), or 5'-AAA GUU CUU-3' (miR-372-G6,9U). More preferably, the RNA interference-inducing nucleic acid is an RNA interference-inducing nucleic acid that suppresses a non-canonical target gene of miR-372, and is 5'-AAA UUG CUG CGA CAU UUG AGC GU-3' (miR-372-G4U ), 5'-AAA GUU CUG CGA CAU UUG AGC GU-3' (miR-372-G6U), 5'-AAA GUG CUU CGA CAU UUG AGC GU-3' (miR-372-G9U), 5'-AAA UUU CUG CGA CAU UUG AGC GU-3' (miR-372-G4,6U), 5'-AAA UUG CUU CGA CAU UUG AGC GU-3' (miR-372-G4,9U), or 5'-AAA GUU The RNA interference-inducing nucleic acid may be an RNA interference-inducing nucleic acid having a base sequence of CUU CGA CAU UUG AGC GU-3' (miR-372-G6,9U).

In addition, the present invention provides that a partial sequence of a specific microRNA is modified in one or more single strands of the double strands of a nucleic acid that induces RNA interference (RNA interference), so that a non-canonical target gene of the microRNA ( An RNA interference-inducing nucleic acid that suppresses a noncanonical target gene,
The RNA interference-inducing nucleic acid includes a sequence of four bases from the second to the fifth from the 5' end of the specific microRNA, the sixth and seventh bases are the same, and the sixth and seventh bases are , has a complementary base sequence to a base that can be aligned with the 6th base of the specific microRNA, and the base that can be aligned with the 6th base of the specific microRNA includes G:A, G:U wobble. A bulge is created in the target gene between the 5th and 6th positions of the microRNA so that all complementary bases containing the sequence are included, and the bulge disappears at the non-canonical target base sequence that binds, creating a continuous sequence. It is characterized by binding with a specific base sequence,
The specific microRNA provides an RNA interference-inducing nucleic acid characterized in that a methyl group (2'OMe) is added to the 2' position of the ribose ring of the 6th nucleotide from the 5' end.

The RNA interference-inducing nucleic acid according to the present invention includes a sequence of four bases from the second to the fifth from the 5' end of a specific microRNA, the sixth and seventh bases are the same, and the sixth and seventh bases are the same. The base has a complementary base sequence to a base that can be aligned with the 6th base of the specific microRNA, and the base that can be aligned with the 6th base of the specific microRNA has a G:A, G:U wobble. Contains all complementary bases including the sequence, excluding cases where a methyl group (2'OMe) is added to the 2' position of the ribose ring at the 6th nucleotide from the 5' end of the microRNA. The remaining base sequences are not limited and can include any base sequence, and in this case, the RNA interference-inducing nucleic acid can include 6 to 24 base sequences, preferably 6 to 24 base sequences. The length of the nucleic acid is not particularly limited, although it can contain 15 base sequences, more preferably 6 to 8 base sequences.

The RNA interference-inducing nucleic acid according to the present invention includes a base sequence that has been chemically modified by adding a methyl group (2'OMe) to the 2' position of the ribose ring of the 6th nucleotide from the 5' end of microRNA. The RNA interference-induced nucleic acid in which the 2'OMe modification has occurred may selectively suppress only the expression of its canonical seed target gene, but may not suppress the expression of the non-canonical seed target gene. As a result, the RNA interference-induced nucleic acid in which the 2'OMe modification has occurred is freed from newly generated non-canonical nuclei while maintaining the purpose of the present invention to specifically suppress only the non-canonical nuclear protrusion site of a specific microRNA. Nuclear prominence sites can be completely removed.

In the RNA interference-inducing nucleic acid according to the present invention, there is no restriction on the type of microRNA to which the methyl group (2'OMe) can be added, and the methyl group (2'OMe) can be added to the 2' position of the ribose ring of the 6th nucleotide from the 5' end of the micro RNA. 2'OMe can be added to any microRNA if present; however, according to a specific embodiment of the present invention, the microRNA to which 2'OMe can be added is miR-124 or miR-1. obtain.

In addition, the present invention provides that a partial sequence of a specific microRNA is modified in one or more single strands of the double strands of a nucleic acid that induces RNA interference (RNA interference), so that a non-canonical target gene of the microRNA ( An RNA interference-inducing nucleic acid that suppresses a noncanonical target gene,
The RNA interference-inducing nucleic acid includes a sequence of four bases from the second to the fifth from the 5' end of the specific microRNA, the sixth and seventh bases are the same, and the sixth and seventh bases are , has a complementary base sequence to a base that can be aligned with the 6th base of the specific microRNA, and the base that can be aligned with the 6th base of the specific microRNA includes G:A, G:U wobble. A bulge is created in the target gene between the 5th and 6th positions of the microRNA so that all complementary bases containing the sequence are included, and the bulge disappears at the non-canonical target base sequence that binds, creating a continuous sequence. Provided is an RNA interference-inducing nucleic acid, which is characterized in that it binds with a unique base sequence.

The RNA interference-inducing nucleic acid according to the present invention includes a sequence of four bases from the second to the fifth from the 5' end of a specific microRNA, the sixth and seventh bases are the same, and the sixth and seventh bases are the same. The base has a complementary base sequence to a base that can be aligned with the 6th base of the specific microRNA, and the base that can be aligned with the 6th base of the specific microRNA has a G:A, G:U wobble. If it contains all the complementary bases containing the sequence, there is no restriction on the remaining base sequence other than this, and it can include any base sequence in its entirety; in this case, the RNA interference-inducing nucleic acid is It can contain a 6 to 24 base sequence, preferably 6 to 15 base sequences, more preferably 6 to 8 base sequences, but the length of the nucleic acid has a special value. There are no restrictions.

The RNA interference-inducing nucleic acid according to the present invention includes a sequence of four bases from the second to the fifth from the 5' end of a specific microRNA, the sixth and seventh bases are the same, and the sixth and seventh bases are the same. The base has a complementary base sequence to a base that can be aligned with the 6th base of the specific microRNA, and the bases that can be aligned with the 6th base of the specific microRNA include G:A, G:U wobble. A bulge is created in the target gene between the 5th and 6th positions of the microRNA, and the bulge is generated in the non-canonical target base sequence that binds. By disappearing and binding with a continuous base sequence, only the non-canonical nuclear protuberance target site is selectively suppressed, but the normal target gene of the microRNA is not suppressed.

The RNA interference-inducing nucleic acid according to the present invention can include a base sequence represented by any one or more of SEQ ID NOs: 103 to 528 (see Table 3).

In addition, the present invention provides that a partial sequence of a specific microRNA is modified in one or more single strands of the double strands of a nucleic acid that induces RNA interference (RNA interference), so that a non-canonical target gene of the microRNA ( An RNA interference-inducing nucleic acid that suppresses only the expression of a noncanonical target gene,
The RNA interference-inducing nucleic acid has a base sequence between the second to ninth bases from the 5' end of a specific microRNA, or a guanine base in the second to seventh base from the 5' end of the 5' end, and uracil ( An RNA characterized by having a modified base sequence in which at least one base (Uracil) is substituted so that the G:A wobble at the site becomes the regular base sequence of U:A. Interference-inducing nucleic acids are provided.

The RNA interference-inducing nucleic acid according to the present invention is a sequence between the second base to the ninth base from the 5' end of a specific microRNA, or a guanine base from the second to seventh base from the 5' end. may be substituted with at least one Uracil base, and the RNA interference-inducing nucleic acid may have a guanine between the second base and the ninth base from the 5' end of the specific microRNA. If the modified base sequence includes at least one base substituted with a Uracil base, there is no restriction on the remaining base sequence, and any base sequence may be included. At this time, the base sequence between the second base to the ninth base from the 5' end of the specific microRNA or the sequence from the second to the seventh base from the 5' end is replaced with a uracil (U) base. In addition to the base, the remaining bases may be the same as the bases of the specific microRNA.

An RNA interference-inducing nucleic acid that suppresses a non-canonical target gene recognized by a specific microRNA according to the present invention, which is a base sequence between the second to ninth bases from the 5' end of the specific microRNA, or a 5' end standard. An RNA interference-inducing nucleic acid characterized by having a modified base sequence in which at least one guanine base is substituted with a uracil base in the second to seventh sequences has 6 to 24 bases. It can contain a base sequence, preferably 6 to 15 bases, more preferably 6 to 8 bases, starting from the second base from the 5' end of the specific microRNA. If it is an RNA interference-inducing nucleic acid that includes a modified base sequence in which at least one guanine base is replaced with a uracil base among the first 6 to 8 consecutive base sequences, the length of the nucleic acid There are no special restrictions. At this time, in the RNA interference-inducing nucleic acid, among the 6 to 8 consecutive base sequences starting from the second base from the 5' end of the specific microRNA, a guanine base is replaced by a uracil base. As long as it contains a modified base sequence with at least one substitution, there are no restrictions on the types of the remaining base sequences, and any base sequence can be included.

In the RNA interference-inducing nucleic acid according to the present invention, at least one guanine (G) base is replaced with a uracil (U) base in a sequence of 6 to 8 consecutive bases starting from the second base from the 5' end. By making the G:A wobble sequence at the site become the regular base sequence of U:A with the modified base sequence, the non-specific microRNA that binds with the G:A wobble sequence can be Only the regular target genes are selectively suppressed, and the regular target genes of microRNAs are not suppressed.

The RNA interference-inducing nucleic acid according to the present invention can include a base sequence represented by any one or more of SEQ ID NOs: 529 to 863 (see Table 4).

Using the above-described RNA interference-inducing nucleic acids according to the present invention, non-canonical target genes of microRNAs can be specifically suppressed.

Accordingly, the present invention provides a composition for suppressing the expression of a non-canonical target gene of a microRNA containing an RNA interference-inducing nucleic acid or a kit for suppressing the expression of a non-canonical target gene of a micro RNA containing an RNA interference-inducing nucleic acid.

Moreover, when the above-mentioned RNA interference-inducing nucleic acid according to the present invention is used, by specifically suppressing the non-canonical target gene of microRNA, the effect caused by suppressing the expression of the non-canonical target gene of microRNA can be selectively regulated. be able to.

More specifically, since the RNA interference-inducing nucleic acid according to the present invention specifically suppresses the non-canonical target gene of microRNA, the above-mentioned RNA interference-inducing nucleic acid according to the present invention suppresses the non-canonical target gene of microRNA. Effects of gene expression (eg, cell cycle, differentiation, reverse differentiation, morphology, migration, division, proliferation, or death) can be specifically modulated.

Accordingly, the present invention provides a composition or kit for regulating cell cycle, differentiation, reverse differentiation, morphology, migration, division, proliferation, or death, which includes an RNA interference-inducing nucleic acid.

In the present invention, the "cell cycle" is a continuous process in which cells grow, divide, and further grow, and in which cells undergo M phase (cell division phase), G1 phase (first division preparation phase), It means repeating four phases: S phase (DNA synthesis phase) and G2 phase (second division preparation phase). The G1 phase is a preparatory phase for DNA synthesis from immediately after cell division to the start of DNA synthesis, and the G2 phase is a preparatory phase for cell division from the end of DNA synthesis to the start of cell division.

In the present invention, the "cell cycle regulation" includes inducing cells to fluctuate between the four phases, promoting or arresting the fluctuations, for example, G2/M Examples of cell cycle regulation may include inducing cells in the G1/G2 phase to transition.

In the present invention, "cell differentiation" is a process in which cells acquire morphological and functional specialities, and through differentiation, cells change size, shape, membrane potential, metabolic activity, and signal response. The reaction changes.

In the present invention, the "cell differentiation regulation" includes promoting or delaying the rate at which cells differentiate, inducing differentiation to begin, and suppressing differentiation from starting, for example, when nerve cells Examples of regulating cell differentiation may include inducing them to differentiate and acquire morphological specialities (eg, neurite differentiation). Alternatively, examples of cell differentiation regulation may include, for example, inducing muscle cells to differentiate and have morphological specificities (eg, myofiberization of muscle cells).

In the present invention, the above-mentioned "reverse differentiation" refers to the phenomenon of reversing the progression of differentiation or transforming cells that have almost completed differentiation into cells that acquire the full differentiation potential of the undifferentiated stage before differentiation and further change into intermediate-stage cells. say.

In the present invention, the "reverse differentiation regulation" includes inducing and promoting the reverse differentiation of cells, and suppressing and delaying the progress of reverse differentiation, and the results of the reverse differentiation regulation include, for example, cell growth morphology (e.g. , community formation, etc.), and the activity of reverse differentiation factors (Oct3/4, Sox2, c-Myc, Klf4).

In the present invention, the above-mentioned "cell morphology" refers to phenotypic characteristics including cell morphology, structure, size, etc. Cell morphology varies depending on the type of organism, and even within the same organism, tissue and There are many different types depending on the type of organ.

In the present invention, the "cell morphology regulation" refers to inducing changes in phenotypic characteristics including cell morphology, structure, size, etc., and refers to promoting, delaying, inducing, or inducing changes in the natural morphology of cells. This includes suppressing and promoting or inducing unnatural changes in cell morphology. For example, one example may include inducing myocardial hypertrophy in cardiomyocytes.

In the present invention, "cell migration" refers to the movement of cells, and is an important process for the growth and physiological activities of animals. Cells mainly respond rapidly to applied stimuli, and usually They migrate in a non-collective manner, whereas other cell types can only migrate during specific periods of growth or under special circumstances. Cell migration occurs primarily in the wound healing process of neural tube and vascular seeds and epithelial tissues, and cell population migration contributes to many types of tumor metastasis.

In the present invention, the term "cell migration regulation" means delaying, promoting, inducing, or suppressing (inhibiting) cell migration.

In the present invention, "cell division, proliferation" refers to a process in which a mother cell divides into two daughter cells, and refers to a process in which the number of cells increases.

In the present invention, "cell division and proliferation regulation" includes delaying, promoting, suppressing, and inducing cell division and proliferation. When inducing the cell phase in the M phase (cell division phase) of the cell cycle, cell division and proliferation can be induced. For example, decreasing the number of cells distributed in the G0/G1 phase and increasing the number of cells distributed in the G2/M phase can be included as examples of cell division and proliferation regulation.

In the present invention, "cell death (apoptosis)" refers to a stage in which cells die due to their genetic properties while maintaining their functional role, and includes both early cell death and late cell death. According to cell death, the entire cell atrophies, new proteins are synthesized, and death is induced by the cell's suicide gene.

In the present invention, "cell death regulation" includes inducing, delaying, promoting or suppressing cell death.

More specifically, the present invention provides a composition for inducing cancer cell death, comprising an RNA interference-inducing nucleic acid (preferably miR-124BS) that suppresses a non-canonical target gene of miR-124;
A composition for inducing neurite branching differentiation, comprising an RNA interference-inducing nucleic acid (preferably miR-124BS) that suppresses a non-canonical target gene of miR-124;
A composition for inducing cell cycle arrest in hepatoma cells, comprising an RNA interference-inducing nucleic acid (preferably miR-122BS) that suppresses a non-canonical target gene of miR-122;
A composition for promoting muscle cell differentiation or muscle fibrosis, comprising an RNA interference-inducing nucleic acid (preferably miR-1BS) that suppresses a non-canonical target gene of miR-1;
A composition for inducing muscle cell death, comprising an RNA interference-inducing nucleic acid (preferably miR-155BS) that suppresses a non-canonical target gene of miR-155;
A composition for promoting cell death of neuroblastoma, comprising an RNA interference-inducing nucleic acid (preferably miR-124-G5U) that suppresses a non-canonical target gene of miR-124;
A composition for promoting cell division and proliferation of neuroblastoma, comprising an RNA interference-inducing nucleic acid (preferably miR-124-G4U) that suppresses a non-canonical target gene of miR-124;
A composition for inducing myocardial hypertrophy comprising an RNA interference-inducing nucleic acid (preferably miR-1-G2U, miR-1-G3U, miR-1-G7U) that suppresses a non-canonical target gene of miR-1;
A composition for inducing myocardial hypertrophy, comprising an RNA interference-inducing nucleic acid (preferably miR-133-G4U) that suppresses a non-canonical target gene of miR-133;
A composition for inducing cell cycle arrest in cancer cells, comprising an RNA interference-inducing nucleic acid (preferably let-7-G2U, let-7-G2,4U) that suppresses a non-canonical target gene of let-7;
A composition for inducing cell cycle progression activity of hepatocytes, comprising an RNA interference-inducing nucleic acid (preferably let-7-G4U) that suppresses a non-canonical target gene of let-7;
A composition for promoting reverse differentiation comprising an RNA interference-inducing nucleic acid (preferably miR-302a-G4U) that suppresses a non-canonical target gene of miR-302a;
A composition for promoting reverse differentiation comprising an RNA interference-inducing nucleic acid (preferably miR-372-G4U) that suppresses a non-canonical target gene of miR-372; or an RNA interference-inducing nucleic acid that suppresses a non-canonical target gene of miR-122 (preferably miR-122-G2U or miR-122-G2,3U), a composition for inhibiting cell migration of hepatoma cells.

Furthermore, by using the RNA interference-inducing nucleic acid according to the present invention, the action of a non-canonical target gene of microRNA can be selectively regulated by specifically suppressing the non-canonical target gene of microRNA. For example, it can be utilized in pharmaceutics, cosmetology, cytology, etc.).

Furthermore, the RNA interference-inducing nucleic acid according to the present invention can be utilized in a method of screening test substances for regulating cell cycle, differentiation, reverse differentiation, morphology, migration, division, proliferation, or death.

More specifically, introducing the RNA interference-inducing nucleic acid according to the present invention into target cells;
A step of treating the target cells with a test substance; and a step of confirming the expression level or presence or absence of the non-canonical target gene of the microRNA suppressed by the RNA interference-inducing nucleic acid in the target cells.
Provided are methods for screening test substances for modulating cell cycle, differentiation, reverse differentiation, morphology, migration, division, proliferation, or death.

In the present invention, the target cells are not limited as long as they are derived from mammals including humans, and there are no restrictions on the tissue or type from which the target cells are extracted, such as nerve cells, cardiomyocytes, Cancer cells, muscle cells, etc. may be included.

In the present invention, the RNA interference-inducing nucleic acid can be introduced into the target cell by various methods known in the art. For example, the introduction can be carried out by transfection using calcium phosphate (Graham, FL, etc. Virology, 52:456 (1973)), transfection with DEAE dextran, transfection by microinjection (Capecchi, MR, Cell, 22:479 (1980)), transfection with cationic lipids (Wong, TK et al., Gene , 10:87 (1980)), electroporation (Neumann E et al., EMBO J, 1:841 (1982)), transduction or transfection (Basic methods in molecular biology, Davis et al., 1986 and Molecular Cloning: A Laboratory Manual, Davis et al., 1986), which is well known to those skilled in the art to which the present invention pertains. Preferably, it can be done by transfection. Transfection efficiency can vary greatly depending on cell type as well as cell culture conditions, transfection reagents, etc.

In the present invention, test substances can be used in screens to examine whether they modulate the expression of non-canonical target genes of microRNAs to affect cell cycle, differentiation, reverse differentiation, morphology, migration, division, proliferation, or death. Refers to the unknown substance used and includes, but is not limited to, compounds, extracts, proteins, or nucleic acids.

In the present invention, the expression level or the presence or absence of expression of non-canonical target genes of microRNA can be confirmed by various expression analysis methods known in the art, such as RT-PCR, ELISA (Reference: Sambrook, J. et al, Molecular Cloning A Laboratory Manual, 3rd ed Cold Spring Harbor Press (2001)), western blot, FACS analysis, etc. may be used, but are not limited thereto.

In the present invention, the expression level or presence or absence of the non-canonical target gene of the microRNA was confirmed, and as a result, the expression level or expression of the non-canonical target gene of the microRNA was determined by treating only the RNA interference-inducing nucleic acid without a test substance. If there is a difference compared to the case, it can be determined that the test substance affects the expression of the non-canonical target gene of the microRNA. Consequently, it can be determined that the test substance has a cell cycle, differentiation, reverse differentiation, morphology, migration, division, proliferation, or death regulating function.

The present invention also provides a method for producing the above-mentioned RNA interference-inducing nucleic acid.

More specifically, the partial sequence of a specific microRNA is modified in one or more single strands of the double strands of the nucleic acid that induces RNA interference, including the following steps. Provided is a method for producing an RNA interference-inducing nucleic acid that suppresses a noncanonical target gene:
Contains the four base sequences from the second to the fifth from the 5' end of the specific microRNA, the 6th and 7th bases are the same, and the 6th and 7th bases are the 6th base sequence of the specific microRNA. The bases that can be aligned with the 6th base of the specific microRNA include all complementary bases including G:A, G:U wobble sequences. A step of preparing an RNA interference-inducing nucleic acid to contain a base, or replacing at least one guanine base with uracil or adenine in the base sequence between the 9th base from the 5' end of the specific microRNA. A step of producing an RNA interference-inducing nucleic acid having a modified base sequence.

In addition, the present invention includes the following steps, in which a part of the sequence of a specific microRNA is modified in one or more single strands of the double strands of a nucleic acid that induces RNA interference, resulting in microRNA A method for producing an RNA interference-inducing nucleic acid that suppresses the expression of a noncanonical target gene, the method comprising:
Contains the four base sequences from the second to the fifth from the 5' end of the specific microRNA, the 6th and 7th bases are the same, and the 6th and 7th bases are the 6th base sequence of the specific microRNA. The bases that can be aligned with the 6th base of the specific microRNA include all complementary bases including G:A, G:U wobble sequences. A bulge is created in the target gene between the 5th and 6th nucleotides of the microRNA, and the bulge disappears at the non-canonical target nucleotide sequence, and the bulge is combined with a continuous nucleotide sequence. and the step of adding a methyl group (2'OMe) to the 2' position of the ribose ring of the 6th nucleotide from the 5' end of the specific microRNA. , provides a method for producing RNA interference-inducing nucleic acids.

In addition, the present invention includes the following steps, in which a part of the sequence of a specific microRNA is modified in one or more single strands of the double strands of a nucleic acid that induces RNA interference, and the microRNA is transformed into a microRNA. A method for producing an RNA interference-inducing nucleic acid that suppresses the expression of a noncanonical target gene, the method comprising:
Contains the four base sequences from the second to the fifth from the 5' end of the specific microRNA, the 6th and 7th bases are the same, and the 6th and 7th bases are the 6th base sequence of the specific microRNA. The bases that can be aligned with the 6th base of the specific microRNA include all complementary bases including G:A, G:U wobble sequences. A bulge is created in the target gene between the 5th and 6th nucleotides of the microRNA, and the bulge disappears at the non-canonical target nucleotide sequence, and the bulge is combined with a continuous nucleotide sequence. Provided is a method for producing an RNA interference-inducing nucleic acid, the method comprising the step of producing an RNA interference-inducing nucleic acid as described above.

In addition, the present invention includes the following steps, in which a part of the sequence of a specific microRNA is modified in one or more single strands of the double strands of a nucleic acid that induces RNA interference, and the microRNA is transformed into a microRNA. A method for producing an RNA interference-inducing nucleic acid that suppresses the expression of a noncanonical target gene, the method comprising:
A modified base sequence in which at least one guanine base is replaced with a uracil base among 6 to 8 consecutive base sequences starting from the second base from the 5' end of a specific microRNA. Provided is a method for producing an RNA interference-inducing nucleic acid, the method comprising the step of producing an RNA interference-inducing nucleic acid having the following properties.

Since the RNA interference-inducing nucleic acid has already been described, its explanation will be omitted to avoid redundant description.

RNA interference-induced nucleic acid production follows a variety of methods known in the art and is not limited to any particular method.

The present invention also provides an interference-inducing nucleic acid modified by inserting a methyl group (2'OME) at the 2' position of the ribose ring of the 6th nucleotide from the 5' end of a specific microRNA.

The modified interference-inducing nucleic acid maintains the objective of the present invention, which is to specifically suppress only the non-canonical nuclear protrusion site of the miRNA, while completely eliminating any non-canonical nuclear protuberance binding that may newly appear. effective.

By using the interference-inducing nucleic acid according to the present invention, the biological functions exhibited by conventional microRNAs by suppressing non-canonical target genes can be efficiently increased, and some of the functions exhibited by conventional microRNAs can be improved. That is, by suppressing the non-canonical target gene, it is possible to have the effect of selectively demonstrating only the biological function exhibited. In addition, since it is possible to control the cell cycle, differentiation, reverse differentiation, morphology, migration, division, proliferation, or death through such interference-inducing nucleic acids, it is expected that it can be used in various fields such as drugs and cosmetics. .

FIG. 1 schematically shows the mechanism of action of interference-inducing nucleic acids that suppress non-canonical targets of microRNAs. Figures 2a to 2c show target gene repression of miR-124's canonical target (Seed) and non-canonical nuclear bulge target (nucleation bulge) by measuring with a luciferase reporter enzyme. The effect of suppressing expression specific to a non-canonical target was confirmed. Figures 3a and 3b show that miR-124-BS induces cell death induced by miR-124's regular target (Seed) and non-canonical nuclear bulge target (nucleation bulge) in cervical cancer cells (HeLa). This shows the results confirmed through flow cytometry (fluorescence-activated cell sorting: FACS) analysis. Figures 4a and 4b show the neurite differentiation induced by miR-124's noncanonical nuclear bulge target (nucleation bulge) in miR-124-BS human neural oncoblastoma (Sh-Sy-5y). This figure shows the results observed in terms of cell morphology and marker expression through the expression of . Figures 5a and 5b show miR-124-BS, a natural form of miR-124-BS that modulates the nuclear bulge target (nucleation bulge) of miR-124 with the canonical target (Seed). This figure shows the results of observing cell morphology and molecular biological characteristics of neurite branching differentiation induced by miR-124-(3714), which has the same seed sequence as miR-124 (4753) and miR-124. Figures 6a to 6c show the mechanism by which neurite branching differentiation induced by the non-canonical nuclear bulge target (nucleation bulge) of miR-124 is mediated by regulation of MAPRE1 gene expression in mouse neuroblastoma (N2a). This is the result of observation based on cell morphology and molecular biological characteristics. Figures 7a to 7c show miR-124-BS-induced suppression of expression of the non-canonical nuclear bulge target (nucleation bulge) gene in mouse neuroblastoma (N2a) cells by RNA-Seq analysis at the transcript level. The results are shown below. Figures 8a to 8c show cell cycle arrest induced by miR-122's non-canonical nuclear bulge target (nucleation bulge) in human hepatoma cell line (HepG2) observed through miR-122-BS and flow cytometry analysis. The results are shown below. Figures 9a to 9e show the thickening effect of muscle cells induced by miR-1 non-canonical nuclear bulge target (nucleation bulge) and miR-155 non-canonical nuclear bulge target (nucleation bulge) in muscle cell lines. This shows the results of cell morphology, molecular biological characteristics, and flow cytometry analysis of muscle cell differentiation inhibition and cell death induction induced by miR-1-BS and miR-155-BS, respectively. be. Figures 10a to 10d show the results of bioinformatic analysis of Ago HITS-CLIP results, which are microRNA and target RNA data that bind to Argonaute protein in human brain tissue. This figure shows the results of confirming that not only a non-canonical GA sequence seed (G:A wobble) target exists naturally, but also a non-canonical GA sequence seed (G:A wobble) target. Figures 11a to 11d show that miR-124 non-canonical GA sequence seed target No. 4 and non-canonical GA sequence seed target No. 5 have completely different functions than miR-124 in brain cell differentiation in mouse neuroblastoma (N2a) cells. This figure shows the results of cell morphology observation through miR-124-G4U and miR-124-G5U and cell death observed by flow cytometry analysis. Figures 12a-12b show the biological functions of miR-124 suppression of non-canonical GA sequence seed target No. 4 and non-canonical GA sequence seed target No. 5 in human neuroblastoma (SH-sy-5y) cells. This figure shows the results observed by flow cytometry analysis (fluorescence-activated cell sorting: FACS) for cell proliferation and cell cycle analysis through miR-124-G4U and miR-124-G5U. Figures 13a to 13d show that the non-canonical GA sequence seed target 7 of miR-1 functions as a gene suppressor in cardiomyocyte cell lines (fluorescence-activated cell sorting: FACS) using a fluorescent protein reporter. h9c2). Figures 14a to 14c show the functions of non-canonical GA sequence seed targets 2, 3, and 7 of miR-1 in neonatal rat cardiomyocytes of miR-1-G2U, miR- The results show the results confirmed through observation of cell morphology and molecular biological characteristics regarding the myocardial hypertrophy inducing effect after expressing 1-G3U and miR-1-G7U. Figures 15a and 15b show the function of the 4th non-canonical GA sequence seed target of miR-133 after expressing miR-133-G4U in cardiomyocytes (h9c2), and the cell morphological characteristics on the myocardial hypertrophy-inducing effect. This shows the observed results. Figures 16a and 16b show the results observed by measuring the enzyme activity of a luciferase reporter in a human hepatoma cell line (HepG2), which shows that gene expression is suppressed through seed targets of non-canonical GA sequences 2 and 3 of miR-122. This is what is shown. Figures 17a to 17d show that inhibition of expression of the second non-canonical GA sequence seed target, second and third non-canonical GA sequence seed targets by miR-122 inhibits miR-122's ability to inhibit the migration of human hepatoma cell line (HepG2). This figure shows the results confirmed by cell morphology observation through the expression of 122-G2U and miR-122-G2,3,U. Figures 18a and 18b show that miR-122-mediated suppression of expression of non-canonical GA sequence seed targets 2 and 3 induces cell cycle arrest in a human hepatoma cell line (HepG2). This shows the results confirmed by flow cytometry analysis regarding the expression of G2,3U. Figures 19a and 19b show that inhibition of expression of non-canonical GA sequence seed targets 2 and 3 and non-canonical GA sequence seed targets 2 and 7 by miR-122 results in cell cycle arrest in human hepatoma cell line (HepG2). This figure shows the results of flow cytometry analysis confirming that miR-122-G2,3U and miR-122-G2,7U induce the following. FIGS. 20a and 20b show that inhibition of expression of the 2nd or 2nd and 4th non-canonical GA sequence seed target by let-7 induces cell cycle arrest in a human hepatoma cell line (HepG2); Results confirmed by flow cytometry analysis through the expression of let-7a-G2U, let-7a-G2,4U, and let-7-G4U that inhibition of the expression of the non-canonical GA sequence seed target number 4 promotes the cell cycle. It showed that. Figures 21a to 21c show that miR-372-G4U or miR-302a-G4U together with miR-372 or miR-302a induce inhibition of non-canonical 4 GA sequence seed target expression by miR-372 or miR-302a. This shows the results of confirmation using an Oct4 gene expression reporter that differentiation is promoted. FIGS. 22a and 22b show the results of confirming the phenomenon that 2'OMe modification at the 6th position of the 5' end standard does not suppress the irregular nuclear protrusion target of the RNA interference derivative. Figures 23a and 23b show the results confirming that the 2'OMe modification at the 6th position of the 5' end standard does not suppress the entire non-canonical nuclear protuberance target mRNA in the transcript. FIGS. 24a and 24b show the results of analyzing microRNAs that bind to irregular nuclear protrusion sites through Ago HITS CLIP experiments. Figures 25a to 25f show the results of analyzing sequence variations in the Cancer Patient MicroRNA Sequencing Database (TCGA) to identify G>U mutations in tumor microRNAs that recognize non-canonical GA sequence seed targets as canonical targets. It is something that

Hereinafter, the present invention will be explained in more detail through Examples. However, the following examples are only examples of the present invention, and the present invention is not limited to the following examples.

[Example 1] Mechanism of action of interference-inducing nucleic acids that suppress non-canonical targets of microRNAs Through Ago HITS CLIP experiments, the present inventors demonstrated that when microRNAs align with targets using Argonaute protein, microRNAs In addition to the regular seed target that has a completely complementary sequence to the RNA seed, the RNA complex sequence result that binds to the Argonaute protein ( Confirmed through analysis of Ago HITS-CLIP). Moreover, as a result of analyzing the base sequence of RNA with these characteristics, it was found that the 6th base of the microRNA induces a nuclear bulge in the target body and the target body (microRNA irregular nucleus) that sequences the bases. It was confirmed that a raised target site) and a target body that allows a GA sequence (wobble) in the microRNA seed region (microRNA non-canonical GA sequence seed target site) naturally exist (Figure 1).

Looking at the diagrammatic example shown in Figure 1, miR-124 transfers the seed nucleotide sequence (5'-UAAGGCAC-3') to the target body's nucleotide sequence 5'-GUGCCUUA-3' through Argonaute protein. It has been reported that a target with such a base sequence is a canonical seed target site of microRNA. Yes (Nature, 2009, 460(7254): 479-86).

In addition, among the RNA complexes bound to Argonaute, miR-124 seed nucleotide sequence (5'-UAAGGCAC-3') and microRNA non-canonical nuclear protuberance target site (5'-GUGGCCUU-3') We confirmed a target with a complementary base sequence so that the G of the target messenger RNA was arranged in a bulge between the 5th and 6th 5' end standards of the microRNA. The seed nucleotide sequence of miR-124, which can be sequenced complementary to the target site, has a nucleotide sequence (5-UAAGGCCA-3) in which the 6th nucleotide is repeated once more between the 6th and 7th nucleotides of the miR-124 seed. A contiguous and perfect complementary sequence to this was used as miR-124BS (BS: Bulge site) siRNA to suppress the non-canonical nuclear bulge target of miR-124.

When we analyzed the sequence of the target messenger RNA bound to the microRNA in the RNA complex bound to Argonaute, we found that in addition to the conventional complementary base sequence, the G:A wobble base sequence was the seed sequence of the microRNA and the target. We confirmed that this occurs when messenger RNA binds. Therefore, in the case of miR-124, the fourth G base of the 5' end standard in the seed base sequence (5'-UAAGGCAC-3') forms a G:A wobble sequence and the non-canonical GA sequence of the microRNA seed target site (5'-UAAGGCAC-3'). '-UGGCAUU-3'), and based on this, we designed siRNA with a sequence that is continuous and completely complementary to the non-canonical GA sequence seed target site of miR-124, and invented miR-124-GU. This siRNA was then used as an siRNA to inhibit the expression of the non-canonical GA sequence seed target of miR-124.

[Example 2] Confirmation of suppressive ability of microRNA-BS whose seed region contains a base sequence that is continuous and perfectly complementary to a non-canonical nuclear protuberance target site Canonical seed target of naturally occurring miR-124 The site (seed target site) that is complementary to the 2nd to 7th bases of the 5' end standard of miR-124 is the 5'-GUGCCUU-3' base sequence. The target site will have a completely complementary base sequence to the miR-124 seed (5'-UAAGGCAC-3') with a minimum of 6 consecutive bases (Figure 2a).

The non-canonical nuclear bulge target site (Nuc site) of miR-124 discovered through the Ago HITS CLIP experiment is 5'-GUGGCCUU-3', and base 6 of miR-124 is located between the target and nuclear bulge. As a result, a bulge of G of the target RNA arranged between the 5th and 6th positions of the 5' end of miR-124 is formed. Based on this, we designed siRNA with a microRNA seed containing a nucleotide sequence (5'-UAAGGCCA-3') that binds continuously and completely complementary to the non-canonical nuclear bulge target site (nucleation bulge site) to detect miR-124. -BS siRNA (Figure 2a). miR-124-BS recognizes the nuclear protuberance target site (Nuc site), which is a non-canonical target of miR-124, as a canonical seed target, and specifically controls the gene expression of the non-canonical target that has this. This fact was confirmed through luciferase reporter experiments (Figures 2b-c).

First, in order to confirm how much miR-124 can suppress nuclear protuberance target sites, which are non-canonical targets, compared to conventional target seed sites, miR-124 was injected into a luciferase reporter. After inserting the relevant site and introducing miR-124 at various concentrations (0, 0.01, 0.1, 0.5, 2.5, 15 nM) into the cells, the IC50 As a result of measuring the (inhibitory concentration 50), it was confirmed that the normal seed site was about 0.5 nM and the irregular nuclear protrusion site was about 0.2 nM, which were similar ( Figure 2b). However, looking at the highest inhibition ratio, the regular seed site is about 50%, and the irregular nuclear protrusion site is about 80%, indicating that inhibition through the irregular nuclear protrusion site is somewhat weak. Ta. In particular, when looking at the point at which gene expression inhibition begins, luciferase activity measurement shows that gene suppression begins at concentrations of approximately 0.01 nM or higher for regular seed targets, and at concentrations of 0.1 nM or higher for non-canonical nuclear protuberance targets. (Figure 2b). Therefore, it was found that miR-124 regulates the expression of canonical targets and non-canonical nuclear protuberance targets, and has a high efficiency in regulating the expression of canonical targets.

At this time, the luciferase reporter experiment was carried out by cloning the corresponding miR-124 target site sequence into the Renilla luciferase gene 3'UTR (3'untranslated region) part of the psi-check2 vector (Promega) twice in a continuous manner. It was carried out. As the non-canonical bulge site sequence, a non-canonical nuclear bulge target site that naturally exists in the 3'UTR portion of MINK1 was used. miR-124 is a human-derived sequence, and according to the miRBase database, the corresponding guide strand and passenger strand were chemically synthesized and produced by Bioneer, separated by HPLC, and manufactured by the above company. A duplex of a guide strand and a passenger strand was prepared by the method provided in . miR-124 and luciferase reporter vector were co-transfected into approximately 10,000 cervical cancer cells (HeLa: ATCC CCL-2) using Lipofectamine 2000 reagent (Invitrogen) according to the manufacturer's protocol. . HeLa cells were cultured in Dulbecco's modified Eagle's medium (Invitrogen) supplemented with 10% FBS (fetal bovine serum), 100 U/ml penicillin, and 100 μg/ml streptomycin, and were treated with antibiotics during transfection. Cells were cultured in complete medium without agents. 24 hours after transfection, luciferase activity was measured using Promega's Dual-luciferase reporter assay system according to the manufacturer's protocol. The experiments were repeated more than once and were normalized and calculated by the activity of firefly luciferase.

In order to confirm the function of miR-124-BS, which was invented to specifically suppress only the non-canonical nuclear protuberance target of miR-124, experiments were conducted using the same luciferase reporter. As a result, miR-124-BS did not inhibit the activity of the luciferase reporter enzyme at any concentration (0-10 nM) against the miR-124 canonical seed target, but against the miR-124 non-canonical nuclear protuberance target reporter. As a result, it was possible to confirm the effect of inhibiting the activity of the luciferase reporter enzyme at a concentration of 0.1 nM or higher (Fig. 2c). When the gene suppression efficiency at this time was measured by IC50, it was shown that the suppression against the non-canonical nuclear protuberance target was about 0.5 nM. From this point of view, it can be analyzed that the function of miR-124-BS in regulating expression of miR-124 non-canonical nuclear protuberance target site is specific to the non-canonical nuclear protuberance target site (Figure 2c). In this luciferase reporter experiment, miR-124-BS was synthesized and produced by Bioneer in the same manner as before, and it was transfected (co-transfected) into cervical cancer cells (HeLa: ATCC CCL-2) together with the luciferase reporter vector. The activity of luciferase enzyme was measured 24 hours after the transfection.

In the above embodiments, the inventors have provided an example in which the miRNA- When we invented BS and applied it to miR-124 to verify its effect through a luciferase reporter experiment, we found that miR-124-BS did not suppress the expression of regular seed targets, but suppressed the expression of non-canonical raised targets. We were able to verify the characteristic that expression can be effectively suppressed.

[Example 3] Observation of cell death phenomenon in cervical cancer cells induced through inhibition of expression of non-canonical nuclear protuberance targets of miR-124. After confirming the fact that only the target is suppressed in the previous example, we attempted to confirm whether only the specific function of miR-124 could be demonstrated through this. In the case of miR-124, it is generally expressed specifically only in nerve cells, and is therefore known to play a role mainly related to nerve cells. However, miR-124 expression is low in tumor-generated gliomas, and when miR-124 expression is artificially induced in tumor cells, tumor cell death can occur. This fact was observed. Therefore, in order to confirm whether the diverse and different functions of miR-124 differ depending on the type that recognizes its target, miR-124-BS was introduced into cervical cancer cells (HeLa), and the cells were killed. was observed by flow cytometry analysis (Fig. 3a).

In this experiment, 75 nM miR-124 or miR-124-BS duplex was transfected into cervical cancer cells (HeLa: ATCC CCL-2) using RNAiMAX reagent (Invitrogen) according to the manufacturer's protocol. ) After 72 hours, the cells were detached from the culture dish by treatment with trypsin, and treated with BD Pharmingen's Annexin V:FITC Apoptosis Detection Kit II by the method provided by the manufacturer. ), apoptosis was measured using Annexin V, and necrosis was measured using PI (Propidium Iodide). At this time, as a control group, a beautiful small nematode (C. elegans) microRNA, cel-miR-67, was synthesized with the same sequence and used.

As a result of comparing these experiments by dividing them into a control group, miR-124 expression, and miR-124-BS expression, it was found that when miR-124 was expressed, cervical cancer cells (HeLa) showed a higher level of expression than the control group. We observed that the rate of apoptosis (the number of cells stained with Annexin V) increased from about 30% to about 78%, and similarly for miR-124-BS, it was about 73%. It was confirmed that the amount increased by more than 2 times (Fig. 3a). In light of the results of the previous Example 2, in which miR-124-BS did not suppress the canonical seed site of miR-124, but only the irregular protuberance site, cell death in cervical cancer was caused by the irregular nucleus. It can be seen that this occurs by suppressing the expression of the protuberance target.

miR-124 is a brain tissue-specific microRNA, and it has been reported that it does not have brain tissue-specific functions in completely different cells that lack tissue cell-specific transcript expression (Farh K et al, 2005 , Science, 310 (5755) 1817-21). Therefore, in order to observe in neurons the function that miR-124 causes by suppressing non-canonical protuberance targets, we introduced miR-124-BS into C17.2, a cerebellar neural stem cell line, and then controlled cell differentiation. It was observed morphologically (Fig. 3b, upper panel). As a result, when miR-124 was introduced, neurite branching differentiation of C17.2 cells was promoted with long formation, but when miR-124-BS was introduced, differentiation was observed but branching were observed differently in the short form. Therefore, it can be seen that general neurite branching differentiation caused by miR-124 occurs by suppressing canonical seed site targets. At this time, miRNA was introduced by synthesizing 50 nM miRNA using the same method as in Example 2 and using RNAiMax reagent.

In addition, since miR-124-BS previously induced cell death in HeLa cells, flow cytometry analysis was performed on C17.2 cells using the same method as in Example 2 to determine whether such a function also appears in nerve cells. I observed it. As a result, in the case of miR-124 expression, the rate of cell death was 22.4%, which was similar to 21% in the control group, whereas miR-124-BS 33.4%, indicating a slight increase (FIG. 3b, middle diagram). This shows the same tendency in cervical cancer cells in that cell death occurs through suppression of miR-124 non-canonical bulge target, but the increase rate is much higher than that in cervical cancer, which appears at approximately twice the rate of cell death. It turned out to be small. miR-124 mainly plays a role related to differentiation in neural stem cells, and in this case, cell cycle regulation can play an important role. Therefore, after introducing miR-124 and miR-124-BS into C17.4 cells, the cell cycle was observed by applying flow cytometry analysis through PI (Propidium Iodide) staining (Figure 3b, bottom panel). At this time, miR-124 was shown to induce cell cycle arrest in G0/G1 at 83%, which was slightly increased compared to 69% in the control group. On the other hand, it was observed that the expression of miR-124-BS did not significantly cause cell cycle arrest. In this experiment, the microRNA was transfected in the same manner as in the previous example, and after culturing for 48 hours, the cells were separated and fixed with ethanol, and 1 mg/ml propidium iodide (PI; Sigma -Aldrich) was reacted with 0.2 mg/ml Rnase A at 37°C for 30 minutes, followed by flow cytometry analysis using BD FACSCalibur (BD Biosciences).

Summarizing the results of the above examples, even when miR-124-BS inhibits the expression of miR-124 non-canonical nuclear protuberance target, it can kill cervical cancer cells (Hela) as well as miR-124. The inducing and inhibiting function of the non-canonical target of miR-124 through this is shown to be the killing of cancer cells. In addition, in the case of neural stem cells, expression of miR-124 causes neurite branching differentiation, resulting in some cell death and cell cycle arrest, but miR-124-BS, which inhibits only the non-canonical target of miR-124, , such functionality appears in somewhat different forms. Therefore, we found that the neuronal differentiation function of miR-124 mainly occurs through the repression of conventionally known canonical seed target genes, and we consider that a different form of miR-124 may occur through non-canonical protuberant targets. was completed.

[Example 4] Confirmation of the function of miR-124-BS in inducing different forms of neural differentiation (branched neurite outgrowth) with many branches and short processes by inhibiting miR-124 non-canonical nuclear protuberance target expression miR-124-BS After confirming in the previous example that miR-124 causes a different form of neurite differentiation, in order to investigate this in more detail, we used human neuroblastoma sh-sy-5y cells. The experiment was performed using the strain (CRL-2266). At this time, in addition to miR-124-BS, the microRNA sequence has the same seed sequence that can recognize and suppress the irregular nuclear prominence site of miR-124, but other sequences have different sequences. The experiment proceeded by synthesizing miR-124-BS (4753), which had the same as miR-124, by the method described in the previous example. At this time, the sequence of miR-124-BS (4753) used was 5'-CAAGGCCAAAGGAAGAGAACAG-3', and as a control, cel, a beautiful small C. elegans microRNA, was used. -It was synthesized and used with the same sequence as miR-67 (NT; non-targeting). The relevant miRNAs were transfected using the same method described in the previous example, and the cells were cultured for 60 hours, after which cell morphological changes were observed by light microscopy (Fig. 4a).

As a result, the expression of miR-124 can be observed in the morphological changes that make human neuroblastoma (Sh-sy-5y) neurites long and differentiated, while the non-canonical protuberance target of miR-124 In the case of miR-124-BS (4753), which has the same seed sequence as miR-124-BS, it has a cell morphology in which many neurite branches occur in one cell, although it is short. neurite outgrowth) was observed (Figure 4a). Based on this, we proceeded with the same experiment using additional catecholamine nerve cells (CAD, CRL-11179). At this time, the cells were transfected with the microRNA, cultured for 52 hours, and then observed (FIG. 4b). As a result, miR-124 formed long protrusions, although the numbers were small, and miR-124-BS ( 4753), we were able to observe a differentiation phenomenon in which many neurite branches were elongated, although they were short. (Figure 4b). Additionally, when both miR-124 and miR-124-BS (4753) were delivered into cells at the same rate, cells with long neurites and cells with many neurite branches were observed at the same time. . This suggests that when expressed together with the invented miR-124-BS and conventional miR-124, it is possible to artificially promote various forms of neural differentiation similar to brain cells exhibiting more complex neural networks. Show your gender.

Additionally, neuronal differentiation of CAD cell lines promoted by such microRNAs was confirmed through immunostaining for Tuj1, a neuron-specific marker (FIG. 4b, bottom panel). In this experiment, 52 hours after transfection with the microRNA, the CAD cells were fixed with 4% paraformaldehyde, and the primary antibody Tuj1 (MRB-435P, Covance Antibody Products) was used. A secondary antibody (Abcam: ab150076) to which three Alexa 594 fluorescent substances were bound at a ratio of 1:1000 was used for antibody staining (immune-staining) at a ratio of 1:1000, followed by staining the nucleus with DAPI. Ta.

In view of the results of the above examples, miR-124 causes the formation of short but many neurite branches, which is a different form from the conventional function of miR-124, by suppressing only the non-canonical nuclear protuberance targets. It can be seen that it has a function. In particular, this fact was demonstrated using miR-124-BS (4753), which has a different sequence from miR-124-BS but has the same seed region sequence and can suppress only non-canonical nuclear protuberance targets. observed, which reveals the fact that RNA interference derivatives containing seed sequences capable of recognizing non-canonical nuclear prominence targets exhibit the same effect as miR-124-BS. We also show that the complex neurite branches generated by miR-124-BS can be utilized as a method to promote various forms of neural differentiation similar to human brain cells, which exhibit complex neural networks. .

[Example 5] Confirmation of morphology and molecular biological characteristics of branched neurite outgrowth induced by expression of miR-124-BS using siRNA and shRNA vector Expression of miR-124-BS In order to further confirm the specific neurite differentiation phenomenon induced in rat neuroblastoma N2a (CCL-131), miR-124, miR-124-BS ( After introducing 4753) into the cells, changes in cell morphology were observed while culturing for 72 hours (Fig. 5a). At the same time, miR-124 (3714), which has the same seed sequence of miR-124 but has a different sequence in other parts, was also synthesized with the 5'-GAAGGCAGCAGUGCUCCCCUGU-3' sequence in the form of siRNA. After forming the duplex, it was introduced into cells for experiments. At this time, as a control group, a beautiful small nematode (C. elegans) microRNA, cel-miR-67 (NT; non-targeting), was synthesized with the same sequence and used.

As a result, in the experimental group in which miR-124 was transmitted, neurons with long neurites similar to those seen in CAD, which are catecholamine-based neurons, were observed, and miR-124-BS (4753 ) transfected cells had morphological features of short neurites with branches in various directions (Fig. 5a, top). Furthermore, in the experimental group transfected with miR-124 (3714), many cells exhibiting relatively long cell processes similar to miR-124 could be observed (Figure 5a, top). Such cell morphological changes can be detected by antibodies against the protein expressed in differentiated nerve cells, Tuj1 (Covance Antibody Products, MRB-435P), vGLUT1 (synaptic systems, 135-303), MAP2 (Millipore, MAB3418). ), it was confirmed through immunoblotting that the expression level was increased compared to the expression of four control proteins (ACT-B, TUB-A, GAPDH, H3B). (Figure 5a, bottom). Through this, neuronal differentiation by miR-124-BS (4753), which is similar to miR-124 but has a different morphology, induces differentiation in the same manner based on gene expression markers, although the morphology is somewhat different. As expected, miR-124 (3714), which has the same seed sequence as miR-124 and is found to primarily suppress canonical seed targets, It was found that the branching of nerve cells exhibits characteristics of long differentiation. Furthermore, when miR-124-BS (4753) and miR-124 (3714) were expressed, the protein expression levels observed in differentiated neurons increased; We were able to confirm that it had the molecular characteristics of a nerve cell.

In the above examples, expression of microRNA was carried out by introducing a synthesized duplex into cells, but expression of microRNA was performed by expressing shRNA, which is a hairpin-shaped RNA that can be produced. It is also possible to introduce a vector that causes Therefore, a vector for expressing miR-124, miR-124-BS (4753), and miR-124 (3714) in shRNA form using the pCAG-miR30 plasmid (addgene #14758), which is a vector for expression with pre-miRNA. was produced. At this time, the pCAG-miR30 vector used uses the CAG promoter to strongly express microRNA, and is designed to express the backbone of microRNA-30 to maximize microRNA expression. (Paddison Pet al, Nature methods, 2004, 1(2): 163-7).

pCAG vectors created to express miR-124, miR-124-BS (4753), and miR-124 (3714), respectively, were introduced into N2a cells using pCAG vectors that do not express anything as a control group. and then observed the cell morphology (Fig. 5b). The experimental group of hairpin structures introduced into neuroblastoma N2a was similar to the cell morphological changes observed in miR-124, miR-124-BS (4753), and miR-124 (3714) duplexes. It showed a change. When cultured for 72 hours after transfection, neuroblastomas with long neurites could be observed in the experimental group in which miR-124 and miR-124(3714) were expressed. In contrast, in the experimental group in which miR-124-BS (4753) was expressed, neurocytomas with short neurites extending in various directions could be observed (Figure 5b, top). Such changes in cell morphology are caused by the expression of Tuj1 (O. von Bohlen et al. 2007 Cell and Tissue Research, 329, 3, 409-20), a protein expressed in differentiated nerve cells, and synaptophysin (SYP). It was confirmed that the expression of miR-124, miR-124-BS(4753), and miR-124(3714) hairpin vectors was increased compared to the control group. It was confirmed that the cells had the characteristics of a differentiated form of nerve cells. The increase in TUJ1 was observed by immunoblotting experiment in the same manner as in the previous example, and the increase in Synaptophysin was observed in the previous paper (S.E. Kwon et al. 2011 Neuron 70, 84) through qPCR (Quantitative Polymerase Chain Reaction) experiment. 7- We proceeded using the primer sequence described in 54). In this experiment, the vector was transfected into N2a cells using Lipofectamine 3000 reagent (Invitrogen) according to the company's protocol, and 24 hours later, total RNA was extracted using the RNeasy kit (Qiagen). According to the protocol, reverse transcription was performed using Superscript III RT (Invitrogen), qPCR was performed using SYBR Green PCR Master Mix (Applied Biosystems), and the amount of SYP messenger RNA was measured using the primers, and the GAPDH messenger RNA value was determined. It was standardized.

Inferring from the results of the above examples, even if a vector for expressing microRNA in shRNA form is used, if only the sequence of the seed part of miR-124 is the same (miR-124 ( 3714)), it was found that long neurite differentiation generally occurs through suppression of the expression of canonical target genes similar to miR-124. In addition, when the non-canonical protuberance target of miR-124 is expressed in shRNA form with only the seed portion identical to miR-124-BS (miR-124-BS (4753)), it induces neurite differentiation with more branches. I can wake you up. In this way, both canonical target inhibition and non-canonical bulge target inhibition by miR-124 showed the expression of molecular gene markers of the neurite differentiation form of neurons, and through this it was possible to confirm that they had all differentiated into neurons. did it.

[Example 6] Confirmation that miR-124-BS induces neurite differentiation of mouse neuroblastoma (N2a) through regulation of MAPRE1 expression In addition, miR-124, miR-124-BS (4753), miR-124 ( We observed the induction of neurite differentiation in neuroblastoma (N2a) of No. 3714) by cell morphology, and analyzed this quantitatively (Fig. 6a). During quantitative analysis, in order to distinguish neural branches derived from each neuron, the pUltra (addgene #24129) plasmid that expresses green fluorescent protein (GFP) and the microRNA expression vector were used in the previous example. Co-transfection was carried out in the same manner as in the previous method, and while the cells were cultured for 72 hours, the number of neurite branches was measured by observing the neurites using a fluorescence microscope (Leica). The length of the neurites was analyzed through the Image J program.

As a result, for 100 cells, the number of neurite branches expressed per cell was found to be higher in miR-124, miR-124-BS (4753), and miR-124 (3714) than in the control group. It was confirmed that the amount increased by about 3 to 5 times (Fig. 6a, lower left). However, when miR-124-BS (4753) was expressed, significantly more branches were observed to be formed than when miR-124 or miR-124 (3714) was expressed. . It was also confirmed that the length of neurites generated per cell was increased 4 to 6 times in the miR-124-BS (4753) and miR-124 (3714) experimental groups compared to the control group. More specifically, we observed that when miR-124 or miR-124(3714) was expressed, longer neurite branches were formed than when miR-124-BS(4753) was expressed. It was completed (Fig. 6a, lower right side). This is the same result as observed cell morphology in the previous examples (FIGS. 4, 5, and 6).

In order to investigate whether the specific neural differentiation phenomenon caused by miR-124-BS is caused by the non-canonical protuberance site of any gene, we created a non-canonical protuberance site of miR-124 in the 3'UTR region of a gene related to neuronal differentiation. I tried searching for the target sequence. As a result, we searched for the MAPRE1 (Microtubule-associated protein RP/EB family member 1: Elena Tortosa et al. 2013 EMBO 32, 1293-1306) gene, which has been reported to regulate neuronal differentiation. MAPRE1 is a protein that attaches to the ends of microtubules that constitute neurites when they are generated, and can determine the morphology of neurites through regulating the formation of the microtubules. Therefore, in order to confirm whether this MAPRE1 gene is recognized as a non-canonical target of miR-124 and its expression is inhibited, after introducing miR-124-BS into N2a cells, the amount of MAPRE1 messenger RNA was was measured by carrying out a qPCR experiment using the method performed in Example 5b (Fig. 6b). When qPCR was performed using two different primers, both results confirmed that MAPRE1 expression decreased to a level of 70-40% in the experimental group introduced with miR-124-BS compared to the control group. We were able to. Through this, it was confirmed that the expression of MAPRE1 was suppressed at the non-canonical protrusion site of miR-124.

If MAPRE1 is a non-canonical protuberance target of miR-124 and is a very important target, the neuronal differentiation pattern manifested by suppression of the non-canonical protuberance target of miR-124-BS should be further promoted by decreased expression of MAPRE-1. Must be. Therefore, in order to confirm this point, we created two siRNAs that can inhibit MAPRE1, transfected them with miR-124 into N2a cells, and conducted an experiment to see if they exhibited changes in cell morphology. (Fig. 6c). As a result, it was observed that the number of neurite branches increased in the experimental group in which miR-124 and siRNA against MAPRE1 were expressed together, compared to the control group in which only miR-124 was expressed. The same observation was made in experiments using MAPRE1 siRNAs with two different sequences (Figure 6c). In this experiment, RNA was introduced into cells using the same method as used in the previous example, and the morphology was observed while culturing the cells for 48 hours.

Through this, miR-124-BS-induced short and branched neurite differentiation may be manifested, at least in part, by suppressing the expression of the MAPRE1 gene through the non-canonical protuberant target site of miR-124. Confirmed that it is a function.

[Example 7] Analysis of inhibition tendency of miR-124 non-canonical target genes upon introduction of miR-124-BS into cells at the transcript level Suppressed through miR-124-BS in mouse neuroblastoma (N2a) It was confirmed that the gene ultimately induces neuronal differentiation that leads to the production of many neurite branches in neuroblastoma (Figure 5). Such a phenomenon is thought to occur through a mechanism in which the seed sequence of miR-124-BS recognizes and suppresses the non-canonical target of miR-124. Confirmed (Figure 6). However, in reality, this phenomenon probably occurs when hundreds of miR-124 non-canonical target genes are suppressed.

In order to confirm only the non-canonical target genes of miR-124 at the transcript level where the miR-124-BS invented by the present inventors actually expresses all genes, miR-124 and miR-124 - RNA-Seq analysis was performed after introducing BS into N2a cells. As a control group, we used a duplex (miR-124-6pi) with the same nucleotide sequence as miR-124 and a non-base (pi) in 5'-terminal reference number 6. It was reported that there is no base at the 6th position of the 5' end standard, so there is no sequence at all with the target messenger RNA ((Lee HS et. Al. 2015, Nat Commun. 6:10154).

The RNA-Seq experiment used the sequence of cel-miR-67, which is a beautiful small C. elegans microRNA, as an experimental control group, with the 5' end reference number 6 converted to a non-base (NT-6pi). miR-124, miR-124-BS, and miR-124-6pi duplexes were each delivered to N2a cells at 75 nM concentration using A library was created using Otogenetics, and next-generation sequencing was performed. After that, the FASTAQ file, which is the sequence data obtained in the above experiment, was mapped to the mouse dielectric array (mm9) using the TopHat2 program, the expression value (FPKM) was determined using the Cufflink and Cuffdiff programs, and the control group NT-6pi was introduced. The analysis was performed by normalizing the results with the mouse neuroblastoma (N2a) cells obtained using the Fold change, log2 ratio.

In order to analyze using RNA-Seq results whether the amount of messenger RNA of a gene that has a microRNA target site is inhibited by the expression of the microRNA, we analyzed the canonical seed site of miR-124 (5' end standard). Genes with a phastCons score of 0.9 or higher are selected in order to select genes that have a 7mer (base sequence from 2nd to 8th nucleotides) in their 3'UTR, and the sequence of this site is conserved in multiple species. were selected, and the profile results were compared and analyzed based on the cumulative fraction in the order in which the expression of the microRNA was inhibited in a dependent manner. In addition, a non-canonical raised site (nuc, 7mer) of miR-124 was also identified using the same method, and the suppression ratio of the gene was analyzed in the same cumulative ratio. In this case, the gene that has the non-canonical raised site of miR-124 also has a regular seed site, and it may be difficult to judge its suppressive effect. In contrast to the case where there is no sequence (nuc only), the case where there is only a regular seed site and no non-canonical raised site (seed only) was also analyzed.

When the fold change due to miR-124 expression was analyzed by the cumulative fraction in the RNA-Seq sequence analysis of the experimental group that expressed miR-124, it was found that the conserved canonical seed site of miR-124 was 3' A phenomenon in which genes that are located in the UTR (miR-124 seed) or genes that only have this site (miR-124 seed only) are suppressed much more often than the distribution of the entire gene (total mRNA). (Fig. 7a, top), and in contrast, we could observe that genes with miR-124 non-canonical raised sites were significantly repressed and very weakly inhibited ( Figure 7a, bottom). However, such suppression phenomenon was not observed in cells transfected with miR-124-6pi as a control group (Fig. 7b).

When miR-124-BS developed by the present inventor was expressed, when the fold change due to miR-124-BS expression was analyzed by cumulative fraction using RNA-Seq sequence analysis, miR -124 conserved canonical seed site in the 3'UTR (miR-124 seed) showed a slight inhibitory effect, but there was no miR-124 non-canonical raised site, and the canonical seed site It was confirmed that there was no change at all in the gene having only the site (miR-124 seed only) (Fig. 7c, top). However, in the case of genes with a non-canonical raised target of miR-124 (miR-124 nuc) and genes with only this target (miR-124 nuc only), both are strong and effective. It was found that a gene suppression phenomenon occurred in (Figure 7c, bottom). When miR-124-BS is expressed in Figure 7c, the gene that has only the canonical seed site of miR-124 (miR-124 seed only) is the canonical seed site of miR-124, as no inhibition occurs. It can be concluded that miR-124-BS inhibited the seed site target of miR-124 to some extent, although it can be concluded that miR-124-BS did not inhibit miR-124 seed site targets at all. It is inferred that irregular upheaval sites exist together and exhibit some suppressive effect.

Inferred from the results of the above examples, miRNAs at the transcript level suppress the repression of conventional canonical seed target genes very strongly and efficiently, but inhibit non-canonical raised targets very weakly. It was confirmed that miRNA-BS suppresses the expression of miRNA non-canonical protuberance targets at the transcript level, but not the conventional canonical seed sequence.

[Example 8] Confirmation of cell cycle arrest induced by miR-122-BS in human hepatoma cell line (HepG2) through flow cytometry analysis miR by non-canonical nuclear protuberance target of miR-124 Based on the results of observing the induction of neuronal differentiation of -124, we conducted experiments on the biological functions of non-canonical nuclear protuberance targets of other microRNAs. miR-122 has 5'-UGG AGU GU-3' as a seed base sequence, and miR-122-BS, which is the base sequence of siRNA that has a base sequence with this non-canonical nuclear protuberance target site, has 5'-UGG AGU GU-3' as a seed base sequence. may be repeated once again, and in this case, it is composed of 19 bases with a 5'-UGG AGU U GUG ACA AUG GUG-3' sequence at the 5' end, and the 20th and 21st The base consists of dt (Deoxy Thymine Nucleotide). In particular, in the duplex structure, the dt portion was composed of a guide and a passenger strand so that it could form a two nucleotide overhang at the 3' end. At this time, the passenger strand is configured to form a completely complementary base bond with the guide strand, modify both the first and second 5' end standards with 2'OMe, and include two dts at the end of the base sequence. (Fig. 8a). The guide chain and passenger chain of miR-122-BS were chemically synthesized by Bioneer, separated by HPLC, and produced as a duplex of the guide chain and passenger chain by the method provided by the company.

The thus prepared miR-122-BS (FIG. 8a) was introduced into a human liver cancer cell line (HepG2), and changes in cell cycle were then observed through flow cytometry analysis. In this experiment, a control group of a duplex of NT-6pi, miR-122, and miR-122-BS was transfected into HepG2 cells at a concentration of 50 nM using RNAiMAX (Invitrogen) reagent. After culturing for 24 hours, cells were synchronized to G2/M (mitotic preparation phase/mitosis phase) by treatment with nocodazole at 100 ng/ml for 16 hours, and then treated with Muse ^TM Cell Cycle Kit (Catalog No. MCH100106 (Milipore) and the cell cycle was analyzed with a Muse Cell Analyzer (Milipore) according to the experimental method provided by the company.

As a result, compared to the cell cycle analysis of the control group to which NT-6pi was transmitted, the miR-122-BS experimental group had about twice as many cells in the G0/G1 phase, from 10.7% to 24.3%. It was confirmed that the cell cycle arrest of HepG2, a liver cancer cell, increased (Figures 8b-c). Such an increase in cells in the G0/G1 phase was not observed in miR-122-transfected cells. Next, the G2/M phase cell distribution of the human liver cancer cell line (HepG2) to which miR-122-BS was transmitted decreased from 83.4% to 67.3% when compared with the control group, and these results was not observed in miR-122-transduced cells (Fig. 8b,c).

Through this, miR-122-BS exhibits a biological function to induce cell cycle arrest in human hepatoma cell lines through the regulation of non-canonical nuclear bulge target expression of miR-122, which is due to miR-122 acting We were able to observe that this function is completely different from biological function.

[Example 9] Confirmation of muscle fibrosis function induced by miR-1-BS in skeletal muscle cells (C2C12) and cell killing function of miR-155-BS miRNA-BS suppresses expression of regular seed target of miRNA We observed a new biological function that is different from the function that appears in muscle cells, so in order to observe how such a function appears in muscle cells, we investigated miR-1, which is expressed in muscle cells and has important functions. We applied miRNA-BS to miR-155.

First, miR-1 is a muscle tissue-specific microRNA and has been reported to function in muscle cell differentiation (Chen J et.al, Nature genetics, 2006, 38(2): 228- (233), miR-1-BS was expressed in the skeletal muscle cell line C2C12, and then cell morphological changes (Figure 9a) and the presence or absence of expression of gene markers expressed during differentiation (Figure 9b) were investigated. At this time, miR-1-BS was synthesized by the same method used in the previous example, transfected into cells, and incubated with 10% FBS (fetal bovine serum), 100 U/ml penicillin, and 100 U/ml penicillin during cell culture for 48 hours. and Dulbecco's modified Eagle's medium (Invitrogen) supplemented with 100 μg/ml streptomycin, which was grown under growth conditions (GM) conditions (Fig. 9a, top) and thus After that, the cells were further cultured for 48 hours under deprivation media conditions (DM) in which FBS was removed and observed separately (FIG. 9a, bottom). Thereafter, the cells were fixed with 4% paraformaldehyde (PFA), and myosin 2 heavy chain protein expressed in differentiated muscle cells was treated with primary antibody (MF20: Developmental studies hybridoma bank). After reaction, cells were stained with mouse secondary antibody (ab150105) tagged with Alexa 488 fluorescent substance to observe cell morphology and differentiation (Figure 9a).

As a result, rat skeletal muscle cells (C2C12) showed even more expression of miR-1-BS under growth culture conditions (GM) compared to control group NT and miR-1 duplex-transfected cells. A number of differentiated myocytes could be confirmed by MF20 staining results. Under deprivation media conditions (DM) in which serum is reduced during culture, we observed that differentiation into muscle fibers progressed, but miR-1-BS A thicker morphology could be observed in the expressed skeletal muscle cells (Fig. 9a). This may be a result of miR-1-BS inducing myocyte differentiation faster under proliferation culture conditions. Such differentiation of muscle cells can be molecularly confirmed by using markers to determine the expression level of genes whose expression increases during differentiation. RT (reverse transcription)-PCR was performed using primers to detect and compare the amount of b-actin messenger RNA, which has no difference in expression level even during differentiation (FIG. 9b). As a result, it was confirmed that the transcription levels of sk-actin and myogenin increased when miR-1 (1) and miR-1-BS (1BS) were all expressed; It could be observed that miR-1 was increased by 1-BS even more than miR-1.

miR-155 is a microRNA that suppresses muscle cell differentiation (Seok Het. Al, JBC, 286(41):35339-46 2011), and miR-155 was The effect of -155-BS was observed through the differentiation of skeletal muscle cells (C2C12) (Fig. 9c). As a result, after expressing NT, miR-155, and miR-155-BS as a control group, we did not observe any particular morphological differences in the cells under growth culture conditions (GM), but the differentiation of myocytes Under deficient culture conditions (DM) that induce myosin 2, the control group differentiates and is suppressed when miR-155 is expressed, but when miR-155-BS is introduced, differentiation is actually abolished. This was confirmed through an immunostaining experiment using heavy chain protein (Figure 9c). In particular, when C2C12 cells expressing miR-155-BS were closely examined, it was observed that miR-155-BS induced cell death. In contrast, no cell death was observed in miR-155-transfected myocytes (Figure 9c, bottom). To investigate this in more detail, C2C12 cells expressing the microRNA were grown in proliferative culture conditions for 48 hours, and then the same flow cytometry analysis as in Example 3 was applied to examine cell death. (Figure 9d). As a result, it was confirmed that cell death in the experimental group to which miR-155-BS was transmitted increased by about 1.5 to 2.5 times compared to the control group (Figures 9d and e).

Through the above examples, miR-1-BS promotes the differentiation of skeletal muscle cells and induces thick muscle fiber differentiation, and miR-155-BS induces the death of skeletal muscle cells and promotes such functions. We were able to confirm that the function of miR-1, which causes conventional muscle cell contraction, and the function of miR-155, which suppresses muscle cell differentiation, are different.

[Example 10] Discovery of a non-canonical target site that allows wobble base sequences at microRNA seed positions using Ago HITS CLIP analysis A method that can analyze microRNA targets at the transcript level is called Ago HITS CLIP. Experimental methods have been developed and are widely used (Nature, 2009, 460(7254): 479-86). The Ago HITS CLIP experiment involves irradiating cells and tissue samples with UV to induce covalent bonds between RNA and Argonaute proteins within the cells, and then converting the thus generated RNA-Argonaute complexes into Argonaute-specific molecules. In this method, RNA is separated by immunoprecipitation using an antibody that recognizes the RNA, and then the separated RNA is analyzed by next-generation sequencing. The base sequence data obtained in this way not only allows for the identification of the target mRNA of microRNA through bioinformatic analysis, but also allows for accurate analysis of its binding position and sequence (Nature, 2009, 460). (7254):479-86). Ago HITS-CLIP was first applied to rat cerebral cortex tissue to map microRNA targets in brain tissue, and the method has since been applied to various tissues to date. Through such various Ago HITS-CLIP analyses, microRNA binding locations in various tissues have been revealed, and in particular, the seed binding of microRNAs at such target sequences is similar, but the position of microRNA binding at target mRNA sequences is similar. It has been learned that there are many non-canonical binding targets with different forms (Nat Struct Mol Biol. 2012 Feb 12; 19(3): 321-7).

It has been observed that some of the revealed non-canonical binding rules exist in the form of wobbles through G:U base sequences in addition to conventionally known base sequences. Wobble base sequences are different from conventional base sequences and have been discovered in specific RNAs, and it has been proposed that the well-known G:U wobble can also be sequenced in non-canonical targets of microRNAs. However, the binding is weaker than this, and it is not observed frequently.In particular, in the case of G:A wobble, a base sequence can be formed with a specific RNA, but nothing else has been investigated. However, in cases such as microRNA seed sequences, the free energy of the base sequence is structurally stabilized by the Argonaute protein, so generally the weak G:A base sequence is relatively weak. The present inventors focused on this fact and conducted the following analysis.

First, in order to confirm whether the wobble base sequence containing G:A exists in microRNA targets in human brain tissue, data obtained by performing Ago HITS-CLIP on gray matter of postmortem brain tissue ( Boudreau RL et al, Neuron, Vol. 81(2), 2014, 294-305) was analyzed (Figure 10a). At this time, the analysis was performed using a FASTQ file sequenced with the RNA sequence combined with Argonaute-microRNA using the method used when Ago HITS-CLIP was developed (Nature, 2009, 460 (7254): 479-86). The program was used to sequence the human gene sequence. At the same time, the sequencing results were arranged into human microRNA sequences, and 20 types of microRNAs (top 20 miRNAs: Figure 10b) that frequently bind to Argonaute protein in the brain tissue were also analyzed. Finally, for the top20 miRNA identified in this way, a canonical target site having a complementary sequence to the seed sequence from the 2nd to 8th positions from the 5' end was created. investigated (Fig. 10b). At this time, we observed that a considerable number of regular seed target sites were found at the microRNA binding position (Figure 3b, median value 1292.5 sites, error range +/-706.62). Then, using Ago HITS-CLIP data, it was investigated whether there was a non-canonical target site that could be bound by the G:U or G:A wobble base sequence using the microRNA seed base sequence. At this time, as a control group for the analysis, a target sequence having the same base sequence as the microRNA seed base sequence and in which a complementary sequence of bases cannot be formed was used for the analysis.

As a result of the analysis, targets where G:A wobble was allowed (median value 1119.5 sites, error range +/-526.48) and targets where G:U wobble was allowed (median value 995 sites, error range +/- 523.22) All sites showed a higher distribution compared to the control group (median 891 sites, error range +/-410.71), and in particular, microRNA target sites where G:A wobbles were present were G:U wobbles. It was shown that the amount was about 1.1 times more than that of the 1.1-fold amount (Fig. 10b). miR-124, which is specifically expressed in brain tissue, has two G's at the fourth and fifth positions from the 5' end in the seed part, and the G:U,G:A wobble base sequence through these. (Figure 10c). Therefore, when we analyzed the G:A and G:U wobble base sequences caused by G at specific positions (Figure 10d), we found that the most regular seed target sites (seeds) of miR-124 were discovered. (1,992 pieces). However, we discovered that the fourth one formed a G:U wobble (1,542 pieces) to the extent that it could be compared with this, and then observed that the fourth one formed a G:A wobble (1,542 pieces). did. In addition, for the fifth G, there were many target sites with G:U wobbles (1,800) and G:A wobbles (1,198), and all sites investigated in this way were compared to the control group. It was confirmed that there were more than 647 pieces (Fig. 10d). Therefore, when microRNA recognizes target mRNA through the base sequence of the seed part, it not only recognizes the regular base sequence through the seed part but also non-canonical G:U or G:A wobble base sequence. It was found that the G:A wobble base sequence in particular can bind to target mRNA through such binding. Such target recognition of microRNAs through G:A wobble bases is a new result that has never been reported, and therefore, as observed in the above examples, non-canonical targets can be recognized in various microRNAs. I was able to confirm that it was recognized.

Through the above examples, there are cases where a G:A wobble sequence is allowed in the seed sequence for non-canonical target recognition of microRNA, and through this, many microRNAs bind to the messenger RNA of the target gene, leading to the recognition of that gene. It is thought to suppress the expression of

[Example 11] Development of mIRNA-GU that recognizes the G:A seed sequence site, which is a non-canonical target of microRNA, and confirmation of the effect of miR-124-G5U on increasing neuronal death when miR-124 is applied In Example 10, We discovered that a G:A wobble sequence may be allowed in the seed sequence for microRNA non-canonical target recognition, and we termed this the non-canonical G:A seed sequence site. We invented miRNA-GU, which is a new RNA interference-inducing substance that can be sequenced, as follows. When such sequencing technology was applied to miR-124, the seed sequence of miR-124 was 5'-AAGGCAC-3', and the non-canonical GA sequence seed target was the 4th G base. , the 5th G base is a base sequence that allows a G:A wobble base sequence, and therefore, the miRNA-GU sequence, which is a format that changes such a G:A wobble sequence to a conventional complementary base sequence. When changed, it can be changed to miR-124-G4U (5'-AAUGCAC-3') or miR-124-G5U (5'-AAUGUCAC-3'). miR-124-G4U and miR-124-G5U modified in this way bind non-canonical target sites, which conventional miR-124 weakly binds and recognizes in the form of a G:A wobble sequence, with complementary sequences. can demonstrate the function of non-canonical G:A seed sequence targets on miR-124.

Therefore, to investigate the biological functions mediated by miRNA non-canonical GA sequence seed targets, experiments were performed by applying non-canonical GA sequence seed targets to miR-124. For this, we previously introduced NT (denoted as N2a in Figure 11b), miR-124, miR-124-G4U, and miR-124-G5U into neuroblastoma N2a as a control group by the same method in Example. The cell morphology was observed while culturing for 72 hours (Fig. 11b). As a result, when miR-124 is expressed, a differentiation phenomenon occurs in which long neural branching processes are formed, but in the experimental group in which miR-124-G4U and miR-124-G5U are transmitted, differentiation does not occur. The phenomenon was observed (Fig. 11b). In this way, the neuronal differentiation ability, which is a function of conventional miR-124, was not observed when miRNA-GU was used, so we investigated changes in cell death in the human neuroblastoma Sh-sy-5y cell line. Observed. The flow cytometry analysis at this time was performed using the Muse Annexin V and Dead Cell Assay Kit (Millipore) using the Muse Cell Analyzer (Millipore) in the same manner as in Example 9. As a result, we confirmed that the expression of miR-124-G-5U increased cell death by more than two times compared to the control group (Figure 11c), indicating that this was associated with early cell death (early apoptosis) and late cell death. When analyzed subdivided into (late apoptosis), compared to the experimental group where miR-124 was delivered, the experimental group where miR-124-G-5U was delivered showed early cell death (early apoptosis) and slow cell death. Both cases of late apoptosis were analyzed as significantly increased (Figure 11d). In the experimental group to which miR-124-G-4U was delivered, late cell death (late apoptosis) was significantly suppressed compared to the experimental group to which miR-124 was delivered (Fig. 11c, d), and the cells remained alive. It was also possible to observe that there were many cells present (Fig. 11b).

Based on this, miR-124 non-canonical GA sequence seed target suppression by miR-124-GU abolishes the ability of miR-124-induced neuronal differentiation and instead induces cell death, which has a different biological function. We were able to confirm that it exhibited a regulatory function.

[Example 12] Promotion of cell division of neuroblasts induced by miR-124-G4U In the case of miR-124-G4U in the previous example, the ability to differentiate into neurons was lost, and even when the cells died, they had special functions. Since it did not appear, we investigated cell division. That is, control groups NT, miR-124, miR-124-G4U, and miR-124-G5U were introduced into neuroblastoma cells N2a using the same method as in the previous example, and then Flow cytometry analysis was performed for the mitotic proliferation phenomenon (Fig. 12a). In this experiment, cells were treated using the Muse Ki67 Proliferation Kit (Millipore) according to the experimental method provided by the manufacturer, and then cell division was analyzed using the Muse Cell Analyzer (Millipore). This method is a method for quantitatively analyzing the number of cells with increased cell division and proliferation by measuring the number of cells stained with Ki67. When miR-124 is expressed in N2a cells, the number of cells with reduced Ki67 staining intensity increases as the cells differentiate compared to the control group NT. However, in the experimental group to which miR-124-G4U was transferred, the number of Ki67-stained cells, which are cells with increased cell division, increased from 61% to 65% compared to the control group (NT). On the other hand, it was observed that there was no difference in miR-14-G5U compared to the control group.

In order to additionally investigate the effect of miR-124-G4U on the cell cycle, flow cytometry analysis was performed using the human neuroblastoma sh-sy-5y cell line (Figure 12b). In this analysis, in order to examine the functions of miR-124-G4U and miR-124-G5U in each cell cycle, ^Muse Cell Cycle Kit (Catalog No. MCH100106, Milipore) was used. e) It was analyzed in As a result, in the case of miR-124, cell cycle arrest, in which the number of cells in the G0/G1 cycle increased, was observed, but miR-124-G4U and miR-124-G5U It was observed that the cell division arrest phenomenon caused by -124 disappeared, and the cell cycle progressed in the same manner as in the control group.

Through the above examples, it was found that miR-124-G4U, which is an RNA interference-induced modified sequence that suppresses miR-124 non-canonical GA sequence seed target, has the function of increasing cell division and proliferation of neuroblasts.

[Example 13] Using a fluorescent protein reporter, we confirmed that miR-1 can suppress the expression of a non-canonical GA sequence seed target gene in a cardiomyocyte cell line. After discovering the fact that A-wobble sequences can bind to target mRNAs and that this can have a new function, we investigated whether such binding can actually cause repression of target genes in cells. We proceeded with a reporter experiment to confirm the level. At this time, confirmation experiments targeted miR-1, which is known to be highly expressed in muscle cells such as h9c2, which is a cardiomyocyte, and focused on G, which is located at the 7th position from the 5' end of miR-1. Then, the G:A wobble base sequence obtained through this was used as the target (Fig. 13). In order to more precisely measure gene suppression by microRNA at the individual cell level, a fluorescent protein expression reporter system was constructed and used (FIG. 13). The fluorescent protein expression reporter system is configured so that two colored fluorescent proteins are expressed in one vector. At this time, green fluorescent protein (GFP) is expressed in the SV40 promoter. A plurality of microRNA target sites are consecutively arranged in the 3'UTR (3'untranslated region) part, so that the gene suppression effect of microRNA can be measured through the intensity of green fluorescent protein, and at the same time, the gene suppression effect of microRNA can be measured through the intensity of green fluorescent protein. A red fluorescent protein (RFP) was expressed by a TK promoter, and the green fluorescent signal was standardized according to its intensity (FIG. 13a, top).

The fluorescent protein expression reporter vector constructed in this way is complementary to the bases from the 2nd to the 8th base from the 5' end of miR-1, and the 7th base is a G;A wobble. After constructing a reporter by inserting a non-canonical target site (7G:A-site) in the nucleotide sequence (GFP-7G:A site), it was confirmed through experiments whether it was suppressed by miR-1. In this experiment, 500 ng GFP-7G:A site reporter vector and 25 uM miR-1 were co-introduced (co-introduced) into the cardiac muscle cell line H9c2 using lipofectamine 2000 (Invitrogen) reagent according to the protocol provided by the company. 24 hours later, each fluorescence signal was measured by flow cytometry analysis using Attune NxT from Life Technology. As a result, we found that the 7G:A site of miR-1 was suppressed very efficiently by the expression of miR-1 (Fig. 13a, middle) in cells into which a control group nucleic acid (cont; NT) had been introduced (Fig. 13a, Confirmed by comparing with the left side). That is, as a result of analyzing the cardiomyocyte cell line h9c2 in four parts (Q5-1, Q5-2, Q5-3, Q5-4) depending on the expression level of the two fluorescent proteins, it was found that cells into which miR-1 had been introduced In this case, the distribution of cells showing 10 ³ intensity red fluorescent protein and 10 ³ intensity green fluorescent protein increased from 59.51% (control: Q5-3) to 41.80% when the control group nucleic acid was introduced. % (miR-1:Q5-3) was observed.

It was observed in the control group that the GFP-7G:A site reporter vector was suppressed to some extent in h9c2 cells even without miR-1 introduction. This can be expected to be suppressed by miR-1 already expressed in h9c2 cells through the G;A wobble sequence with the reporter target mRNA. To confirm this point, miRIDIAN microRNA Hairpin inhibitor (miR-1 inhibitor, Fig. 13a, right), which can suppress the expression of miR-1 in h9c2 cells, was purchased from Dharmacon and transfected into cells at a concentration of 50 uM. It was used with an injection. These results show that compared to the control group (Fig. 13a, left), when miR-1 inhibitor was introduced (Fig. 13a, right), red fluorescent protein with an intensity of 10 ³ and green fluorescent protein with an intensity of 10 ³ was observed. Considering that the distribution of cells increased to 81.56% (miR-1 inhibitor: Q5-3), miR-1 present in cells suppresses gene expression through the 7G:A site of miR-1. It was confirmed that this was possible (Fig. 13a).

This is a control group in which a microRNA target site is not included in the fluorescent protein expression reporter (GFP-no site) and a GFP-7G:A site, which is a reporter in which the miR-1 7G:A site is included, in H9c2 cells. The positive control group was confirmed by quantitative comparison with the case in which miR-1 was introduced together (Figure 13b). In this analysis, the red fluorescent protein (RFP) values measured with a flow cytometry analyzer were divided into sections, and the green fluorescent protein (GFP) values were averaged and converted using a log ratio. The green fluorescent protein value of the control group GFP-no site was set to 1 (relative log GFP) and relative calculations were made. At this time, if there is a site where the base sequence is complementary to mR-1 and No. 7 G base through G:A wobble (GFP-7G:A site), especially the 1.5-3 value. It was confirmed that the expression of green fluorescent protein (relative log GFP) through G:A wobble was about 10% lower than 1 in the interval where the expression of red fluorescent protein (log RFP) was observed. This kind of suppression is particularly consistent with the pattern in which the expression of red fluorescent protein (log RFP) in the 1.5-4 interval is suppressed by about 40% to 10% of the expression of green fluorescent protein (relative log GFP). We observed that it appears identical to .

In order to confirm that the results observed in the above example are not affected by the different promoter strengths used in the fluorescent protein reporter and the difference in fluorescence activity of the two different fluorescent proteins, we tested two fluorescent proteins. The fluorescent protein reporter p. UTA. 3.0 (Lemus-Diaz N et al, Scientific Reports, (7), 2017) was purchased from Addgene (plasmid #82447) and used. At this time, the reporter experiment was conducted on p. UTA. After inserting the miR-1-7G;A sequence 5 times into the 3'UTR region of red fluorescent protein (RFP) regulated by the SV40 promoter using the 3.0 vector, the reporter vector (RFP-7G : A site) was prepared and proceeded, and at this time, the red fluorescent protein expression value was standardized to reflect the degree of introduction into the cells of green fluorescent protein (GFP) expressed by the Tk promoter. It was modified and used (Fig. 13c, top). As a result, we confirmed that the 7G;A site of miR-1 is suppressed by miR-1 expressed in h9c2, as in the previous example, in both the control group of a reporter with no miRNA target position (RFP-no site) and the miR- It was confirmed by comparing the results of the reporter (RFP-7G: A site) containing 1 of 7G; observed (Fig. 13c-d).

From the above, the non-canonical targets of microRNAs discovered through Ago HITS-CLIP analysis can actually not only bind to the microRNA seed sequence through the G:A wobble sequence, but also suppress the expression of the target gene. I confirmed that it can be done. Therefore, inferring from this point, it is possible to sufficiently bind to microRNA using the target site through the G;A wobble sequence, and this point can be similarly induced through miRNA-GU. It is thought that only the biological functions of microRNAs can be exploited through non-canonical target suppression.

[Example 14] Confirmation of the hypertrophy-inducing function of cardiomyocytes through non-canonical GA sequence seed target gene regulation for G bases 2, 3, and 7 of miR-1 Such non-canonical gene suppression phenomenon is caused by In order to confirm the possibility that the A-wobble base sequence can also act as an atypical target of microRNA and regulate biological functions through miRNA-GU, we investigated the possibility that the A-wobble base sequence can also act as an atypical target of microRNA and regulate biological functions. The experiment focused on miR-1.

To investigate whether non-canonical G:A wobble targets have specific biological functions, miR-1 does not recognize canonical targets but recognizes non-canonical G:A wobble targets. Since it is necessary to recognize only the sequence target site, we used miRNA-GU produced by replacing G in the seed region of miR-1 with U so that the base sequence is A, which is not C, in the experiment. did. In this experiment, primary rat neonatal cardiomyocyte culture was cultured from rat heart tissue in order to conduct the experiment under conditions more physiologically similar to those of the heart. At this time, the cardiomyocytes were cultured by separating the cardiomyocytes from the heart tissues of 1-day-old rat neonates through an enzyme reaction (Cellutron, neomyt kit), and the cardiomyocytes were incubated with 10% FBS (fetal bovine serum). ), Dulbecco's modified Eagle's medium (Invitrogen) and M199 (WellGene) medium supplemented with 5% HS (horse serum), 100 U/ml penicillin, and 100 μg/ml streptomycin were mixed at a ratio of 4:1. BrdU was added to the culture medium to differentiate the cells from other cells with a cell cycle present in the heart tissue and to culture them.

It was preferentially confirmed whether myocardial hypertrophy was induced in the primary cultured cardiomyocytes thus obtained (FIG. 14a). Cardiomyocytes have the characteristic of stopping the cell cycle and increasing their size, which is called myocardial hypertrophy. Myocardial hypertrophy plays a role in compensating for the decline in cardiac muscle cell function, and is also known as an intermediate stage in the pathology of heart disease, and is a heart disease model system with well-known cell morphological observations and changes in gene expression profiles. . In a cell model of myocardial hypertrophy, treatment of adrenergic receptor agonists such as phenylephrine (PE) to myocardial cells can induce myocardial hypertrophy (Molkentin, JD et al, 1998, Cell 93(2), 215-228). Therefore, we treated cultured cardiomyocytes with 100 uM phenylephrine to induce myocardial hypertrophy, which resulted in an increase in cardiomyocyte size compared to the untreated control group (Fig. 14a, rCMC). This was confirmed scientifically (Figure 14a, +PE). Furthermore, when miR-1 was introduced into primary cardiomyocytes, we observed that the size of the cardiomyocytes decreased (Fig. 14a, upper right side). This is consistent with the well-known myocardial hypertrophy phenomenon and the function of miR-1 in cardiomyocytes (Molkentin, JD et al, 1998, Cell 93(2), 215-228, Ikeda S et al, Mol cell boil , 2009, vol29, 2193-2204).

miR-1 contains three G's in the seed region, which allows it to recognize only non-canonical G:A wobble sequence target sites without recognizing canonical targets. We designed miR-1 in which G is replaced with U, and determined the position of G at the second position (miR-1-G2U) and third position (miR-1-G3U) based on the 5' end. , was applied to the seventh position (miR-1-G7U) and synthesized. At this time, the substituted base sequences of miR-1 used in FIG. 14 are as follows (miR-1-G2U: 5'p-UUGAAUGUAAAGAAGUAUGUAU-3'; 3'; miR-1-G7U: 5'p-UGGAAUUUAAAGAAGUAUGUAU-3'), which is synthesized with a guide strand, and the passenger strand is designed from that of conventional miR-1 and chemically synthesized at Bioneer. After production, it was separated by HPLC, and a duplex of a guide strand and a passenger strand was prepared and used according to the method provided by the company. Transfection of the microRNA into the primary cardiac muscle cell line was performed at a concentration of 50 uM using RNAimax (Invitrogen) reagent.

As a result, when miR-1-G2U, miR-1-G3U, or miR-1-G7U was introduced into primary cardiomyocyte culture, the morphology of cardiomyocytes was similar to that of cells that induced myocardial hypertrophy (Fig. 14a, +PE). It was possible to observe that the size had increased to the level shown in Figure 14a. At this time, in order to quantitatively analyze the size of each cell, more than 100 cells were quantified using the Image J program (NIH), and only the G:A wobble sequence site of miR-1 was detected. The miR-1 substituted products (miR-1-G2U, miR-1-G3U, miR-1-G7U) synthesized to recognize the protein were all approximately 1.5% higher than when the control group (NT) nucleic acid was introduced. It was analyzed that the size had increased by about 1.8 times. This is at a level similar to the size of cardiomyocytes in a cardiomyocyte hypertrophy model induced by phenylephrine (PE) drugs, and miR-1 itself shows the effect of reducing cell size on cardiomyocyte morphology. We observed that the canonical target suppression effect exhibited a different function in cell morphology from the target suppression through G;A wobble (Fig. 14b). Additionally, in order to confirm the myocardial hypertrophy phenomenon observed in this example at a molecular level, the expression of the ANP (atrial natural peptide) gene, which is known to increase its expression, was measured using qPCR (Quantitative Polymerase Chain Reaction). ) Through experiments, relative values were measured in comparison with GAPDH (Figure 14c). As a result, miR-1 substitution products (miR-1-G2U, miR-1-G3U, miR-1-G7U) synthesized to recognize only the G:A wobble sequence site of miR-1 were introduced into cardiomyocytes. Through repeated experiments four times, it was confirmed that all ANP expression levels were significantly increased by about 1.2 to 1.5 times compared to the control group (NT). Although this increase in ANP expression was smaller than the increase in the size of cardiomyocytes in the experimental group treated with phenylephrine (PE), which was about twice as large as that in the control group, it was found that the original miR-1 was introduced. On the contrary, ANP expression is significantly reduced, and miR-1 can be seen to exhibit completely different biological functions through the G:A wobble sequence site.

From the above, it was finally found that the non-canonical target suppressed through the G:A wobble sequence in the microRNA seed region can exhibit a biologically different function from the conventional canonical target recognition. These findings indicate that the functions of microRNAs through canonical targets and those through non-canonical targets are different from each other, and when inferred from this point, the G:A of microRNAs is If only the wobble target can be suppressed, only the biological function caused by the G:A wobble target can be selectively controlled.

[Example 15] Observation of induction of cardiac hypertrophy through miR-133-G4U expression in cardiomyocyte cell line (H9c2) Non-canonical target phenomenon that G:A wobble base sequence acts on and recognizes the seed portion of microRNA In addition to miR-1, which was investigated in the previous example, in order to additionally confirm its biological function in cardiomyocytes, we used miRNA-133, which is known to function in cardiomyocytes. I tried applying GU. miR-133 is known to regulate muscle cell development and disease pathology, and to have the function of suppressing myocardial hypertrophy (Nat Med. 2007, 13(5): 613-618; Ikeda S et al, Mol cell boil , 2009, vol29, 2193-2204). Therefore, the present inventors have developed an interference-inducing nucleic acid, miR-133-G4U (5 '-UUUUGUCCCCUUCAAACCAGCUG-3') was synthesized and produced by Bioneer as in the above example, and the duplex at a concentration of 50 nM was delivered to the cardiac muscle cell line h9c2 using RNAiMAX reagent (Invitrogen), and the cell morphology was determined. observed changes in (Fig. 15a). As a result, the expression of miR-133-G4U increased the size of H9c2 cells compared to the control group (NT) (Fig. 15a), and the size of more than 100 cells was quantitatively determined through the Image J (NIH) program. As a result of analysis, it was possible to confirm that the amount was significantly increased by about 2 times (FIG. 15b).

Therefore, the decreased expression of the non-canonical GA sequence seed target of miR-133 caused by miR-133-G4U induces cardiac hypertrophy, which is different from the canonical seed target inhibitory function of miR-133 that suppresses cardiac hypertrophy. It can be inferred that this is a new and different function.

[Example 16] Confirm the expression inhibition effect of non-canonical GA sequence seed site gene by miR-122 in hepatoma cells through luciferase reporter In the previous Example 10, miRNA non-canonically binds to G:A wobble sequence seed site This discovery led us to invent miRNA-GU that specifically recognizes only such non-canonical target binding, and when applied to miR-124, miR-1, and miR-133, It was observed in Examples 11, 12, 13, and 14 that effects different from conventional functions appeared. Furthermore, in order to confirm whether it is actually possible to inhibit the expression of a target gene that has a non-canonical G:A wobble sequence seed site in microRNA, in Example 13, we targeted miR-1 in myocardial tissue. Its function was confirmed using cell lines. Additionally, we attempted to use a luciferase reporter system to confirm whether the expression of a non-canonical G:A wobble sequence seed target gene could be inhibited by targeting miR-122, which is specifically expressed in liver tissue and hepatoma cells (Fig. 16).

miR-122, which is expressed and functions specifically in liver cancer or liver tissue, has a seed sequence (bases 1-8 of the 5' seed reference: 5'-UGG AGU GU-3') with the second 5' end reference, It has G that can form a G:A wobble arrangement at the 3rd, 5th, and 7th positions. Therefore, preferentially, in order to measure the suppression efficiency of the target site where the second G can be non-canonically recognized through the G:A wobble sequence, the same method proceeded in Example 2 was used. The IC50 (inhibitory concentration 50) was measured and compared with the regular seed site (FIG. 16a).

The luciferase reporter vector for proceeding with the experiment was designed to detect the suppression of expression of a non-canonical GA sequence seed target that can have a GA base sequence for the 2G base of miR-122. The non-canonical GA sequence seed site (2G:A) sequence (5' seed reference bases 1-9: 5'-CAC ACU CAA-3') was added to the Renilla luciferase gene in the psi-check2 (Promega) vector. was introduced into the 3'UTR (3'untranslated region) part of the 3'UTR (3'untranslated region) so that it was arranged five times consecutively. At the same time, a luciferase reporter vector for the regular seed site (bases 1 to 9 of the 5' seed standard: 5'-CAC ACU CCA-3') was similarly produced.

With the luciferase reporter vector constructed in this way, the canonical seed site and the non-canonical GA sequence seed site (2G:A The inhibitory effect on the expression of the target gene with the luciferase reporter site was measured by the change in the activity of the luciferase reporter, and the IC50 (inhibitory concentration 50) value was examined. .5 nM for a non-canonical GA sequence seed site (2G:A site), miR-122 suppresses the expression of genes with a non-canonical GA sequence seed site (2G:A site). It was confirmed that the efficiency was lower than that of the regular seed site (Fig. 16a). Furthermore, when the concentration at which gene expression suppression begins was investigated using a luciferase reporter using a non-canonical GA sequence seed target, it was confirmed that luciferase enzyme activity was inhibited at a concentration of 1.5 nM or higher (FIG. 16a). This showed that miR-122 suppresses the expression of the regular seed target site, and the suppression efficiency was about twice as high.

In this experiment, miR-122 duplex was synthesized using the same method used in the above example, and it was mixed with Lipofectamine at various concentrations (0, 1, 5, 10, 25 nM) together with the psi-check2 vector (50 ng). Approximately 10,000 hepatoma cell lines (Huh7, KCLB: 60104) were co-transfected using 2000 reagent (Invitrogen) according to the manufacturer's protocol, and cultured in 96 wells. Luciferase activity was measured and carried out in the same manner as described above.

After confirming the fact that miR-122 suppresses the expression of non-canonical GA sequence seed site genes, we investigated whether miR-122 is present in cells to confirm whether this is identically regulated by miR-122 present in cells. Using the Huh7 cell line, which is a hepatoma cell line with a 5' terminus, we performed a luciferase reporter experiment against a non-canonical GA sequence seed site (2G:A) caused by the second G and third G of the 5' end standard (Figure 16b). . To proceed with such experiments, we additionally prepared a luciferase reporter vector (luc-3G:A site) for a non-canonical GA sequence seed site (3G:A) caused by the third G of the 5' end standard, 5' end standard 2. Luciferase reporter vector for non-canonical GA sequence seed sites (luc-2G:A, 3G:A site) co-occurring by the 3rd and 3rd G, luciferase reporter vector to measure a sequence that is completely complementary to the entire miR-122 sequence A luciferase reporter vector (luc-PM site) was created in the same manner as described above and a luciferase reporter vector (luc-2G:A site) was generated using the same method as described above and a luciferase reporter vector (luc-2G:A site) directed against the non-canonical GA sequence seed site (2G:A) caused by the second G of the 5' end standard. ) was introduced into a hepatoma cell line, and the luciferase activity was measured for an experiment (FIG. 16b).

As a result, when we introduced the luc-PM site vector into Huh7 cells, which are known to contain miR-122, we observed that its activity decreased by approximately 50% compared to a control group in which only the luciferase vector itself was introduced. We confirmed the presence of miR-122 in Huh7 cells, and at the same time, miR-122 is associated with non-canonical GA sequence seed target reporter number 3 (Luc-3G:A), non-canonical GA sequence seed target reporter number 2, and number 3. It was confirmed that the activity of the reporter (Luc-2G:A, 3G:A) was suppressed. In addition, in order to confirm whether such a suppressive effect was induced by miR-122, we used an expression inhibitor of miR-122 (hsa-miR-122-5p inhibitor, IH-300591-06-0010) from Dharmacon Co., Ltd. It was confirmed that all such suppressive phenomena disappeared upon treatment with 50 nM.

Through the above examples, we confirmed the fact that miR-122 can suppress the expression of the 5'-end standard No. 2 non-canonical GA sequence seed target, even though it is weaker than the canonical seed site, and also suppresses the expression of the non-canonical GA sequence seed target in liver cancer. It has been found that miR-122, which naturally exists in Huh7 cells, suppresses the expression of the 5'-end standard No. 3 non-canonical GA sequence seed target gene and the No. 2 and 3 non-canonical GA sequence seed target genes. Ta. In particular, the suppressive effect exerted on Huh7, a liver cancer cell, was abolished by treatment with a miR-122 expression inhibitor, confirming that it was regulated by miR-122 (Figure 16b). Therefore, it can be seen that miR-122 has a regulatory function to suppress the expression of non-canonical GA sequence seed target genes.

[Example 17] Confirmation of cell migration inhibition phenomenon in liver cancer cell lines through suppression of expression of a specific non-canonical GA sequence seed target gene of miR-122 To investigate the intracellular function of the non-canonical GA sequence seed target of miR-122 in hepatoma cells After applying miRNA-GU that complementary recognizes and inhibits the non-canonical GA sequence seed target site and introducing it into hepG2 hepatoma cells, we investigated the properties of hepatoma cells that are important for cancer metastasis. A wound healing assay was performed to examine how cell migration ability changed.

Wound healing analysis in hepG2, a liver cancer cell line, was performed using miR-122 5' end standards, No. 2 non-canonical GA sequence seed target, No. 3 non-canonical GA sequence seed target, and No. 2 and 3 non-canonical GA sequence seeds. After synthesizing miRNA-GU that recognizes the target complementary to the target sequence, it was introduced into hepG2 cells using the same method performed in the above example, and after culturing for 24 hours, the cell culture layer ( A scratch was made on the cell layer, and the cells were cultured for up to 48 hours, and the migration of cells in each experimental group was observed in comparison with the control group in which NT-6pi was introduced. At this time, the sequence of the interference-inducing nucleic acid used in the experiment is as follows (miR-122:5'-UG GAG UGU GAC AAU GGU GUU UG-3'; miR-122-G2U:5'-UU GAG UGU GAC AAU GGU GUU UG-3';miR-122-G3U:5'-UG UAG UGU GAC AAU GGU GUU UG-3';miR-122-G2,3U:5'-UU UAG UGU GAC AAU GGU GUU UG-3').

As a result, when miR-122 was introduced in the same manner as in the case of the control group NT-6pi, it was possible to observe cell migration that almost filled the created scratch during cell culture for 48 hours. However, the experimental group to which miR-122-G2U and miR-122-G2,3U siRNAs were delivered showed that such cell migration was inhibited, and this inhibition phenomenon was due to miR-122-G2, It was most strongly shown in the experimental group to which 3U siRNA was delivered (Figure 17a). As a result of quantitatively measuring this, it was confirmed that in the case of miR-122-G2U and miR-122-G2,3U siRNA, cell migration was inhibited 2 to 3 times compared to the control group (Figure 17b ). The quantitative analysis was performed by observing cell migration of miR-122 on the cell morphology and then analyzing the observation results using the Image J program (NIH). Additionally, as a result of conducting experiments on cell migration induced by miR-122-G3U siRNA, no cell migration inhibitory function could be observed by miR-122-G3U (FIGS. 17c-d).

Based on this, miR-122's non-canonical GA sequence seed target inhibition inhibits the migration of liver cancer cells, and this is because the GA sequence at base 2 acts preferentially, followed by the GA sequence at base 3 in an additive manner. It can be seen that when the GA sequence is added, enhanced cell migration inhibition function is induced.

[Example 18] Confirmation of increase in cell cycle arrest by non-canonical GA sequence seed target regulation by miR-122-G2,3U Cell migration is closely related to cell division, and especially in cancer cells. (Cancer research, 2004, 64(22):8420-8427). Therefore, in order to confirm whether miR-122-G2,3U-induced inhibition of hepatoma cell migration was related to cell cycle regulation, a flow cytometry analysis experiment was carried out in the same manner as in Example 12. At this time, after delivering NT-6pi, miR-122, miR-122-G2, miR-122-3U, and miR-122-G2,3U to the liver cancer cell line HepG2, the cell cycle of each cell was measured. As a result, it was confirmed that in the experimental group to which miR-122-G2,3U was transferred, the proportion of cells showing G0/G1 distribution increased from an average of 52% in the control group (NP-6pi) to 68%. It was confirmed that the cell distribution in G2/M phase was significantly low (FIG. 18). However, no significant differences were observed in miR-122, miR-122-G2U, and miR-122-G3U compared to the control group. At this time, the cell cycle analysis experiment was performed by transmitting the synthesized siRNA duplex to HepG at a concentration of 50 nM, culturing it for 24 hours, and using the Muse Cell Cycle Kit (Catalog No. MCH100106, Milipore). Experiments were performed according to the provided protocol and measured with a Muse Cell Analyzer (Milipore).

Therefore, we confirmed that cancer cell functions regulated by miR-122-G2,3U reduce cell division (G2/M) and induce cell cycle arrest (G0/G1). As a result, the results of this example show that miR-122 recognizes the non-canonical GA sequence seed site by simultaneously arranging both the second G and third G of the 5' end in a G:A wobble sequence. It can be analyzed to show the function of preventing progression of the liver cancer cell cycle.

[Example 19] Observation of induction of cell cycle arrest state by regulation of non-canonical GA sequence seed target by miR-122-G2,3U and miR-122-G2,7U The function of the non-canonical GA sequence seed target of miR-122 was previously described. In Example 17 of observed that when the regulatory effect of G base 3 is added to the biological function of the non-canonical GA sequence seeded target regulation of G base 2, the function is maximized (Figure 17). Therefore, in order to investigate whether the biological function of non-canonical GA sequence seed target inhibition changes with additional combinations of the 2nd, 3rd, and 7th G bases of miR-122, miR-122-G7U, miR- Experiments were conducted to additionally confirm the cell cycle regulatory function of 122-G3,7U and miR-122-G2,7U siRNAs.

In this experiment, HepG2, a liver cancer cell line, was used, and the miRNA-GU duplex was prepared in the same manner as in Example 18 and transfected into cells at a concentration of 50 nM. However, in this cell cycle observation, after 24 hours of culture, HepG2 cells were synchronized to G2/M (mitotic preparation phase/mitosis phase) by treatment with nocodazole at 100 ng/ml for 16 hours. After that, the amount of cells in cell cycle arrest in G0/G1 was determined based on how many cells were arrested in G2/M using a Muse Cell Analyzer (Milipore) flow cytometry analyzer (FIG. 19).

As a result, miR-122-G2U had no difference in the number of cells in G0/G1, which is a cell cycle arrested state, compared to the control group NT-6pi, but miR-122-G2,3U siRNA, miR When regulating a non-canonical GA sequence seed target site attached to the second G such as -122-G2,7U, it was confirmed that the ratio of cell cycle arrest state (G0/G1) increases. (Figure 19a). However, miR-122-G3,7U showed no particular increase (Fig. 19a). Quantitative analysis of this revealed that in the experimental group to which miR-122-G2,3U siRNA was delivered, the ratio of cell cycle arrest (G0/G1) increased more than twice that of the control group. However, it was subsequently observed that in the miR-122-G2,7U experimental group, the ratio of cell cycle arrest (G0/G1) increased approximately 1.5 times (FIG. 19b).

Based on this, we confirmed that cell cycle arrest can be increased through regulation of non-canonical GA sequence seed targets by miR-122-G2,3U siRNA and miR-122-G2,7U siRNA.

[Example 20] Observation of induction of cell cycle arrest induced by non-canonical GA sequence seed target regulation by let-7a-G2U and let-7a-G2,4U Representative microRNAs that function as anti-cancer regulatory (tumor suppressor) An example of this is the let-7 family. After its function in regulating the developmental process of beautiful small nematodes was reported (Nature, 2000, 403(6772):901-906), its expression was suppressed in various cancer cells (Cancer Res., 2004, 64(11):3753-3756) and anti-cancer function through regulation of tumorigenesis (Cell, 2005, 120(5): 635-647; Genes Dev. 2007, 21(9): 1025-1030) ) has been reported, and based on this, it has been studied as an important gene with the potential for cancer diagnosis and anticancer drug development. This led us to conduct experiments to investigate the biological functions induced by inhibition of the non-canonical GA sequence seed target of let-7.

The seed sequence from the 1st to 9th positions at the 5' end of let-7a is 5'-UGA GGU AGU-3', and G, which can form a G:A wobble base sequence, is at the 2nd and 4th positions. , 5th and 8th. The present inventor focused on the second and fourth Gs, synthesized them by modifying them with miRNA-GU, and after introducing this into HepG2 cells, a liver cancer cell line, there was no effect on the cell cycle. An experiment was conducted in the same manner as in the previous Example 19 to determine whether At this time, the nucleotide sequence for let-7 synthesized and used is as follows (let-7a:5'-U GAG GUA GUA GGU UGU AUA G UU-3'; let-7a-G2U:5' -U UAG GUA GUA GGU UGU AUA G UU-3'; let-7a-G4U:5'-U GAU GUA GGU UGU AUA G UU-3'; let-7a-G2,4U:5'-U UAU GUA GUA GGU UGU AUA G UU-3').

As a result, let-7a showed no particular difference compared to the control group NT-6pi, but let-7a-G2U and let-7a-G2,4U It was observed that let-7-G4U increased the state of cycle arrest and instead increased the amount of cells in G2/M, causing the cell cycle to progress faster (FIG. 20a). When this was quantified in four repeated experiments, it was found that in the let-7a-G2U experimental group, the distribution of G1/G0 cells increased by about 1.5 times compared to the control group (Nt-6pi). , it could be observed that the distribution of G2/M cells decreased (Fig. 20b). Through this, it was confirmed that suppression of the expression of let-7a's second non-canonical GA sequence seed target induces cell cycle arrest in HepG2 cells. Subsequently, in the case of the 4th G base present in the let-7a seed, a single GA sequence (let-7a-G4U) reduces the induction of HepG2 cell cycle arrest, and instead accelerates the progression to the G2/M state. The results showed that a large number of cells remained after nocodazole drug treatment. Furthermore, when No. 2 and No. 4 had the GA sequence at the same time (let-7a-G2, 4U), induction of HepG2 cell cycle arrest state could be observed. Such cell cycle arrest was not observed in let-7a transduced HepG2 cells (Figure 20b). Therefore, suppression of the expression of let-7a non-canonical GA sequence seed targets 2 and 2 and 4 induces cell cycle arrest in HepG2 cells, and this is because let-7a We confirmed that this function is completely different from that seen in cell regulation.

Through the above embodiments, let-7 plays a role in promoting cancer cell cycle arrest by suppressing the gene at the non-canonical GA seed sequence site through the 5' end standard No. 2G, and more preferably, let-7 It can be seen that the effect is greatest when both the 2nd and 4th end standards G and 4 act in the G;A wobble arrangement. In addition, the non-canonical GA seed sequence site through the 5' end reference number 4 G of let-7 is thought to promote cell cycle progression of cancer cells and induce cancer cell proliferation.

[Example 21] Observation of reverse differentiation induced by inhibition of non-canonical GA sequence seed targets by miR-302-4GU and miR-372-4GU using OCT4 promoter reporter. Cells that can further acquire differentiation ability through expression and finally acquire totipotent differentiation ability such as embryonic cells are called induced pluripotent stem cells. Such technology introduces four factors (Oct3/4, Sox2, c-Myc, Klf4) into rat embryonic fibroblasts (mouse embryonic fibroblasts: MEMs), which allows them to have differentiation diversity like stem cells. It has been reported that it is possible to create inducible pluripotent stem cells (iPS cells) having the following characteristics (Cell, 2006, 126(4): 663-676). Similarly, it has been reported that induced pluripotent stem cells can be created through transmission of four factors (OCT4, SOX2, NANOG, and LIN28) to human somatic cells (Science, 2007, 318(5858): 1917- 1920). This is a field with very high potential for application in that any cell, including human cells, can be regenerated and can be differentiated into a versatile variety through the process of dedifferentiation. , efforts have continued to increase the efficiency of induced pluripotent stem cell generation. As part of this, microRNA-induced pluripotency (mirPS), which has been reported, is one of the methods to efficiently induce pluripotency, and microRNAs such as miR-302a and miR-372 are The function of reverse differentiation of cells together with four factors (Oct3/4, Sox2, c-Myc, Klf4) has been reported (RNA, 2008 14(10): 2115-2124; Nat Biotechnol. 2011, 29(5) ):443-448). Therefore, the present inventors conducted experiments to investigate whether inhibition of non-canonical GA sequence seed target expression of miR-302a and miR-372 could promote the process of reverse differentiation of cells.

First, in order to monitor the induction of pluripotency, we purchased a plasmid containing the Oct4 promoter, which is reported to be activated in stem cells, and the green fluorescent protein that is expressed depending on this promoter (Addgene #21319) (Fig. 21a). When such Oct4 expression reporter vector (pOct4:GFP) is introduced into differentiated cervical cancer cells HeLa, green fluorescent protein is not expressed, but when the cells reversely differentiate and acquire differentiation ability, Oct4 Green fluorescent protein will be expressed in the expression reporter. Experiments were conducted using miR-302a and miR-372, which are microRNAs known to cause reverse differentiation together with such an Oct4 expression reporter vector. Among the seed sequences of miR-302a and miR-372, the 2nd to 8th sequences of the 5' end standard are "AA GUG CU". Therefore, the non-canonical GA seed sequence is It is possible through G and the 6th G. However, the present inventors focused on the fourth G and conducted experiments by synthesizing miR-302-G4U and miR-372-G4U.

Preferentially, experiments were performed based on human miR-302 and miR-372 sequences after transfecting HeLa cells with Oct4 expression reporter vector (pOct4:GFP) using Lipofectamine 2000 (Invitrogen) reagent according to the manufacturer's protocol. The guide strand and passenger strand were synthesized in Bioneer as in the above example, prepared as a duplex, and sequentially transferred at a concentration of 50 nM using RNAimax (Invitrogen) reagent. In addition, non-canonical GA sequence seed target-specific siRNAs for miR-302 and miR-372 were prepared in the same manner as in the above example by replacing the G base in the seed with U, and delivered to cells at a concentration of 50 nM. The base sequence of the nucleic acid used for this is as follows (miR-302: 5'-UAAGUGCUUCCAUGUUUUGGUGA-3'; miR-302-4GU: 5'-UAAUUGCUU CCAUGUUUUGGUGA-3'; miR-302 passenger strand: 5'-ACUUAAACGUGGAUGUACUUGCU-3';miR-372:5'-AAAGUGCUGCG ACAUUUGAGCGU-3';miR-372-G4U:5'-AAAUUGC UGCGACA UUUGAGCGU-3', miR-372 passenger Chain: 5'-CCUCAAAUGUGGAGCACUAUUCU-3' ). HeLa cells transfected with the reporter vector and RNA were incubated for 14 days in Dulbecco's modified Eagle's medium (Invitrogen) supplemented with 10% FBS (fetal bovine serum), 100 U/ml penicillin, and 100 μg/ml streptomycin. During transfection, cells were cultured in complete medium without antibiotics.

As a result, the experimental group to which miR-302 or miR-372 was transmitted alone showed colony-like cell growth compared to the control group, but the expression of green fluorescent protein, which indicates Oct4 promoter activity, was lower than that of the control group. It was not observed until the day-culture period. However, in the experimental group in which miR-302 and miR-372 non-canonical GA sequence seeded target-suppressing siRNAs were introduced, not only cluster-like cell growth but also green fluorescent protein expression indicating Oct4 promoter activity was observed (Fig. 21b -c, red arrow), which observed an even stronger and larger green fluorescent protein-expressing cell population when miR-302 and miR-372 and their respective non-canonical GA sequence seeded target inhibitory siRNAs were simultaneously delivered to the cells. (Fig. 21b-c). Based on this, it can be seen that inhibition of miR-302 and miR-372 non-canonical GA sequence seed targets can promote cell reverse differentiation and acquisition of differentiation potential through Oct4 expression.

Through the above examples, miR-372-G4U and miR-302a-G4U were shown to be more effective in reverse differentiation of cells than the conventional expression of miR-372 and miR-302 alone. It was observed that miR-372-G4U and miR-302a-G4U can be used as materials for promoting reverse differentiation of cells.

[Example 22] Confirming the phenomenon that 2'OMe modification at the 6th position of the 5' end standard does not suppress the non-canonical nuclear protuberance target of the RNA interference derivative In the above example, the non-canonical nuclear protuberance target is completely different from the regular seed target. We have invented and confirmed the function of miRNA-BS, an RNA interference-inducing nucleic acid that exhibits a different new biological function and therefore only modulates the inhibition of expression of non-canonical nuclear protuberance targets. However, in the case of miRNA-BS as well, the seed sequence was changed to complement the non-canonical nuclear protrusion site of conventional miRNA, but it was confirmed that new nuclear protrusion sites were generated through the changed seed sequence. We were able to. Therefore, it has been found that a technique is required in which an RNA interference-inducing nucleic acid such as miRNA-BS recognizes only the canonical seed sequence and does not recognize the non-canonical nuclear prominence site. As a result, the present inventors found that the base at position 6 of the 5' end standard in the seed sequence is important for non-canonical nuclear protuberance binding, and that depending on the sequence strength of the base sequence, the base at position 6 is important. Focusing on the fact that the degree to which normal nuclear protuberance binding occurs changes, we conducted the following experiment with the aim of reducing the strength of the base sequence at position 6. That is, miR-124 is modified by inserting a methyl group (2'OMe) into the 2' position of the ribose ring, and the modified interference-inducing nucleic acid (miR-124-6me) is a non-canonical member of miR-124. The ability to inhibit nuclear protuberance targets was observed using a luciferase reporter experiment. 2'OMe-modified miR-124 was produced through synthesis (Bionia), and luciferase reporter experiments were carried out in the same manner as previously performed in Example 2, and gene silencing efficiency was measured by IC50 (Fig. 22a ). At this time, the luciferase reporter vectors used were those directed to the regular seed site (Luc-seed) and irregular nuclear bulge site (Luc-nucleation bulge) of miR-124.

As a result, the inhibitory effect of miR-124 on non-canonical protuberant target genes shown in Example 2 disappeared, whereas gene expression inhibition through the regular seed site had an IC50 value of 0.3 nM, and 2'OMe Even when added to the 6th nucleoid, it was observed that it still showed excellent inhibitory effect (Figure 22a). Additionally, in the same manner, 2'OMe was applied to the 6th nucleoid of miR-1, which is another microRNA, instead of miR-124 (miR-1-6me), and this was added to the canonical seed site of miR-1 ( When we investigated the effect of inhibiting gene expression on the target using a luciferase vector containing a Luc-seed and a non-canonical nuclear bulge (Figure 22b), we found that miR- It was confirmed that the inhibitory effect of the non-canonical protuberance target gene by 1 was eliminated, and on the other hand, gene expression suppression through the regular seed site was excellent with an IC50 value of 0.7 nM. Through the above examples, when the 2'OMe modification is added to the 6th 5' end standard of the RNA interference inducing nucleic acid, the expression suppression effect of the regular seed target gene induced by the RNA interference inducing nucleic acid is maintained, and the We were able to confirm that the expression suppression of normal nuclear protuberance target genes disappeared.

[Example 23] RNA-Seq analysis confirmed that 2'OMe modification at the 6th position of the 5' end standard does not suppress the entire non-canonical nuclear protuberance target mRNA in the transcript In Example 22 above, the 6th position of miR-1 It was confirmed that when 2'OM modification was added to the nucleoid (miR-1-6me), the effect of suppressing the expression of the canonical seed target gene was maintained, and the suppression of the expression of the non-canonical nuclear protuberance target gene was reduced. Therefore, miR-1-6me, which we invented, is actually a non-canonical target of miR-1 in the transcript of h9c2, a cardiomyocyte cell line in which all genes related to miR-1 function are expressed. To confirm whether the gene suppression function was reduced, RNA-Seq analysis was performed after miR-1 and miR-1-6me were introduced into h9c2 cells. The RNA-Seq experiment used the sequence of cel-miR-67, which is a beautiful small C. elegans microRNA, as an experimental control group, with the 5' end reference number 6 converted to a non-base (NT-6pi). An experiment was conducted in the same manner as in Example 7 using . At this time, the RNA-Seq library was constructed using Lexogen's SENSE Total RNA-Seq Library Kit, and then sequenced using Illumina's MiniSeq.

After that, the FASTAQ file, which is the sequence data obtained in the above experiment, was mapped to the rat gene sequence (rn6) using the TopHat2 program, the expression value (FPKM) was determined using the Cufflink and Cuffdiff programs, and the control group NT-6pi was introduced. The analysis was performed by normalizing the results using h9c2 cells and expressing the results as a log ratio (fold change, log2 ratio). At this time, in order to analyze by RNA-Seq results whether the amount of messenger RNA of a gene that has a microRNA target site is inhibited by the expression of the microRNA, the canonical seed site of miR-1 (5 Select genes that have a 7mer in their 3'UTR that has a base sequence from the 2nd to 8th base of the terminal standard, and calculate the cumulative ratio of such profile results in the order of inhibition depending on the expression of the microRNA. Comparative analysis was performed using (cumulative fraction) (Fig. 23a). As a result, miR-1 and miR-1-6me all suppress genes (seeds) that have the canonical seed site of miR-1 in their 3'UTRs very well compared to the distribution of total genes (total mRNA). I confirmed that it can be done. Subsequently, using the same method, we analyzed the cumulative ratio of genes that have a non-canonical raised site (nuc, 7mer) of miR-1 in their 3'UTR, and found that miR-1 non-canonical bulge sites (nuc, 7mer) due to miR-1-6me. We compared the relative amounts of target mRNA in cells into which miR-1 and miR-1-6me were introduced to determine whether inhibition of the expression of normal nuclear protuberance target genes was reduced (Fig. 23b). As a result, we observed that miR-1 still significantly inhibits non-canonical nuclear prominence target genes compared to total mRNA when compared to miR-1-6me expression. Through this, it was confirmed that miR-1-6me reduced the expression suppression of the non-canonical nuclear protuberance target gene.

Inferring from the results of the above examples, conventional microRNAs at the transcript level efficiently repress both conventional canonical seed target genes as well as non-canonical protuberance target genes; We found that the 'OMe-modified microRNA (miRNA-6me) still represses canonical seed target genes, but not non-canonical protuberance target genes. Therefore, the 2'OMe modification added to the 6th 5' end standard maintains the original intention of applying it to miRNA-BS to specifically suppress only the irregular nuclear protrusion site of the miRNA, while newly appearing. By completely eliminating possible irregular nuclear protuberance binding, its side effects can be minimized.

[Example 24] Identification of microRNAs that bind to non-canonical nuclear protrusion sites through Ago HITS CLIP experimental analysis Identification of microRNAs to which the invention created so that microRNAs can recognize and suppress only non-canonical nuclear protrusion sites can be applied. To identify, microRNAs that bind to non-canonical nuclear protuberance sites were analyzed by analyzing the results of an Ago HITS CLIP experiment, which is a method that allows microRNA targets to be analyzed at the transcript level. First, Ago HITS-CLIP data obtained from the cerebral cortex of a mouse (p13) aged 13 days after birth was sequenced in the same manner as in Example 10, and the top 20 expressions that bind with Argonaute were identified. It was confirmed that the microRNA of 100% binds to the target mRNA and the non-canonical nuclear protuberance site (Fig. 24a). Therefore, when the microRNA (Fig. 24b) recognizes the target mRNA through the base sequence of the seed part, it not only recognizes the target mRNA through the normal base sequence through the seed part but also non-canonically through the nuclear protrusion site. It turns out that it can be combined with

Expanding from the mouse Ago HITS-CLIP data analysis of the above example, other Ago HITS-CLIP results performed on human brain and heart tissue (boudreau RL et al, 2014, Neuron, 81(2) 294-305, Spengler RM et al, 2016, Nucleic Acids Res, 44 (15) 7120-7131) was analyzed using the same method as in the above example to determine the frequency of regular seed sites and irregular nuclear prominence sites where Ago and the microRNA bind. The numbers were calculated (human brain microRNA, Table 1; human heart microRNA, Table 2).

As a result, it was confirmed that the microRNAs (Tables 1 and 2) described in the result table were bound to non-canonical nuclear protrusion sites in the tissue. Therefore, if the present invention is applied to the microRNA excavated by integrating all the data analyzed by Ago HITS-CLIP in the above example, it will be transformed so that the irregular nuclear protrusion site is recognized as a regular seed site. As shown in Table 3 below, a total of 426 sequences (BS sequences) constitute the nucleoids from the 2nd to 7th 5' end standards of the RNA interference nucleic acid, and thus the modified RNA interference The nucleic acid will bind only the non-canonical raised target of the microRNA and exhibit only the function of interest.

Through the Ago HITS-CLIP experiment sequencing analysis of the above example, we identified microRNAs that bind to non-canonical protuberance targets in various tissue cells and modified them to recognize non-canonical nuclear protuberance sites as canonical seed sites. By applying the present invention, a total of 426 sequences (BS sequences) were created, thereby completing a technique that only shows the non-canonical target suppressing function of microRNA.

[Example 25] Analyzing sequence variations in the Cancer Patient MicroRNA Sequencing Database (TCGA) and identifying microRNAs that bind to non-canonical G:A wobble seed sequence sites. We confirmed the fact that G:A wobble site inhibits target gene expression, and through this, the present invention uses a microRNA sequence conversion method to transform the sequence so that non-canonical G:A wobble site is recognized as a regular seed site. (miRNA-BS) was developed. In this way, if binding to the A of the target mRNA in the wobble sequence through the G in the conventional microRNA seed sequence changes the function of the microRNA, such a mechanism may cause microRNA sequence mutations in diseases such as tumors. We analyzed the results of microRNA sequencing (miRNA-Seq) in tissues of cancer patients based on the idea that cancer can appear through cancer. At this time, 8,105 microRNA sequences were obtained from the TCGA database related to cancer patient microRNA sequence experiments, and an additional 468 cancer-related microRNA sequences were obtained from the Gene Expression Omnibus (GEO) database, for a total of 8,573 tumor microRNA sequences. The results were mapped using the bowtie2 program in the same manner as used in Example 10, and the site where the mutation occurred was analyzed. The microRNA sequence data used in the analysis for each cancer type is the same as shown in FIG. 25a.

The frequency of mutations (fraction) in the results was divided by microRNA 5' end reference position (position), and the 10,000 mutations that occurred most frequently (top 10,000) and the 10,000 mutations that occurred least frequently (top 10,000) As a result of examining the distribution of 10,000 mutations (bottom 10,000) (Figure 25b), we observed that the most frequent mutations (10,000) occur mainly in the seed part (between the second and eighth positions) of the microRNA. We were able to. Furthermore, when we examined the 100 mutations that occurred the most and the 100 mutations that occurred the least on the heatmap (Figure 25c), we found that the mutations were concentrated in the seed region, as in the example above. . Sequence variations in tumor microRNAs that tend to appear in the seed region were analyzed by reverse transcription and DNA sequence analysis, and the base composition was divided into A, T, C, and G. In this case, only G showed a tendency to be formed in the seed sequence (Fig. 25d). This tendency was further confirmed that mutations mainly occur in G, not only for the top 100 mutation rates, but also in all cases where mutations occur beyond this (Figure 25e). ).

This may be a phenomenon that occurs when the G of the microRNA that recognizes the non-canonical G:A wobble that the present inventor focused on is mutated to U in order to better bind the A of the target mRNA on the opposite side. As a result of analyzing what kind of base was changed by the mutation that occurs in G, which appears in the highest mutation rate (Fig. 25f), it was found that most of the bases were reverse transcribed and sequenced using DNA. Observed. These results suggest that microRNA actually changes its own G to U to recognize the non-canonical G:A wobble seed target as a regular seed, which is present in the seed part in cancer tissue. Therefore, this type of mutation means that the sequencing method that recognizes the non-canonical G:A wobble seed sequence developed in this invention as a regular seed sequence can be used for any microRNA. Informs whether the position G must be applied. Therefore, by integrating all of these results, microRNAs with a normalized mutation frequency of 2 or more per cancer patient were selected (see Table 4). As shown in Table 4, a total of 335 sequences (sequences (G>U)) constitute a nucleoid from the second 5' end standard of the RNA interference nucleic acid, and the RNA interference nucleic acid modified in this way is , will bind only the non-canonical G:A wobble target of the microRNA and exhibit only the function.

Through the microRNA sequencing mutation analysis of the above example, we identified mutant microRNAs that recognize the non-canonical G:A wobble seed target as the regular target in various cancer patients, and added the non-canonical G:A wobble seed site to the regular seed. When the present invention (miRNA-GU), which is modified to recognize sites, was applied, a total of 335 sequences (sequences (G>U)) were created (Table 4), which enabled the suppression of non-canonical targets of microRNAs. We have completed the technology that shows only the functions.

By using the RNA interference-inducing nucleic acid according to the present invention, the biological functions exhibited by microRNAs by suppressing non-canonical target genes can be efficiently increased, and some of the functions exhibited by conventional microRNAs, namely It has the effect of selectively showing only the biological function shown by suppressing non-canonical target genes, and regulates cell cycle, differentiation, reverse differentiation, morphology, migration, division, proliferation, or death through the interference-inducing nucleic acid of the present invention. It is expected that it can be used in a variety of fields such as drugs and cosmetics.

Claims (3)

A composition for suppressing expression of a non-canonical target gene of microRNA, comprising an RNA interference-inducing nucleic acid,
The RNA interference-inducing nucleic acid is one in which a partial sequence of a specific microRNA is modified in one or more of the double strands of the nucleic acid that induces RNA interference (RNA interference), resulting in a non-canonical target gene of the microRNA. (noncanonical target gene),
Contains the 4 base sequence from the 2nd to 5th base from the 5' end of the specific microRNA, the 6th and 7th bases are the same, and the 6th and 7th bases are the 6th base sequence of the specific microRNA. The base that has a complementary base sequence to a base that can pair with the specific microRNA, and the base that can pair with the 6th base of the specific microRNA includes all complementary sequences including G:A and G:U wobble sequences. a target base, and
A composition for suppressing the expression of a non-canonical target gene of a microRNA, wherein the RNA interference-inducing nucleic acid contains a sequence from the second to the seventh of the 5' end selected from the following :
An RNA interference-inducing nucleic acid that suppresses a non-canonical target gene of miR-124, the RNA interference-inducing nucleic acid (miR-124-BS) having a base sequence of 5'-AA GGC C-3';
An RNA interference-inducing nucleic acid that suppresses a non-canonical target gene of miR-122, the RNA interference-inducing nucleic acid (miR-122-BS) having a base sequence of 5'-GG AGU U-3';
an RNA interference-inducing nucleic acid that suppresses a non-canonical target gene of miR-155, the RNA interference-inducing nucleic acid having a base sequence of 5'-UA AUG G-3'(miR-155-BS); and
An RNA interference-inducing nucleic acid (miR-1-BS) that suppresses a non-canonical target gene of miR-1 and having a base sequence of 5'-GG AAU U-3'. 2. The composition according to claim 1, wherein the RNA interference-inducing nucleic acid selectively suppresses a non-canonical target gene of a microRNA, but does not suppress a canonical target gene of a microRNA. The composition according to claim 1 , wherein the RNA interference-inducing nucleic acid has one of the following sequences :
An RNA interference-inducing nucleic acid (miR-124-BS) that suppresses a non-canonical target gene of miR-124, the RNA interference-inducing nucleic acid having a base sequence of 5'-UAA GGC CAC GCG GUG AAU GCC-3';
An RNA interference-inducing nucleic acid (miR-122-BS) that suppresses a non-canonical target gene of miR-122, the RNA interference-inducing nucleic acid having a base sequence of 5'-UGG AGU UGU GAC AAU GGU GUU-3';
An RNA interference-inducing nucleic acid that suppresses a non-canonical target gene of miR-155, the RNA interference-inducing nucleic acid (miR-155-BS) having a base sequence of 5'-UUA AUG GCU AAU CGU GAU AGG-3'; or
An RNA interference-inducing nucleic acid (miR-1-BS) that suppresses a non-canonical target gene of miR-1 and having a base sequence of 5'-UGG AAU UGU AAA GAA GUA UGU-3'.