KR20190137015A - RNA interference inducing nucleic acids suppressing noncanonical targets of microRNA and use thereof - Google Patents

Abstract

The present invention relates to RNA interference-inducing nucleic acids in which some sequences of a particular microRNA are modified to inhibit noncanonical target genes of the microRNA. The use of RNA interference-inducing nucleic acids according to the present invention has the effect of efficiently increasing the biological function of microRNAs derived by suppressing noncanonical target genes, and has the effect of selectively showing only some functions represented by existing microRNAs, that is, biological functions exhibited by suppressing noncanonical target genes. Through the interference-inducing nucleic acids of the present invention, cell cycle, differentiation, dedifferentiation, morphology, migration, division, proliferation or death can be controlled, thus the application in various fields such as drugs and cosmetics is expected.

Description

RNA interference inducing nucleic acids suppressing noncanonical targets of microRNA and use approximately}

FIELD OF THE INVENTION The present invention relates to RNA interference inducing nucleic acids and their use which inhibit gene expression, and to interference interference nucleic acids and the use thereof having useful effects exerted by selectively inhibiting an irregular target of a microRNA.

RNA interference refers to a phenomenon of inhibiting the expression of genes in the post-transcriptional stage. Naturally occurring RNA interference is caused by microRNA (miRNA). MicroRNAs are small RNAs consisting of about 21 bases that are complementary to the messenger RNAs (mRNAs) of the target gene through the Argonaute protein. In the case of animal microRNAs, it binds to the agon nut protein and forms a partial nucleotide sequence with the target messenger RNA, wherein the seed region defined as the first to eighth nucleic acids based on the 5 'end of the microRNA. At least six consecutive nucleotide sequences in the gene can be recognized as a target, and their expression is inhibited by inhibiting the degradation or translation of the mRNA (Lewis BP, et.al, 2003, Cell, 115). (7), 787-98). Since microRNAs recognize messenger RNAs of target genes through partial nucleotide sequences, one microRNA can usually affect the expression of hundreds to thousands of genes.

Inhibition of target gene expression of microRNAs is one of the important mechanisms of gene expression, which is involved in cell differentiation and growth in normal situations, and in the event of dysfunction, causes cancer, degenerative disease, diabetes, etc. It is attracting attention as. Therefore, in order to artificially induce the gene expression inhibitory action of the microRNA, RNA interference material (siRNA or shRNA) containing the starting region of the microRNA is designed and introduced into the cell to artificially differentiate or alter the function of the cell, In some cases, it can be used as a cure for disease. Therefore, complementary nucleotide sequences in the microRNA initiation region that are mediated by agon nuts to recognize the target messenger RNA are important for the function of the RNA interference material, including the microRNA. In particular, in order to utilize such RNA interference materials, it is necessary to understand the function of each microRNA, and the function of the microRNA is determined by which target genes are inhibited. Sieve analysis is needed.

At the transcript level, the study of microRNA targets was first performed by the inventors through the Ago HITS CLIP experiment. The Ago HITS CLIP test method involves irradiating UV light to a cell or tissue sample to form a complex through covalent bonding between RNA and agon nut protein in the cell, and using the RNA-agon nut complex with an agon nut specific recognition antibody. After separation by immunoprecipitation, RNA is analyzed by next-generation sequencing. Through this, it is possible to accurately analyze the microRNA bound to the agonut and the target messenger RNA group complementary to the base (Chi S et al, Nature, 2009, 460 (7254): 479-86).

As a result, the inventors have found that when a microRNA binds to a target messenger RNA, it may bind even if it is not exactly complementary to the microRNA seed region. More specifically, the canonical target is that the microRNA seed region defined by the 1st to 8th base sequence of the 5 'end of the microRNA binds the messenger RNA in an array of at least 6 consecutive and complete base sequences. Non-canonical targets, called canonical target recognition, deviate from the above rules and recognize and bind to microRNA targets, even if they are not continuous and accurate complementary sequence relationships with the microRNA seed region. recognition). The results of the Ago HITS-CLIP analysis show that the frequency of microRNA recognition of targets irregularly through the initiation region is about 50% of the frequency of regular recognition.

Thus, an RNA interference material (eg, siRNA or shRNA) designed to include the sequence of the origination region of an existing microRNA may be one that exhibits biological function by inhibiting several non-regular target genes as well as regular target genes.

Accordingly, the problem to be solved by the present invention is to solve the limitations of the RNA interference material designed to include the sequence of the origin of the existing microRNA as it is and to increase the efficiency.

More specifically, the RNA interference material designed to include the sequence of the origin of the existing microRNA intactly suppresses both the regular target gene and the non-regular target gene, while strongly inhibiting the regular target gene but very weak the non-regular target gene. Suppress Accordingly, the problem to be solved by the present invention is to efficiently increase the biological function of the existing microRNA by suppressing the non-standard target gene, or to suppress some of the functions of the existing microRNA, that is, by suppressing the non-regular target gene. To provide an RNA interference inducing nucleic acid having the effect of selectively displaying only biological function.

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

More specifically,

In the present invention, in one or more single strands of a nucleic acid inducing RNA interference, some sequences of specific microRNAs are modified to induce RNA interference to suppress noncanonical target genes of the microRNAs. As nucleic acid,

The RNA interference-inducing nucleic acid comprises four to five base sequences from the 5 'end of a particular microRNA, the sixth and seventh bases are identical, and the sixth and seventh bases are the sixth of a particular microRNA. A base having a base sequence complementary to that of the base, and a base sequenceable to the sixth base of the specific microRNA include all complementary bases including G: A, G: U wobble sequences Characterized in that, or

The RNA interference-inducing nucleic acid has a guanine base of the base sequence between the 5 'end and the ninth base of a specific microRNA having a modified base sequence substituted with at least one or more of uracil or adenine (Uracil) or adenine (Adenine) Characterized by

Provide RNA interference inducing nucleic acid.

Preferably, the RNA interference-inducing nucleic acid provides RNA interference-inducing nucleic acid, characterized in that it selectively inhibits the irregular target gene of the microRNA and does not inhibit the regular target gene of the microRNA.

Preferably, the specific microRNA is at least one selected from the group consisting of miR-124, miR-155, miR-122, miR-1, let-7, miR-133, miR-302a and miR-372 , RNA interference induction nucleic acid.

Preferably, the RNA interference inducing nucleic acid provides an RNA interference inducing nucleic acid, characterized in that the 5 'terminal 2 to 7 base sequence is any one or more of the following:

RNA interference-inducing nucleic acid (miR-124-BS) (SEQ ID NO: 1), which exhibits the base sequence of 5'-AA GGC C-3 'as an RNA interference-inducing nucleic acid that inhibits an irregular target gene of miR-124;

RNA interference-inducing nucleic acid (miR-122-BS) (SEQ ID NO: 2), which exhibits the base sequence of 5'-GG AGU U-3 'as an RNA interference-inducing nucleic acid that inhibits an irregular target gene of miR-122;

RNA interference-inducing nucleic acid (miR-155-BS) (SEQ ID NO: 3), which exhibits the base sequence of 5'-UA AUG G-3 'as an RNA interference-inducing nucleic acid that inhibits an irregular target gene of miR-155; or

RNA interference-inducing nucleic acid (miR-1-BS) (SEQ ID NO: 4), which exhibits the base sequence of 5'-GG AAU U-3 'as an RNA interference-inducing nucleic acid that inhibits an irregular target gene of miR-1.

Preferably, the RNA interference-inducing nucleic acid is characterized in that the base sequence of any one or more of the following, RNA interference-inducing nucleic acid:

RNA interference-inducing nucleic acid (miR-124-BS) (SEQ ID NO: 5) which shows the nucleotide sequence of 5'-UAA GGC CAC GCG GUG AAU GCC-3 'as an RNA interference-inducing nucleic acid that inhibits an irregular target gene of miR-124;

RNA interference-inducing nucleic acids (miR-122-BS) (SEQ ID NO: 6) which show the base sequence of 5'-UGG AGU UGU GAC AAU GGU GUU-3 'as an RNA interference-inducing nucleic acid that inhibits an irregular target gene of miR-122;

RNA interference-inducing nucleic acid (miR-155-BS) showing the nucleotide sequence of 5'-UUA AUG GGC UAA U CGU GAU AGG-3 'as an RNA interference-inducing nucleic acid that inhibits an irregular target gene of miR-155 (SEQ ID NO: 7) ; or

RNA interference inducing nucleic acid (miR-1-BS) showing the nucleotide sequence of 5'-UGG AAU UGU AAA GAA GUA UGU-3 'as an RNA interference inducing nucleic acid that inhibits an irregular target gene of miR-1 (SEQ ID NO: 8).

Preferably, the RNA interference inducing nucleic acid provides an RNA interference inducing nucleic acid, characterized in that the base sequence between the 5 'end and the ninth base is any one or more of the following:

As RNA Interference Inducing Nucleic Acids That Inhibit the Irregular Target Gene of miR-124

5'-UAA UGC ACG-3 '(miR-124-G4U) (SEQ ID NO: 9), 5'-UAA GUC ACG-3' (miR-124-G5U) (SEQ ID NO: 10) or 5'-UAA UUC ACG An RNA interference inducing nucleic acid comprising the nucleotide sequence of a -3 '(miR-124-G4,5U) (SEQ ID NO: 11);

As RNA Interference Inducing Nucleic Acids That Inhibit the Irregular Target Gene of miR-1

5'-UUG AAU GUA-3 '(miR-1-G2U) (SEQ ID NO: 12), 5'-UGU AAU GUA-3' (miR-1-G3U) (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'-UGU 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- RNA interference inducing nucleic acid comprising the nucleotide sequence of 1-G2,3,7U) (SEQ ID NO: 18);

RNA Interference-Inducing Nucleic Acids That Inhibit the Irregular Target Gene of miR-122

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: Number 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) RNA interference inducing nucleic acids comprising base sequences;

 As RNA Interference Inducing Nucleic Acids That Inhibit the Minormal Target Gene of miR-133 5'-UUU UGU CCC-3 '(miR-133-G4U) (SEQ ID NO: 34), 5'-UUU GUU CCC-3' (miR-133-G5U) (SEQ ID NO: 35) or 5'-UUU UUU CCC An RNA interference inducing nucleic acid comprising the nucleotide sequence of -3 '(miR-133-G4,5U) (SEQ ID NO: 36);

As RNA Interference Inducing Nucleic Acids That Inhibit the Irregular Target Gene of let-7

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 RNA interference inducing nucleic acid comprising the nucleotide sequence of 5'-UGA GUU AUU-3 '(let-7-G5,8U) (SEQ ID NO: 46);

RNA Interference-Inducing Nucleic Acids That Inhibit the Irregular Target Gene of miR-302a 5'-UAA UUG CUU-3 '(miR-302a-G4U) (SEQ ID NO: 47), 5'-UAA GUU CUU-3' (miR-302a-G6U) (SEQ ID NO: 48), or 5'-UAA UUU An RNA interference inducing nucleic acid comprising the base sequence of CUU-3 '(miR-302a-G4,6U) (SEQ ID NO: 49); or

RNA Interference Inducing Nucleic Acids That Inhibit MiR-372 Irregular Target Genes 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 represented by any one or more of the following:

As RNA Interference Inducing Nucleic Acids That Inhibit the Irregular Target Gene of miR-124

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: No. 57) or an RNA interference inducing nucleic acid showing the base sequence of a 5'-UAA UUC ACG CGG UGA AUG CCA A-3 '(miR-124-G4,5U) (SEQ ID NO: 58);

As RNA Interference Inducing Nucleic Acids That Inhibit the Irregular Target Gene of miR-1

5'-UUG AAU GUA AAG AAG UAU GUA U-3 '(miR-1-G2U) (SEQ ID NO: 59), 5'-UGU AAU GUA AAG AAG UAU GUA U-3' (miR-1-G3U) (SEQ ID NO: Number 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 Base of -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) RNA interference inducing nucleic acids representing sequences;

RNA interference-inducing nucleic acid that inhibits an irregular target gene of miR-122 as 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'-U GG 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) ( RNA interference inducing nucleic acid showing the nucleotide sequence of SEQ ID NO: 80);

5'-UUU UGU CCC CUU CAA CCA GCU G-3 '(miR-133-G4U) (SEQ ID NO: 81), 5'-UUU GUU CCC CUU CAA Base sequence of 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 acids, including;

RNA interference-inducing nucleic acid that inhibits the irregular target gene of let-7 as 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 The nucleotide sequence of -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) RNA interference inducing nucleic acid;

RNA interference-inducing nucleic acid that inhibits an irregular target gene of miR-302a as 5'-UAA UUG CUU CCA UGU UUU GGU GA-3 '(miR-302a-G4U) (SEQ ID NO: 94), 5'-UAA GUU CUA CCA UGU Base of 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) RNA interference inducing nucleic acids representing sequences; or

RNA interference-inducing nucleic acids that inhibit the non-regular target gene of miR-372 as 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: No. 101), or an RNA interference inducing nucleic acid showing the base sequence of 5′-AAA GUU CUU CGA CAU UUG AGC GU-3 ′ (miR-372-G6,9U) (SEQ ID NO: 102).

It provides a composition for inhibiting the expression of the irregular target gene of the microRNA comprising the RNA interference induction nucleic acid of the present invention.

It provides a cell cycle, differentiation, dedifferentiation, morphology, migration, dedifferentiation, division, proliferation or death control composition comprising the RNA interference induction nucleic acid of the present invention.

Preferably, the composition provides a composition, characterized in that one or more of the following:

a composition for inducing cancer cell death comprising an RNA interference inducing nucleic acid that inhibits an irregular target gene of miR-124;

a composition for inducing neurites branching differentiation comprising an RNA interference inducing nucleic acid that inhibits an irregular target gene of miR-124;

a composition for inducing cell cycle arrest of liver cancer cells comprising an RNA interference inducing nucleic acid that inhibits an irregular target gene of miR-122;

a composition for promoting myocyte differentiation or promoting muscle fibrosis, including an RNA interference-inducing nucleic acid that suppresses an irregular target gene of miR-1;

a composition for inducing myocyte death, including an RNA interference-inducing nucleic acid that inhibits an irregular target gene of miR-155;

a composition for promoting cell death of neuroblastoma, including RNA interference-inducing nucleic acid that inhibits an irregular target gene of miR-124;

a composition for promoting cell division proliferation of neuroblastoma, including an RNA interference-inducing nucleic acid that inhibits an irregular target gene of miR-124;

a composition for inducing myocardial hypertrophy comprising an RNA interference inducing nucleic acid that inhibits an irregular target gene of miR-1;

a composition for inducing myocardial hypertrophy comprising an RNA interference inducing nucleic acid that inhibits an irregular target gene of miR-133;

a composition for inducing cell cycle arrest of cancer cells, including an RNA interference inducing nucleic acid that inhibits an irregular target gene of let-7;

a composition for inducing cell cycle progression activity of hepatocytes comprising an RNA interference inducing nucleic acid that inhibits an irregular target gene of let-7;

a composition for promoting reverse differentiation comprising an RNA interference inducing nucleic acid that inhibits an irregular target gene of miR-302a;

a composition for promoting reverse differentiation comprising an RNA interference inducing nucleic acid that inhibits an irregular target gene of miR-372; or

A composition for inhibiting cell migration of liver cancer cells comprising an RNA interference inducing nucleic acid that inhibits an irregular target gene of miR-122.

The present invention provides a noncanonical target gene of a microRNA by modifying some sequences of a specific microRNA in at least one single strand of a double strand of nucleic acid inducing RNA interference, comprising the following steps: There is provided a method for preparing an RNA interference-inducing nucleic acid that inhibits the expression of:

It contains four base sequences from second to fifth from the 5 'end of a particular microRNA, wherein the sixth and seventh bases are identical and the sixth and seventh bases can be aligned with the sixth base of a particular microRNA. A base having complementary base sequences to the base and capable of being aligned with the sixth base of the specific microRNA includes RNA complementary nucleic acids such that all complementary bases, including G: A and G: U wobble sequences, are included. Constructing, or

Constructing an RNA interference inducing nucleic acid such that the guanine base in the base sequence between the 5 'end and the ninth base of the particular microRNA has a modified base sequence substituted with at least one of uracil or adenine.

The present invention also provides a method for screening a test substance for controlling cell cycle, differentiation, reverse differentiation, morphology, migration, division, proliferation, reverse differentiation or death, comprising the following steps:

Transfecting the RNA interference inducing nucleic acid into the subject cell;

Treating the test substance with the subject cell; And

Confirming the expression amount or expression of the non-regular target gene of the microRNA inhibited by the RNA interference induction nucleic acid in the target cell.

Hereinafter, the present invention will be described.

When a microRNA binds to a target messenger RNA, the microRNA may be recognized as a target of the microRNA and bind to the messenger RNA even though it is not exactly complementary to the microRNA seed region. This is called a non-canonical target. recognition). The present inventors have focused on the recognition of non-normal targets of such microRNAs, and have completed the present invention by developing RNA interference-inducing nucleic acids that selectively inhibit the non-regular target genes of the microRNAs by modifying some sequences of the microRNAs and revealing their efficacy. .

The present invention relates to RNA in which at least one of the double strands of a nucleic acid inducing RNA interference is modified, in which some sequences of a particular microRNA are modified to suppress only the expression of a noncanonical target gene of the microRNA. As interference-inducing nucleic acid,

The RNA interference-inducing nucleic acid comprises four to five base sequences from the 5 'end of a particular microRNA, the sixth and seventh bases are identical, and the sixth and seventh bases are the sixth of a particular microRNA. A base having a base sequence complementary to that of the base, and a base sequenceable to the sixth base of the specific microRNA include all complementary bases including G: A, G: U wobble sequences Characterized in that, or

The RNA interference-inducing nucleic acid is RNA interference-inducing nucleic acid, characterized in that having a modified base sequence in which the guanine base of the base sequence between the 5 'end and the ninth base of a specific microRNA at least one or more substituted with uracil or adenine to provide.

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

RNA interference inducing nucleic acids according to the invention are based on two non-canonical target recognition of microRNAs.

More specifically, microRNAs recognize not only regular target genes but also irregular target genes. Irregular target genes recognized by the microRNA do not have a complementary relationship that exactly matches the microRNA.

One aspect of an irregular target gene recognized by a microRNA and an RNA interference-inducing nucleic acid that inhibits the irregular target gene is as follows.

The recognition class of one of the non-normal target genes to which the microRNA binds has a continuous complementary relationship with all the bases from the 5 'end of the microRNA to the fifth. However, a gene recognized by the 6th base at the 5 'end of the microRNA (arranged with the 6th base at the 5' end of the microRNA) has a base complementary to the 5th base at the 5 'end of the microRNA. Immediately contiguous bases, rather than contiguous bases, are pushed out in the shape of a ridge, and then the base complementary to the microRNA is the base recognized by the sixth base at the 5 'end of the microRNA.

An RNA interference-inducing nucleic acid that suppresses an irregular target gene recognized by the microRNA of the above aspect comprises four base sequences from the 5 'end to the fifth to the fifth of the specific microRNA. In addition, two bases consecutive to the four base sequences are identical to each other, and the two base sequences have a base sequence complementary to a base that can be arranged with the sixth base of a specific microRNA, Bases that can be aligned with the sixth base include all complementary bases, including G: A and G: U wobble configurations.

RNA interference-inducing nucleic acids that inhibit the non-canonical target genes recognized by this particular microRNA, as compared with the base sequence of the microRNA, share at least four base sequences from 2 'to 5' from the 5 'end of the particular microRNA. Include as.

In addition, the 6th base and the 7th base sequence from the 5 'end of the RNA interference induction nucleic acid may be identical, and these two base sequences are complementary to bases that can be arranged with the 6th base of a specific microRNA. And bases that can be arranged with the sixth base of the particular microRNA include all complementary bases including G: A, G: U wobble configurations. For example, the sixth base of a specific microRNA: a base that can be arranged with the sixth base: a base complementary to the base that can be arranged with the sixth 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 sixth base of a particular microRNA is A, the sixth and seventh base sequences from the 5 'end of the RNA interference inducing nucleic acid may be AA, CA; When the sixth base of a particular microRNA is G, the sixth and seventh base sequences from the 5 'end of the RNA interference inducing nucleic acid may be UG, AG or GG; When the sixth base of a particular microRNA is U, the sixth and seventh base sequences from the 5 'end of the RNA interference inducing nucleic acid may be CU, UU; Or when the sixth base of a particular microRNA is C, the sixth and seventh base sequences from the 5 'end of the RNA interference inducing nucleic acid may be CC.

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

In addition, the RNA interference-inducing nucleic acid may preferably include a 7th base sequence from the 5 'end of the specific microRNA as the 8th base sequence from the 5' end.

In another embodiment, one aspect of an irregular target gene recognized by a microRNA and an RNA interference inducing nucleic acid that inhibits the irregular target gene is as follows.

The irregular target gene recognized by the microRNA has a wobble relationship of G: A with an array of complementary relations of A: U, G: C, C: G, and U: A with the bases of the microRNA and the target gene recognized by the microRNA. Contains an array of or wobble relations of G: U.

When a microRNA and a target gene recognized by the microRNA are in a wobble relationship of G: A, a base of a specific microRNA: a base of a target gene recognized by the specific microRNA: inhibits a target gene recognized by the specific microRNA The base sequence of the RNA interference inducing nucleic acid is G: A: U. In addition, when a microRNA and a target gene recognized by the microRNA are in a wobble relationship of G: U, a base of a specific microRNA: a base of a target gene recognized by the specific microRNA: a target gene recognized by the specific microRNA The sequence of bases of RNA interference inducing nucleic acids that inhibits is G: U: A.

RNA interference inducing nucleic acids according to the present invention have a modified base sequence in which one or more guanine (G) bases of the base sequences between the 5 'end and the ninth base of a specific microRNA are substituted with uracil (U). Alternatively, an RNA interference inducing nucleic acid according to the present invention has a modified base sequence in which at least one guanine (G) base in the base sequence between the 5 'end and the ninth base of a specific microRNA is substituted with adenine (A). When one or more guanine (G) bases are included in the base sequence between the 5 'end and the 9th base of a specific microRNA, all of the included guanine bases may be substituted with uracil (U) or adenine (A), At least one guanine base is substituted with uracil (U) or adenine (A). When one or more guanine (G) bases are included in the base sequence between the 5 'end and the ninth base of a specific microRNA, the remaining bases are identical to the bases of the specific microRNA, in addition to the base substituted with uracil or adenine base.

RNA interference inducing nucleic acids according to the present invention selectively inhibit the irregular target gene of the microRNA and do not inhibit the regular target gene of the microRNA.

According to specific Example 2 of the present invention (see FIG. 2), the expression regulation function of messenger RNA for a target gene of an RNA interference-inducing nucleic acid according to the present invention is specific for a non-canonical target gene, and inhibited for a regular target gene. It did not show any effect.

Therefore, using the RNA interference inducing nucleic acid according to the present invention, it is possible to selectively suppress only the irregular target gene expression, and thus to selectively induce only the effect expected through RNA interference inducing nucleic acid expression inhibition.

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

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

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

An RNA interference-inducing nucleic acid that suppresses an irregular target gene recognized by a specific microRNA according to the present invention, comprising a second to fifth base sequence from the 5 'end of a specific microRNA, and a sixth and seventh base May be the same, and the sixth and seventh bases have a base sequence complementary to the bases that can be arranged with the sixth base of a particular microRNA, and the bases that can be arranged with the sixth base of the particular microRNA include G: The length of the RNA interference inducing nucleic acid, which includes all complementary bases including the A, G: U wobble sequences, includes, but is not limited to, 6 or more base sequences, but is usually limited to 21 or 24 It may have the following base sequences.

RNA interference inducing nucleic acid according to the present invention is an RNA interference inducing nucleic acid that inhibits an irregular target gene of miR-124 when a specific microRNA is miR-124, and is the second to fifth from the 5 'end of miR-124. It comprises four base sequences, the sixth and seventh base may be the same and the sixth and seventh base has a base sequence complementary to the base that can be arranged with the sixth base of miR-124, the miR- The base that can be aligned with the sixth base of 124 is an RNA interference inducing nucleic acid that includes all complementary bases including G: A, G: U wobble sequences.

For example, the RNA interference induction nucleic acid may be an RNA interference induction nucleic acid (miR-124BS) in which the 2nd to 7th nucleotide sequences of the 5 ′ terminal represent the nucleotide sequence of 5′-AA GGC C-3 ′. More preferably, the RNA interference-inducing nucleic acid may be an RNA interference-inducing nucleic acid (miR-124BS) representing a nucleotide sequence of 5′-UAA GGC CAC GCG GUG AAU GCC-3 ′.

RNA interference inducing nucleic acid according to the present invention is an RNA interference inducing nucleic acid that suppresses an irregular target gene of miR-122 when a specific microRNA is miR-122, which is the second to fifth from the 5 'end of miR-122. It comprises four base sequences, the sixth and seventh base is the same and the sixth and seventh base has a base sequence complementary to the base that can be arranged with the sixth base of miR-122, Bases that can be aligned with the sixth base are RNA interference-inducing nucleic acids that include all complementary bases, including G: A, G: U wobble sequences.

For example, the RNA interference-inducing nucleic acid may be an RNA interference-inducing nucleic acid (miR-122BS) in which the 2nd to 7th base sequences of the 5 ′ terminal represent the 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) representing the nucleotide sequence of 5′-UGG AGU UGU GAC AAU GGU GUU-3 ′.

RNA interference-inducing nucleic acids according to the present invention, RNA interference-inducing nucleic acids that suppress the irregular target gene of miR-155 when a specific microRNA is miR-155, the second to fifth from the 5 'end of miR-155 It comprises four base sequences, the sixth and seventh base is the same and the sixth and seventh base has a base sequence complementary to the base that can be arranged with the sixth base of miR-155, the base of miR-155 Bases that can be aligned with the sixth base are RNA interference-inducing nucleic acids that include all complementary bases, including G: A, G: U wobble sequences.

For example, the RNA interference induction nucleic acid may be an RNA interference induction nucleic acid (miR-155BS) in which the 2nd to 7th nucleotide sequences of the 5 ′ terminal represent the nucleotide sequence of 5′-UA AUG G-3 ′. More preferably, the RNA interference inducing nucleic acid may be an RNA interference inducing nucleic acid (miR-155BS) representing the nucleotide sequence of 5′-UUA AUG GGC UAA U CGU GAU AGG-3 ′.

RNA interference-inducing nucleic acids according to the present invention are RNA interference-inducing nucleic acids that inhibit the irregular target gene of miR-1 when a specific microRNA is miR-1, and is the second to fifth from the 5 'end of miR-1. It comprises four base sequences, the sixth and seventh base is the same and the sixth and seventh base has a base sequence complementary to the base that can be aligned with the sixth base of miR-1, Bases that can be aligned with the sixth base are RNA interference-inducing nucleic acids that include all complementary bases, including G: A, G: U wobble sequences.

For example, the RNA interference induction nucleic acid may be an RNA interference induction nucleic acid (miR-1BS) in which the 2nd to 7th base sequences of the 5 ′ terminal represent the base sequence of 5′-GG AAU U-3 ″. More preferably, the RNA interference inducing nucleic acid may be an RNA interference inducing nucleic acid (miR-1BS) representing the nucleotide sequence of 5′-UGG AAU UGU AAA GAA GUA UGU-3 ′.

An RNA interference-inducing nucleic acid that inhibits an irregular target gene recognized by a particular microRNA according to the present invention, wherein the guanine (G) base in the base sequence between the 5 'end and the 9th base of the specific microRNA is uracil (U) or An RNA interference-inducing nucleic acid having a modified base sequence substituted with at least one or more adenine (A) s has a length of 9 or more bases, but is not limited thereto. Can have.

RNA interference inducing nucleic acids according to the present invention is an RNA interference inducing nucleic acid that inhibits an irregular target gene of miR-124 when a specific microRNA is miR-124, and includes a base between the 5 'end of the miR-124 and the ninth base. An RNA interference-inducing nucleic acid having a modified base sequence in which at least one guanine (G) base is substituted with uracil (U) or adenine (A) base in the sequence.

For example, the RNA interference-inducing nucleic acid has a base sequence between the 5 'end and the 9th base, 5'-UAA UGC AC-3' (miR-124-G4U), 5'-UAA GUC AC-3 '(miR-124). -G5U) or RNA interference inducing nucleic acid indicating 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).

RNA interference inducing nucleic acid according to the present invention is an RNA interference inducing nucleic acid that suppresses an irregular target gene of miR-1 when a specific microRNA is miR-1, and includes a base between the 5 'end of the miR-1 and the ninth base. An RNA interference-inducing nucleic acid having a modified base sequence in which at least one guanine (G) base is substituted with uracil (U) or adenine (A) base in the sequence.

For example, the RNA interference-inducing nucleic acid has a base sequence between the 5 'end and the ninth base, 5'-UUG AAU GUA-3' (miR-1-G2U), 5'-UGU 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 RNA interference inducing nucleic acids representing AAU UUA-3 ′ (miR-1-G2,7U) or 5′-UUU AAU UUA-3 ′ (miR-1-G2,3,7U). 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 ' -UUU AAU UUA AAG AAG AAG UAU GUA U-3 '(miR-1-G2,3,7U) can be an RNA interference inducing nucleic acid.

RNA interference inducing nucleic acid according to the present invention is an RNA interference inducing nucleic acid that inhibits an irregular target gene of miR-122 when a specific microRNA is miR-122, and includes a base between the 5 'end of the miR-122 and the ninth base. An RNA interference-inducing nucleic acid having a modified base sequence in which at least one guanine (G) base is substituted with uracil (U) or adenine (A) base in the sequence.

 For example, the RNA interference-inducing nucleic acid has a base sequence between the 5 'end and the ninth base, 5'-UUG AGU GUG-3' (miR-122-G2U), 5'-UGU 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 RNA interference inducing nucleic acid representing 5'-UGG AGU UUU-3 (miR-122-G7,9U). . More preferably, the RNA interference inducing nucleic acid is 5'-UUG AGU GUG ACA AUG GUG UUU G-3 '(miR-122-G2U), 5'-UGU 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 RNA interference inducing nucleic acid representing the base sequence of -G7,9U).

RNA interference-inducing nucleic acids according to the present invention is an RNA interference-inducing nucleic acid that inhibits an irregular target gene of miR-133 when a specific microRNA is miR-133, and is a base between the 5 'end of the miR-133 and the 9th base. An RNA interference-inducing nucleic acid having a modified base sequence in which at least one guanine (G) base is substituted with uracil (U) or adenine (A) base in the sequence.

For example, the RNA interference-inducing nucleic acid has a base sequence between the 5 'end and the 9th base, 5'-UUU UGU CCC-3' (miR-133-G4U), 5'-UUU GUU CCC-3 '(miR-133). -G5U) or an RNA interference inducing nucleic acid representing 5'-UUU UUU CCC-3 '(miR-133-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) can be an RNA interference inducing nucleic acid.

RNA interference inducing nucleic acid according to the present invention is an RNA interference inducing nucleic acid that inhibits a non-normal target gene of let-7 when a specific microRNA is let-7, a base between the 5 'end and the 9th base of let-7. An RNA interference-inducing nucleic acid having a modified base sequence in which at least one guanine (G) base is substituted with uracil (U) or adenine (A) base in the sequence.

 For example, the RNA interference inducing nucleic acid has a base sequence between the 5 'end and the 9th base, and has a 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, RNA interference inducing nucleic acid). 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 AUU RNA interference inducing nucleic acid showing the base sequence of 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.

RNA interference induction nucleic acid according to the present invention is an RNA interference induction nucleic acid that suppresses an irregular target gene of miR-302a when a specific microRNA is miR-302a, and a base between the 5 'end of the miR-302a and the 9th base. An RNA interference-inducing nucleic acid having a modified base sequence in which at least one guanine (G) base is substituted with uracil (U) or adenine (A) base in the sequence.

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

RNA interference inducing nucleic acid according to the present invention is an RNA interference inducing nucleic acid that inhibits an irregular target gene of miR-372 when a specific microRNA is miR-372, and includes a base between the 5 'end of the miR-372 and the ninth base. An RNA interference-inducing nucleic acid having a modified base sequence in which at least one guanine (G) base is substituted with uracil (U) or adenine (A) base in the sequence.

 For example, the RNA interference-inducing nucleic acid has a base sequence between the 5 'end and the ninth base, 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 RNA interference inducing nucleic acid representing 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 inhibits an irregular target gene of miR-372. 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 CUU CGA CAU UUG AGC RNA interference inducing nucleic acid representing the base sequence of the RNA interference inducing nucleic acid showing the nucleotide sequence of GU-3 '(miR-372-G6,9U).

Using the above-described RNA interference inducing nucleic acid according to the present invention, it is possible to specifically suppress the irregular target gene of the microRNA.

Accordingly, the present invention provides a composition for inhibiting irregular target gene expression of microRNA comprising RNA interference inducing nucleic acid or a kit for inhibiting irregular target gene expression of microRNA comprising RNA interference inducing nucleic acid.

In addition, by using the above-described RNA interference induction nucleic acid according to the present invention, by specifically inhibiting the irregular target gene of the microRNA, it is possible to selectively regulate the action that occurs due to suppression of the expression of the non-regular target gene of the microRNA.

More specifically, using the RNA interference-inducing nucleic acid according to the present invention specifically inhibits the irregular target gene of the microRNA, and using the above-described RNA interference-inducing nucleic acid according to the present invention, expression of the non-regular target gene of the microRNA Action (eg, cell cycle, differentiation, retrodifferentiation, morphology, migration, division, proliferation, or death) can be specifically regulated.

Accordingly, the present invention provides a composition or kit for regulating cell cycle, differentiation, dedifferentiation, morphology, migration, division, proliferation, or death, including RNA interference-inducing nucleic acids.

In the present invention, the 'cell cycle' is a continuous process in which cells grow, divide, and grow again, and the cells form M (cell division), G1 (first division preparation), S group (DNA synthesizer), This means repeating four phases of the G2 group (second cleavage preparation device). The G1 group is a DNA synthesis preparation device from immediately after cell division to the start of DNA synthesis, and the G2 group is a cell division preparation device from the end of DNA synthesis to the beginning of cell division.

In the present invention, the 'cell cycle regulation' includes inducing cells to fluctuate between the four phases, or promoting or arresting the fluctuations, for example, G1 / G2 cells of G1 Inducing variation in the / G2 phase can be included as an example of cell cycle regulation.

In the present invention, the 'cell differentiation (differentiation)' is a process by which cells acquire morphological and functional specificity, and the cells change in size, shape, membrane potential, metabolic activity, and response to signals through differentiation. .

In the present invention, the 'regulation of cell differentiation' includes promoting or delaying the rate at which cells differentiate, inducing differentiation to start, or suppressing differentiation from starting, for example, inducing nerve cells to differentiate. To induce morphological specificity (eg neurite differentiation) can be included as an example of cell differentiation control. Or, for example, inducing myocytes to differentiate and inducing them to have morphological specificities (eg, myofibroblasts).

In the present invention, the term "de-differentiation" refers to a phenomenon of reversing the progression of the differentiation and acquiring the pluripotency of the undifferentiated period before the differentiation of cells is differentiated, and further changing the medium cells.

In the present invention, the 'dedifferentiation control' includes inducing, promoting, or inhibiting and retarding the progression of dedifferentiation so that the cells are dedifferentiated, and the result of dedifferentiation control is, for example, a cell growth form (eg, colony formation) , Can be determined by confirming the activity of dedifferentiation factors (Oct3 / 4, Sox2, c-Myc, Klf4) and the like.

In the present invention, the 'cell morphology' refers to a phenotypic characteristic including the shape, structure, size, etc. of the cell, and the cell morphology depends on the type of organism, and even the same organisms depending on the type of tissue or organ Varies.

In the present invention, the 'cell morphology control' is to induce changes in the characteristics of the phenotype, including the shape, structure, size, etc. of the cells, to promote, delay, induce or inhibit the change in the natural shape of the cells and Promoting or inducing to change the form non-naturally. For example, inducing myocardial hypertrophy of cardiomyocytes may be included.

In the present invention, the 'cell movement' refers to the movement of the cell, and is an important process for the growth and physiological activity of the animal, while the cells mainly move quickly in a given stimulus and usually move to a non-collective shape, Other cell types can only migrate during certain growth periods or in specific situations. Cell migration occurs mainly at the beginning of neural and vasculature or wound healing of epithelial tissues, and cell population migration contributes to multiple tumor metastases.

In the present invention, the 'cell movement control' means to delay, promote, induce or inhibit (inhibition) the movement of the cells.

In the present invention, 'cell division, proliferation' refers to a process of dividing a parent cell into two daughter cells and increasing the number of cells.

In the present invention, 'cell division, proliferation control' includes delaying, promoting, inhibiting or inducing cell division and proliferation. Induction of cell phase into the M phase (cell divider) in the cell cycle can lead to cell division and proliferation. For example, reducing the number of cells distributed in the G0 / G1 phase and increasing the number of cells distributed in the G2 / M phase may be included as examples of cell division and proliferation regulation.

In the present invention, 'apoptosis' refers to a step in which a cell dies by genetic properties while maintaining a functional role, and includes both early cell death and late cell death. In apoptosis, the entire cell shrinks and new proteins are synthesized and killed by the cell's suicide gene.

In the present invention, 'cell death control' includes inducing, delaying, promoting or inhibiting 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 an irregular target gene of miR-124;

a composition for inducing neural branching differentiation, including an RNA interference-inducing nucleic acid (preferably miR-124BS) that suppresses an irregular target gene of miR-124;

a composition for inducing cell cycle arrest of liver cancer cells comprising an RNA interference-inducing nucleic acid (preferably miR-122BS) that inhibits an irregular target gene of miR-122;

a composition for promoting myocyte differentiation or promoting muscle fibrosis, including an RNA interference-inducing nucleic acid (preferably miR-1BS) that suppresses an irregular target gene of miR-1;

a composition for inducing myocyte death, including an RNA interference-inducing nucleic acid (preferably miR-155BS) that suppresses an irregular target gene of miR-155;

a composition for promoting cell death of neuroblastoma, including an RNA interference-inducing nucleic acid (preferably miR-124-G5U) that suppresses an irregular target gene of miR-124;

a composition for promoting cell division proliferation of neuroblastoma, including an RNA interference-inducing nucleic acid (preferably miR-124-G4U) that suppresses an irregular 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 inhibits an irregular target gene of miR-1;

a composition for inducing myocardial hypertrophy comprising an RNA interference-inducing nucleic acid (preferably miR-133-G4U) that suppresses an irregular target gene of miR-133;

compositions for inducing cell cycle arrest of cancer cells, including RNA interference-inducing nucleic acids (preferably let-7-G2U, let-7-G2,4U) that inhibit the non-regular target genes 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 inhibits 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 an irregular target gene of miR-302a;

a composition for promoting reverse differentiation comprising an RNA interference-inducing nucleic acid (preferably miR-372-G4U) that suppresses an irregular target gene of miR-372; or

A composition for inhibiting cell migration of liver cancer cells comprising an RNA interference induction nucleic acid (preferably miR-122-G2U, or miR-122-G2,3U) that suppresses an irregular target gene of miR-122.

In addition, by using the RNA interference induction nucleic acid according to the present invention, by specifically inhibiting the irregular target gene of the microRNA, it is possible to selectively regulate the action of the irregular target gene of the microRNA, various fields (eg, pharmaceutical, Cosmetics, cytology, etc.).

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

More specifically, the step of introducing the RNA interference inducing nucleic acid according to the present invention to the target cell;

Treating the test substance with the subject cell; And

Including the step of confirming the expression level or expression of the irregular target gene of the microRNA inhibited by the RNA interference-inducing nucleic acid in the target cell,

Methods for screening test substances for controlling cell cycle, differentiation, retrodifferentiation, morphology, migration, division, proliferation or death are provided.

In the present invention, the target cell is not limited as long as it is derived from a mammal including a human, and tissues or types from which the target cell is extracted are not limited, and examples thereof include neurons, cardiomyocytes, cancer cells, and muscle cells. Can be.

In the present invention, the introduction of the RNA interference-inducing nucleic acid into the target cell may be appropriately performed according to various methods known in the art, for example, the introduction may be performed by transfection using calcium phosphate (Graham, FL et al., Virology, 52: 456). (1973)), transfection with DEAE dextran, transfection with 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)), such as transduction or transfection (Basic methods in molecular biology, Davis et al., 1986 and Molecular cloning: A laboratory manual, Davis et al., 1986) can be made according to methods well known to those of ordinary skill in the art. Preferably, it may be by transfection. Transfection efficiency can vary greatly depending on cell type as well as cell culture conditions and transfection reagents.

In the present invention, the test substance is an unknown substance used in screening to control the expression of the non-normal target gene of the microRNA, thereby affecting cell cycle, differentiation, reverse differentiation, morphology, migration, division, proliferation or death. It includes, but is not limited to, compounds, extracts, proteins or nucleic acids.

In the present invention, the expression level or expression of the non-regular target gene of the microRNA can be confirmed by various expression analysis methods known in the art, such as RT-PCR, ELISA (see 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 is not limited thereto.

In the present invention, the expression amount or expression of the non-regular target gene of the microRNA is confirmed, and as a result, the expression amount or expression of the non-regular target gene of the microRNA is compared with the case where only RNA interference-inducing nucleic acid is treated without a test substance. If there is a difference, the test substance may be judged to affect the expression of the non-normal target gene of the microRNA. Furthermore, the test substance may be determined to have a function of cell cycle, differentiation, dedifferentiation, morphology, migration, division, proliferation or death.

In addition, the present invention provides a method for producing an RNA interference-inducing nucleic acid described above.

More specifically, in a single strand of one or more of the double strands of nucleic acid inducing RNA interference, some sequences of a particular microRNA are modified so that the noncanonical target of the microRNA is included. Methods of Making RNA Interference Inducing Nucleic Acids That Inhibit Genes

It contains four base sequences from second to fifth from the 5 'end of a particular microRNA, wherein the sixth and seventh bases are identical and the sixth and seventh bases can be aligned with the sixth base of a particular microRNA. A base having complementary base sequences to the base and capable of being aligned with the sixth base of the specific microRNA includes RNA complementary nucleic acids such that all complementary bases, including G: A and G: U wobble sequences, are included. Constructing, or

Constructing an RNA interference-inducing nucleic acid such that the guanine base in the base sequence between the 5 'end and the ninth base of the specific microRNA has a modified base sequence substituted with at least one of uracil or adenine.

Since the RNA interference inducing nucleic acid has already been described, the description is omitted in order to avoid duplicate description.

 RNA interference inducing nucleic acid construction is in accordance with various 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 ribosil ring of the 6th nucleotide from the 5' end of a specific microRNA.

The modified interference-inducing nucleic acid has the effect of completely eliminating the newly appearing nonnuclear nucleus bonds while maintaining the object of the present invention to specifically suppress only the nonnuclear nucleus sites of the miRNA.

By using the interference-inducing nucleic acid according to the present invention, it is possible to efficiently increase the biological function of the existing microRNA by suppressing the irregular target gene, or to suppress some of the functions of the existing microRNA, that is, the biological function of the non-regular target gene. Only functions can have the effect of selectively indicating. In addition, the interference-inducing nucleic acid can control the cell cycle, differentiation, dedifferentiation, morphology, migration, division, proliferation or death, and is expected to be applicable to various fields such as drugs and cosmetics.

1 schematically illustrates the mechanism of action of an interference-inducing nucleic acid that inhibits an irregular target of a microRNA.
FIG. 2 shows non-regular target specific expression of miR-124-BS by measuring the target gene inhibition of the canonical target (initiated: Seed) and the nonnuclear nuclear target (nucleation bulge) of miR-124 with an enzyme of a luciferase reporter. The inhibitory effect was confirmed.
FIG. 3 shows the flow-induced apoptosis of miR-124-BS in the cervical cancer cell (HeLa) -induced apoptosis induced by the normal target (Seed) and the nonnuclear target (nucleation bulge) of miR-124-BS. Cell sorting (FACS) analysis shows the results confirmed.
4 shows the morphology and morphology of neurite differentiation induced by the non-normal nuclear ridge target (nucleation bulge) of miR-124 through expression in human neuronal tumor blastoma (Sh-Sy-5y) of miR-124-BS. The results observed with the expression of the markers are shown.
FIG. 5 shows miR-124-BS (4753) and miR-124, which are natural forms of miR-124-BS, which regulate the nucleation target (nucleation bulge) of miR-124 to a regular target (Seed). The results of observing the differentiation of neurites branched by miR-124 (3714) having the same initiation sequence as the cell morphology and molecular biological characteristics.
6 is a cell morphology and molecular biological feature that the neural dendritic branch differentiation induced by the non-normal nuclear ridge target (nucleation bulge) of miR-124 in mouse neuroblastoma (N2a) is a mechanism through regulation of MAPRE1 gene expression. The observed results are shown.
Figure 7 shows the results confirmed by RNA-Seq analysis at the transcript level to suppress the expression of miR-124-BS-induced nonnuclear target (nucleation bulge) gene in mouse neuroblastoma (N2a) cells .
Figure 8 shows the results observed by miR-122-BS and flow cytometry cell cycle arrest induced by the non-regular nuclear ridge target (nucleation bulge) of miR-122 in human liver cancer cell line (HepG2).
Figure 9 shows the thickening effect of miR-1 induced non-normal nuclear bulge target (nucleation bulge) in muscle cells and the differentiation of miR-155 non-nuclear target (nucleation bulge) induced myocyte differentiation. Inhibition and apoptosis induction were observed by miR-1-BS and miR-155-BS in terms of cell morphology, molecular biological characteristics and flow cytometry, respectively.
FIG. 10 is a bioinformatics analysis of Ago HITS-CLIP, which is a microRNA and target RNA data binding to an agon nut protein in human brain tissue, as well as a normal initiation target (initiation: seed), as well as irregular GA It shows the result of confirming that the target of G: A wobble exists naturally.
FIG. 11 shows that miR-124's 4th irregular GA sequence initiation target and 5th irregular GA sequence initiation target of mouse neuroblastoma (N2a) cells exhibit completely different functions from miR-124 in brain cell differentiation. G4U and miR-124-G5U show cell morphology observations and cell death by flow cytometry.
FIG. 12 shows the biological functions of inhibition of miR-124 4th irregular GA sequence initiation target, 5th nonnormal GA array initiation target in human neuroblastoma (SH-sy-5y) cells. MiR-124-G4U, miR-124- G5U shows the results observed by Fluorescence-activated cell sorting (FACS) for cell proliferation and cell cycle analysis.
Figure 13 shows the results confirmed in the cardiomyocyte cell line (h9c2) through the flow cytometry (Fluorescence-activated cell sorting (FACS)) in the fluorescent protein reporter that the miR-1 seventh non-regular GA sequence initiation target of gene .
14 shows miR-1-G2U, miR-1-G3U, miR in neonatal cardiomyocytes of rats. After the expression of -1-G7U, the results were confirmed through observation of cell morphology and molecular biological characteristics on myocardial hypertrophy induction effect.
Figure 15 shows the results of observing the cell morphological characteristics of the myocardial hypertrophy induction effect after the expression of miR-133-G4U in the cardiomyocytes (h9c2) the function of the 4th non-regular GA array initiation target of miR-133.
FIG. 16 shows that human hepatocellular carcinoma cell lines (HepG2) inhibit the expression of genes through the second and third non-canonical GA sequence initiation targets of miR-122. Shows the results observed by measuring the enzyme activity of luciferase reporter.
FIG. 17 shows that miR-122 inhibits the migration of the non-regular GA sequence initiation target No. 2 and the non-regular GA sequence initiation target No. 2,3 miR-122-G2U, miR- Through the expression of 122-G2,3, U shows the results confirmed by the observation on the cell morphology.
FIG. 18 shows expression of miR-122-G2,3U indicating that inhibition of 2,3 non-regular GA sequence initiation target expression by miR-122 induces cell cycle arrest of human liver cancer cell line (HepG2). The results confirmed by flow cytometry are shown.
19 shows that inhibition of expression of the 2,3 irregular GA sequence initiation target and the 2,7 irregular GA sequence initiation target by miR-122 induces cell cycle arrest of human liver cancer cell line (HepG2). It shows the results confirmed by flow cytometry through the expression of miR-122-G2,3U, miR-122-G2,7U.
20 shows that inhibition of 2 or 2 or 4 irregular GA sequence initiation target expression by let-7 induces cell cycle arrest of human liver cancer cell line (HepG2) and 4 non-regular GA sequence initiation targets. Expression of let-7a-G2U, let-7a-G2,4U, let-7-G4U expression of inhibition of expression promotes the cell cycle shows the results confirmed by flow cytometry.
FIG. 21 shows that miR-372-G4U or miR-302a-G4U, along with miR-372 or miR-302a, promote the differentiation of miR-372 or miR-302a, leading to the inhibition of expression of 4th irregular GA sequence initiation target. The results are confirmed by the Oct4 gene expression reporter.
FIG. 22 shows a result of confirming a phenomenon in which the 2'OMe modification does not inhibit the irregular nuclear ridge target of the RNA interference derivative at the 6th end of the 5 'end.

Hereinafter, the present invention will be described in more detail with reference to 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-Induced Nucleic Acids That Inhibit Non-normal Targets of MicroRNA

In the Ago HITS CLIP experiment, the present inventors have shown that in addition to the normal initiation target which is completely complementary to the microRNA initiation, when the microRNA is nucleotide sequenced with the target in the agon nut protein, the present inventors have completed the complementary arrangement with the microRNA initiation. It was confirmed through the analysis of the RNA complex sequence result (Ago HITS-CLIP) bound to the agon nut protein. In addition, as a result of analyzing the base sequence of the RNA having these characteristics, the target (microRNA non-normal nuclear target site) that base 6 is arranged by inducing nucleation bulge to the target base of the microRNA, In the microRNA initiation region, it was confirmed that a target (microRNA non-regular GA alignment initiation target site) was naturally present (FIG. 1) allowing the GA wobble.

In the illustrated example shown in FIG. 1, miR-124 is a minimum of 6 nucleotides of 5'-GUGCCUUA-3 'of the target through the nucleotide sequence (5'-UAAGGCAC-3') via an agon nut protein. Consecutive complementary nucleotide sequences are generated, and targets having such nucleotide sequences have been reported as canonical seed sites of microRNAs (Nature, 2009, 460 (7254): 479-86).

Also, among the RNA complexes bound to the agonut, the miR-124 nucleotide sequence (5'-UAAGGCAC-3 ') and the microRNA irregular nucleus target site (5'- GUGGCCUU -3') were used as the 5 'terminal standards. Targets with complementary base sequences were identified so that the G of the target messenger RNA was arranged in a bulge between the fifth and the sixth, and based on this, the base sequence was complementary to the microRNA irregular nucleus target site. The starting nucleotide sequence of miR-124 has the nucleotide sequence (5-UAAGGCCA-3) in which 6 bases are repeated once more between 6 and 7 of the miR-124 beginning, with the continuous and complete complementary sequence being miR It was used as miR-124BS (BS: Bulge site) siRNA that inhibits -124 nonnuclear target.

When analyzing the sequence of the target messenger RNA coupled to the microRNA in the RNA complex bound to the agon nut, the G: A wobble base sequence in addition to the conventional complementary nucleotide sequence was used to bind the initiation sequence of the microRNA to the target messenger RNA. It was confirmed that it takes place. Therefore, in the case of miR-124, the 4th G base at the 5 'end of the base nucleotide sequence (5'-UAAGGCAC-3') is arranged in a G: A wobble, and thus the microRNA irregular GA sequence initiation target site (5'- UGGCAUU- 3 ') was invented, and based on this, the siRNA was invented miR-124-GU by designing a siRNA with a sequence that is continuous and perfectly complementary to the miR-124's irregular GA sequence initiation target site. It was used as an siRNA that inhibits the expression of the GA array initiation target.

Example 2 Confirmation of the Inhibitory Activity of the MicroRNA-BS Containing the Nucleotide Target Site Contiguous and Completely Complementary Sequences in the Initiation Region

Of the naturally occurring miR-124's normal seed target sites, the 5'-GUGCCUU-3 'base sequence is complementary to the second through seventh bases of the miR-124 base. The regular initiation target site is completely complementary to the beginning of miR-124 (5'-UAAGGCAC-3 ') and at least six consecutive bases (Figure 2a).

 The nucleation bulge site of miR-124 discovered through Ago HITS CLIP experiment is 5'- GUGGCCUU -3 ', where C base 6 of miR-124 forms nucleus with target. The nucleotide sequence is arranged so that the G of the target RNA arranged between the 5th and 6th bases of the 5 'terminal of miR-124 is formed into a bulge. Based on this, we designed a siRNA whose microRNA originated the nucleotide sequence (5'-UAAGGCCA-3 ') that continuously and completely complements the non-normal nuclear bulge site and named it miR-124-BS siRNA. (FIG. 2A). miR-124-BS recognizes miR-124's nonnuclear target nucleus target site as a regular initiation target and thinks that it will specifically and more strongly inhibit gene expression of non-normal targets with it. This was confirmed through the luciferase reporter experiment (FIG. 2B-C).

First of all, to see how irregular mi target target nucleus target sites can be suppressed compared to the existing regular target seed sites, the Luciferase reporter has the corresponding site for miR-124. After insertion of miR-124 at various concentrations (0, 0.01, 0.1, 0.5, 2.5 and 15 nM) into the cells, IC50 (inhibitory concentration 50) was measured by luciferase activity. Was about 0.5 nM, the irregular nuclear ridge site was confirmed to be similar to about 0.2 nM (Fig. 2b). However, the highest suppression rate was found to be about 50% for regular initiation sites and about 80% for non-regular nuclear sites, which was somewhat weaker. In particular, when the inhibition of gene expression begins, it was confirmed by luciferase activity measurement that the normal initiation target is about 0.01 nM or more and the irregular nuclear ridge target starts gene suppression at a concentration condition of 0.1 nM or more ( 2b). Therefore, miR-124 regulates the expression of the normal target and the irregular nuclear ridge target, and it can be seen that the efficiency of regulating the target regulation is high.

The luciferase reporter experiment performed at this time was performed by cloning the corresponding miR-124 target site sequence in the sequence of 2 consecutive sequences in the 3'UTR (3'untranslated region) region of the renilla luciferase gene of the psi-check2 vector (Promega). . The irregular ridge site sequence was used a non-nuclear ridge target site naturally present in the 3'UTR portion of MINK1. miR-124 is a human-derived sequence prepared according to the miRBase database by chemically synthesizing the corresponding guide strands and the transport strands by Bioneer, and then separating them by HPLC and providing them by the company. According to the duplex of the guide strand and the transport strand. miR-124 and luciferase reporter vectors 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 steptomycin, and the complete medium without antibiotics when transfection was performed. Cells were cultured. After 24 hours of transfection, luciferase activity was measured using Promega's Dual-luciferase reporter assay system according to the manufacturer's protocol, and Renilla luciferase activity measurement was performed using promega's Glomax Luminometer for at least 3 hours. More than one replicate was performed and normalized to the activity of firefly luciferase and calculated.

In order to confirm the function of the miR-124-BS invented to specifically inhibit only the non-normal nuclear ridge target of miR-124, the same experiment was carried out using a luciferase reporter. As a result, miR-124-BS did not inhibit luciferase reporter enzyme activity at any concentration (0-10 nM) against miR-124 normalized target, but concentration of 0.1nM or higher for miR-124 nonnuclear nuclear reporter target reporter. It was confirmed that the effect of inhibiting the luciferase reporter enzyme activity in (Fig. 2c). When the gene suppression efficiency at this time was measured by IC50, the inhibition of the irregular nuclear ridge target was about 0.5 nM. In view of this, it can be interpreted that the function of miR-124 irregular nuclear ridge target expression regulation of miR-124-BS is specific to the irregular nuclear ridge target site (FIG. 2C). The luciferase reporter experiment at this time was prepared by synthesizing miR-124-BS in Bioneer as before, and transfection (co-) into cervical cancer cells (HeLa: ATCC CCL-2) with the corresponding luciferase reporter vector. 24 hours after the transfection, luciferase enzyme activity was measured.

In the above examples, the inventors have invented miRNA-BS comprising a region of origin that is continuous and completely complementary to the site sequence for the purpose of inhibiting only the non-normal organic target of the microRNA, and verifying its effect. When applied to miR-124 and confirmed through the luciferase reporter experiment, it could be verified that miR-124-BS did not inhibit the expression of the normal initiation target and effectively suppressed the expression of the irregular ridge target. .

Example 3 Observation of Apoptosis of Cervical Cancer Cells Induced by Inhibition of Irregular Nuclear Target Expression of miR-124

After confirming in the previous example that miR-124-BS inhibits only non-normal raised target among several targets inhibited by miR-124, it was intended to confirm whether it can show only a specific function of miR-124. In general, miR-124 is specifically expressed in neurons, so it is known to play a role mainly related to neurons. However, the tumor-induced nocturnal glioma showed a lack of miR-124 expression, and correlated with the expression that miR-124 expression could lead to tumor cell death. Therefore, in order to confirm whether the various different functions of miR-124 are different depending on the kind of recognition of the target, miR-124-BS was introduced into cervical cancer cells (HeLa) and cell death was observed by flow cytometry ( 3a).

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

These experiments were divided into control group, miR-124 expression, and miR-124-BS expression, respectively, and when miR-124 was expressed, cervical cancer cells (HeLa) showed apoptosis (the number of cells stained with Annex V) compared to the control group. ), The rate of death was increased from about 30% to about 78%, and miR-124-BS was also increased to about 73% more than twice (Fig. 3a). In light of the results of previous Example 2 where miR-124-BS did not inhibit the normal initiation site of miR-124 but only the irregular ridge site, apoptosis of cervical cancer resulted from inhibition of expression of the irregular nuclear ridge target. Able to know.

miR-124 is a brain tissue-specific microRNA, and has been reported to have no brain-specific function in completely different cells without tissue cell-specific transcript expression (Farh K et al, 2005, Science, 310 (5755). 1817-21). Thus, in order to observe in neurons the function that miR-124 causes by inhibiting irregular ridge targets, miR-124-BS was introduced into the cerebellar neural stem cell line C17.2 and then cell differentiation was observed morphologically ( 3b, above). As a result, the introduction of miR-124 was promoted by the differentiation of neurites of C17.2 cells, but in the case of miR-124-BS, differentiation was observed in the form of short branches. Therefore, it can be seen that the general neural branch differentiation caused by miR-124 occurs by suppressing the regular initiation site target. At this time, the introduction of miRNA was carried out with RNAiMax reagent by synthesizing 50nM miRNA in the same manner as in Example 2.

In addition, since miR-124-BS induced cell death in HeLa cells, it was observed by flow cytometry in C17.2 cells in the same manner as in Example 2 to see whether such a function is also observed in neurons. As a result, in the expression of miR-124, the rate of apoptosis was 22.4%, which is almost similar to that of 21% of the control group, whereas miR-124-BS was slightly increased to 33.4% (FIG. 3B, intermediate figure). ). The same trend was observed in the cervical cancer cells in that apoptosis occurred through the inhibition of miR-124 irregular target, but the increase rate was much smaller than that of cervical cancer, which was about doubled. miR-124 plays a role in differentiation in neural stem cells, and cell cycle regulation may 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 through PI (Propidium Iodide) staining (FIG. 3B, below). MiR-124 induced cell cycle arrest at G0 / G1 to 83%, slightly higher than the control group of 69%. In contrast, it was observed that expression of miR-124-BS did not cause significant cell cycle arrest. In this experiment, the microRNA was transfected in the same manner as in the previous example, cultured for 48 hours, the cells were removed, fixed with ethanol, and 1 mg / ml propidium iodide (PI; Sigma-Aldrich) 0.2 mg / After reacting with ml of RnaseA for 30 minutes at 37 ° C, flow cytometry was performed with BD FACSCalibur (BD Biosciences).

In summary, the results of the above-described embodiments induce death of cervical cancer cells (Hela) in the same manner as miR-124, even if miR-124-BS inhibits the expression of miR-124 irregular nuclear ridge targets. This suggests that the inhibitory function of miR-124's irregular ridge target is the death of cancer cells. In addition, in neural stem cells, differentiation of neurite branches occurs due to the expression of miR-124, and a little cell death and cell cycle arrest occur. In miR-124-BS, which inhibits only the miR-124 irregular target, It appears somewhat different. Therefore, miR-124's neuronal differentiation function is mainly caused by the suppression of known regular initiation target genes, and it can be thought that other forms can occur through irregular ridge targets.

[Example 4] miR-124-BS inhibited the expression of miR-124 irregular nucleus target, confirming branched neurite outgrowth-inducing function with many branches and short processes

After confirming that miR-124-BS causes a different type of neural dendritic differentiation from miR-124, the sh-sy-5y cell line (CRL-2266), which is a human neuroblastoma, was examined in detail. ) Was used. At this time, in addition to miR-124-BS, miR-124 has the same starting sequence for recognizing and inhibiting the irregular nuclear ridge site, but other sequences have the same microRNA sequence as miR-124. One miR-124-BS was synthesized by the method described in the previous example and the experiment was conducted. The sequence of miR-124-BS (4753) used was 5'-CAAGGCCAAAGGAAGAGAACAG-3 '. As a control, cel-67 (NT; non-targeting), a microRNA of a pretty C. elegans, was used as a control. It was synthesized and used in the same manner as in the following. After transfecting the corresponding miRNAs in the same manner described in the previous example and incubating the cells for 60 hours, cell morphological changes were observed by light microscopy (FIG. 4A).

As a result, the expression of miR-124 was able to observe morphological changes that elongate the neurites of human neuroblastoma (Sh-sy-5y), whereas the development of miR-124 was able to inhibit only the irregular bump target. In the case of miR-124-BS (4753) having the same sequence as miR-124-BS, a short but branched neurite outgrowth was observed with a large number of neurites in one cell (FIG. 4A). ). Based on this, catecholamine-based neurons (CAD, CRL-11179) were also conducted in the same experiment. At this time, the microRNA was transfected and observed after culturing the cells for 52 hours (FIG. 4B). As a result, miR-124 has a small number but long protrusions, and miR-124-BS (4753) has a short but large number of nerves, as shown in human neuroblastoma (Sh-sy-5y, CRL-2266) cells. The eruption of the protruding branches could be observed. (FIG. 4B). In addition, when intracellularly delivered miR-124 and miR-124-BS (4753) at the same rate, cells with long neurites and cells with many neurites were observed simultaneously. When expressed with the miR-124-BS and miR-124 invented, this shows the possibility of promoting differentiation of various types of neurons similar to brain cells seen in artificially more complex neural networks.

In addition, neuronal differentiation promoted by these microRNAs was confirmed by immunostaining for Tuj1, a neuron-specific marker (FIG. 4B, below). In this experiment, 52 hours after transfection of the corresponding microRNA, CAD cells were fixed with 4% paraformaldehyde, and then the primary antibody, Tuj1 (MRB-435P, Covance Antibody Products) was 1: 1000. In the ratio of Alexa 594 fluorescence-binding secondary antibody (Abcam: ab150076) was carried out by antibody staining (immune-staining) in a ratio of 1: 1000, and then stained the nucleus with DAPI.

As a result of the above embodiment, it can be seen that miR-124 has a function of forming a short but many neurite branches, which is different from the function of the existing miR-124 by suppressing only irregular nuclear ridge targets. In particular, this was observed using miR-124-BS (4753), which is different from miR-124-BS but has the same region of origin and can only suppress non-nuclear targets. It is revealed that the RNA interference derivative including the starting sequence capable of recognizing has the same effect as miR-124-BS. In addition, the complex neurites produced by miR-124-BS can be used as a way to promote various types of neuronal differentiation, similar to human brain cells showing complex neural networks.

EXAMPLE 5 Expression of miR-124-BS with siRNA and shRNA vectors, followed by confirmation of the morphology and molecular biological characteristics of the induced neurite outgrowth.

Specific neuronal differentiation induced by the expression of miR-124-BS was further confirmed in the mouse neuroblastoma N2a (CCL-131), as in Example 4 miR-124, miR-124-BS (4753) The morphology of the cells was observed while incubating for 72 hours after introducing them into the cells (FIG. 5A). At the same time, miR-124 (3714), which has the same initiation sequence of miR-124 but the other part, is also synthesized by 5'-GAAGGCAGCAGUGCUCCCCUGU-3 'sequence to form duplexes in siRNA form, and then into cells. Introduced and tested. At this time, as a control, the same sequence as that of cel-67 (NT; non-targeting), a microRNA of C. elegans, was used.

As a result, in the experimental group to which miR-124 was delivered, neurons with long neurites similar to those shown in CAD, which are catecholamine-based neurons, were observed. In the cells transfected with miR-124 (4753), they were branched in various directions. It was able to determine the characteristics of the cell morphology with the short neurites extending (Fig. 5a, above). In addition, in the experimental group transfected miR-124 (3714), it was possible to observe a lot of cells showing a relatively long cell projection similar to miR-124 (Fig. 5a, above). This change in cell morphology was attributed to antibodies against the corresponding proteins expressed in differentiated neurons, Tuj1 (Covance Antibody Products, MRB-435P), vGLUT1 (synaptic systes, 135-303), MAP2 (millipore, MAB3418). It was confirmed that the increase in the expression amount by using the immunoblot experiment (immuno blotting) in comparison with the expression of the four control proteins (ACT-B, TUB-A, GAPDH, H3B) (Fig. 5a, below). Through this, neuronal differentiation by miR-124-BS (4753), which is similar to miR-124 but shows a different morphology, was slightly different in morphology, but it was confirmed that differentiation was induced on the gene expression marker. MiR-124 MiR-124 (3714), which has the same initiation sequence and is likely to suppress the regular initiation target, shows that the branch of neurons is long differentiated like miR-124. In addition, when miR-124-BS (4753) and miR-124 (3714) were expressed, the amount of protein expression seen in differentiated neurons was increased, all of which have molecular characteristics of differentiated neurons. It could be confirmed.

The expression of microRNAs in the previous examples has been performed by introducing a synthesized duplex into cells, but the expression of microRNAs can also be introduced by introducing a vector expressing a shRNA form, a hairpin type RNA capable of producing the microRNA. . Therefore, a vector expressing miR-124, miR-124-BS (4753) and miR-124 (3714) in the shRNA form was constructed using pCAG-miR30 plasmid (addgene # 14758), which is a vector expressed by pre-miRNA. . The pCAG-miR30 vector used was a CAG promoter for strongly expressing the microRNA, and designed to express the backbone of the microRNA-30, which has the advantage of maximizing microRNA expression (Paddison P et. al, Nature methods, 2004, 1 (2): 163-7).

PCAG vectors designed to express miR-124, miR-124-BS (4753) and miR-124 (3714), respectively, were introduced into N2a cells using a pCAG vector expressing nothing as a control, followed by cell morphology. Was observed (FIG. 5B). The experimental group of the hairpin structure introduced into the neuroblastoma N2a showed similar changes in the cell morphology of the miR-124, miR-124-BS (4753) and miR-124 (3714) duplexes. In 72 hours after transfection, miR-124 and miR-124 (3714) expressed in the experimental group was observed neuroblastoma with long neurites. On the contrary, in the experimental group in which miR-124-BS (4753) was expressed, neuronal cell tumors with neurites extending in various directions were observed (FIG. 5B, above). This change in cell morphology is due to the expression of Tuj1 (O. von Bohlen et al. 2007 Cell and Tissue Research, 329, 3, 409-20), a protein expressed in differentiated neurons, but with increased expression of Synaptophysin (SYP). (FIG. 5b, below), and thus, the cells into which the miR-124, miR-124-BS (4753) and miR-124 (3714) hairpin vectors were introduced are characterized by the differentiated form of neurons compared to the control group. Could confirm. An increase in TUJ1 was observed by immunoblot experiments in the same manner as in the previous example, and Synaptophysin was a primer described in the previous paper (SEKwon et al. 2011 Neuron 70, 847-54) through qPCR (Quantitative Polymerase Chain Reaction). Proceed using the sequence. In this experiment, the vector was transfected using Lipofectamine 3000 reagent (Invitrogen) according to the company's protocol for N2a cells, and after 24 hours, total RNA was extracted with the RNeasy kit (Qiagen), and the protocol. Reverse transcription with Superscript III RT (Invitrogen), qPCR with SYBR® Green PCR Master Mix (Applied Biosystems) was performed to measure the amount of messenger RNA of SYP with the corresponding primers and normalized to GAPDH messenger RNA values.

In view of the results of the above examples, even when using a vector to express the introduction of microRNA in the shRNA form, miR-124 (3714)) As with -124, it can be seen that the inhibition of the expression of a regular target gene generally causes long neurogenesis differentiation. In addition, if the irregular ridge target of miR-124 is expressed in the shRNA form only the end portion of the miR-124-BS (miR-124-BS (4753)), the branch can cause more neurites differentiation. Both miR-124 normal and non-normal ridge target inhibition showed the expression of molecular gene markers in the form of neurites differentiation of neurons, and thus all of them were differentiated into neurons.

Example 6 miR-124-BS induces neurogenesis differentiation of mouse neuroblastoma (N2a) through regulation of MAPRE1 expression

In addition, the induction of neurite differentiation in neuroblastomas (N2a) of miR-124, miR-124-BS (4753) and miR-124 (3714) was observed in cell morphology and analyzed quantitatively (FIG. 6A). In quantitative analysis, pUltra (addgene # 24129) plasmid expressing a green fluorescent protein (GFP) and a corresponding microRNA expression vector were used to identify the neural branches derived from each neuron. Transfection (co-transfection) and cell culture for 72 hours, while measuring the number of neurite branches while neurite observed by fluorescence microscope (Leica), the length of neurites per cell Was analyzed via the ImageJ program.

As a result, for 100 cells, the number of neurite branches expressed per cell was about 3-5 in miR-124, miR-124-BS (4753) and miR-124 (3714) compared to the control group. It was confirmed that the fold increased (FIG. 6A, bottom left). However, when miR-124-BS (4753) is expressed, it was observed that significantly more branches were produced than when miR-124 or miR-124 (3714) were expressed. In addition, if the length of the neurites generated per cell also increased by 4 to 6 times in the miR-124-BS (4753) and miR-124 (3714) experimental group compared to the control group, more specifically miR-124 or miR-124 When (3714) was expressed, it was observed that longer neurites were made than when miR-124-BS (4753) was expressed (FIG. 6A, bottom right). This is the same result as observed in the cell morphology in the previous example (FIGS. 4, 5, 6).

In order to determine the specific neuronal differentiation caused by miR-124-BS caused by the irregular bump site of the gene, find the non-normal bump target sequence of miR-124 in the sequence of 3'UTR of the gene involved in neuronal differentiation. saw. As a result, 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, was found. MAPRE1 is a protein attached to the ends of the microtubules constituting the neurites, and the shape of the neurites may be determined by controlling the formation of the microtubules. Therefore, in order to confirm whether this MAPRE1 gene is recognized as a non-normal bump target of miR-124 and its expression is inhibited, the method was performed in Example 5b after introducing miR-124-BS into N2a cells and the amount of messenger RNA of MAPRE1. According to the qPCR experiment was measured (Fig. 6b). When qPCR was performed with two different primers, both results showed that MAPRE1 expression was reduced to 70-40% in the miR-124-BS introduced experimental group compared to the control group. This confirmed that MAPRE1 was suppressed by the irregular ridge site of miR-124.

If MAPRE1 is an irregular ridge target of the miR-124 and is a very important target, the neuronal differentiation resulting from the inhibition of the irregular ridge target of miR-124-BS should be further promoted due to the decreased expression of MAPRE-1. Therefore, to confirm this point, two siRNAs capable of inhibiting MAPRE1 were prepared, and experiments were also performed to transfect miR-124 together with N2a cells and to show cytomorphological changes (FIG. 6C). As a result, it was observed that the number of neurite branches increased significantly in the experimental group expressing the siRNAs for miR-124 and MAPRE1, compared to the control group expressing only miR-124, which used MAPRE1 siRNAs of two different sequences. The same was observed in the experiment (FIG. 6C). At this time, if RNA was introduced into the cells in the same manner as in the previous example, the morphology was observed while cell culture for 48 hours thereafter.

Through this, it was confirmed that the short and branched neural differentiation induced by miR-124-BS is a biological function that can be at least partially inhibited the expression of the MAPRE1 gene through the non-normal ridge target site of miR-124.

Example 7 Analysis of Inhibition Tendency of MiR-124 Irregular Uplift Target Genes upon Incorporation of miR-124-BS at the Transcript Level

Genes inhibited via miR-124-BS in mouse neuroblastoma (N2a) ultimately induced neuronal differentiation, which produces many neurites in neuroblastoma (FIG. 5). This phenomenon is thought to be caused by the mechanism by which the beginning sequence of miR-124-BS recognizes and suppresses the irregular ridge target of miR-124, and confirmed the possibility with MAPRE1 found as a target gene (FIG. 6). In practice, however, hundreds of miR-124 irregular ridge target genes may be suppressed.

The miR-124-BS invented by the present inventors introduced miR-124 and miR-124-BS into N2a cells in order to identify only the irregular ridge target gene of miR-124 at the transcript level where all genes are expressed. RNA-Seq analysis was performed later. In this case, if the same nucleotide sequence as miR-124 or a duplex (miR-124-6pi) having a non-base (pi) number 6 based on the 5 'end was used, the modification was the 6th based on the 5' end of the miRNA. It has been reported that it does not align with the target messenger RNA at all because there is no base (Lee HS et. Al. 2015, Nat Commun. 6: 10154).

The RNA-Seq experiment was performed by converting miR into N2a cells using a non-base (NT-6pi) of the 5 'terminal 6 in the sequence of cel-67, a microRNA of C. elegans as an experimental control. -124, miR-124-BS and miR-124-6pi duplexes were delivered at 75 nM concentrations, and after 24 hours of incubation, total RNA was extracted with RNAeasy (Qiagen) kit, libraries were prepared in Otogenetics and next-generation sequencing. Next-generation sequencing was performed. Subsequently, the FASTAQ file, which is the sequence data obtained in the above experiment, was mapped to the mouse genome sequence (mm9) by the TopHat2 program, the expression value (FPKM) was obtained by the Cufflink and Cuffdiff programs, and mouse neuroblastoma cells into which the control NT-6pi was introduced ( N2a) was normalized to the results in the cells and expressed as a log change (Fold change, log2 ratio) was analyzed.

In order to analyze in the RNA-Seq results whether the amount of messenger RNA of a gene having a target site of the microRNA is inhibited by the expression of the microRNA, the normal initiation site of miR-124 (2nd to 8th of the 5 'end) Genes with a phastCons score of 0.9 or higher were selected to select those whose sequences were sequenced in 3'UTRs with 7mer) and base sequences, and inhibited these profile results depending on the expression of the corresponding microRNAs. The cumulative fraction was compared and analyzed in order of success. In addition, the miR-124 irregular bump site (nuc, 7mer) was also identified, and the inhibition rate of the gene was analyzed in the same cumulative ratio. At this time, since the genes having miR-124's irregular ridge site have regular initiation sites at the same time, it may be difficult to judge the inhibitory effect. On the contrary, we analyzed the case where there was only a regular starting site and no irregular ridge site (seed only).

In RNA-Seq sequencing of miR-124-expressed experimental group, when the fold change according to miR-124 expression was analyzed according to the cumulative fraction, the conserved normal initiation site of miR-124 was 3 '. It was confirmed that a gene (miR-124 seed) or a gene having only the corresponding site (miR-124 seed only) in the UTR was significantly inhibited compared to the distribution in the total gene (Total mRNA) (Fig. 7a, above). In contrast, it was observed that genes with non-regular bump sites of miR-124 were significantly inhibited and very weakly inhibited (FIG. 7A, bottom). However, this inhibition was not observed in cells into which the control miR-124-6pi was introduced (FIG. 7B).

In the case of expressing miR-124-BS developed by the present inventors, when miR-124-BS was analyzed, the fold change according to miR-124-BS expression was analyzed according to the cumulative fraction. The gene having 124 conserved normal initiation sites in the 3'UTR (miR-124 seed) showed some inhibitory effects, but there was no miR-124 irregular proliferation site and only the normal initiation site (miR-124 seed). Seed only) was confirmed that there is no change at all (Fig. 7c, above). However, both genes with miR-124's irregular ridge targets (miR-124 nuc) and those with only those targets (miR-124 nuc only) show strong and effective gene suppression. (Figure 7c, below). When miR-124-BS is expressed in FIG. 7C, genes having miR-124 seed only (miR-124 seed only) are not inhibited, and thus do not inhibit miR-124 normal start site at all. It can be concluded that miR-124-BS somewhat inhibited miR-124's initiation site target, but in the presence of a regular site of miR-124, the miR-124's irregular ridge site was also present. It is guessed that it shows the inhibitory effect even a little.

As a result of the above example, it was found that miRNA at the transcript level inhibits the inhibition of the existing regular initiation target gene very strongly and efficiently, but weakly inhibits the irregular ridge target. At the cadaveric level, it was confirmed that miRNA suppressed the expression of the irregular ridge target, but not the existing normal initiation sequence.

[Example 8] Confirmation through flow cytometry of cell cycle arrest induced by miR-122-BS in human liver cancer cell line (HepG2)

Based on the observation of miR-124's neuronal differentiation induced by miR-124's nonnuclear target, an experiment was performed on the biological function of other microRNA's nonnuclear target. miR-122 has 5'-UGG AGU GU-3 'as the base sequence, and miR-122-BS, which is the base sequence of siRNA nucleotide sequence with its irregular nucleus target site, repeats U 6 more times In this case, the 5'-UGG AGU U GUG ACA AUG GUG-3 'sequence consists of 19 bases at the 5' end, and the 20th and 21st bases are dt (Deoxy Thymine Nucleotide). Is done. Particularly, in the duplex structure, the dt portion was composed of a duplex of guide and carrier strands so as to constitute two nucleotide overhangs at the 3 'end. At this time, the carrier strand is completely complementary base bond with the guide strand, the 5 'terminal based on both the first and second modified 2'OMe, and the base sequence was configured to include two dt (Fig. 8a). The guide strand and carrier strand of miR-122-BS were chemically synthesized and separated by HPLC from Bioneer, and prepared as a duplex of the guide strand and the transport strand according to the method provided by the company.

Thus prepared miR-122-BS (Fig. 8a) was introduced into the human liver cancer cell line (HepG2) and then the cell cycle change was observed through flow cytometry. In this experiment, the control group NT-6pi, miR-122, miR-122-BS duplex at 50nM concentration in the HepG2 using RNAiMAX (Invitrogen) reagent, transfection (transfection) to cells, and after 24 hours of incubation, no coder sol (nocodazole) using the 100ng / ml was treated for 16 hours with G2 / M (cleavage preparation machine / beamsplitter), a sync (synchronization) for a next cell, Muse ^TM cell Cycle Kit (Catalog No. MCH100106, Milipore) The cell cycle was analyzed by Muse Cell Analyzer (Milipore) according to the experimental method provided by the company.

As a result, compared with the cell cycle analysis of the control group delivered with NT-6pi, miR-122-BS experimental group increased the G0 / G1 phase cells by 10-fold from 10.7% to 24.3%. It was confirmed that the cell cycle arrest of HepG2 was increased (FIGS. 8B-C). This increase in G0 / G1 phase was not observed in the cells to which miR-122 was delivered. Subsequently, the G2 / M phase cell distribution of human liver cancer cell line (HepG2) to which miR-122-BS was delivered decreased from 83.4% to 67.3% compared to the control group. It was not observed (Figure 8b, c).

 In this way, miR-122-BS exhibits a biological function of inducing cell cycle arrest in human liver cancer cell lines through the regulation of miR-122's irregular nuclear ridge target expression, which is completely different from the biological function that miR-122 acts on. It could be observed that it is a function.

Example 9 Confirmation of Myofibroblast Function Induced by miR-1-BS in Skeletal Muscle Cells ( C2C12) and Cell Death Function by miR-155-BS

Since miRNA-BS observed a new biological function that differs from the function of inhibiting the expression of miRNA's normal initiation target, miR-1 is expressed in muscle cells to play an important role in observing how this function appears in muscle cells. , miRNA-BS was applied to miR-155.

First, miR-1 is a muscle tissue-specific microRNA, which has been reported to function in myocyte differentiation (Chen J et al, Nature genetics, 2006, 38 (2): 228-233), miR-1- BS is skeletal muscle cell owner After expression in C2C12, cell morphological changes (Fig. 9a) and the expression of gene markers expressed at differentiation (Fig. 9b) were investigated. At this time, miR-1-BS was synthesized and transfected into the cells in the same manner used in the previous example, and 10% FBS (fetal bovine serum), 100 U / ml penicillin, and 100 μg / Using Dulbecco's modified Eagle's medium (Invitrogen) supplemented with ml steptomycin, this was followed by growth condition (GM) conditions (FIG. 9A, above) and deprivation media condition (DM) after which FBS was removed. ) Was observed by dividing the culture into a further 48 hours (Fig. 9a, below). The cells were then treated with 4% para-form aldehyde (PFA) to immobilize the cells and react with myosin 2 heavy chain protein expressed in differentiated muscle cells with a primary antibody (MF20: Developmental studies hybridoma bank). Thereafter, the cells were stained with a mouse secondary antibody (ab150105) attached with Alexa 488 fluorescent material to observe cell morphology and differentiation (FIG. 9A).

As a result, the mouse skeletal muscle cells (C2C12) MF20 increased the number of differentiated myocytes when the miR-1-BS was expressed in the growth culture condition (GM), compared to the cells to which NT or miR-1 duplexes were delivered as control. As a result of staining, it could be confirmed. In the deprivation media condition (DM), which reduced serum in culture, all differentiation into muscle fibers was observed, but miR-1-BS was more effective than skeletal muscle cells expressing NT, the control group. A bold morphology could be observed (Figure 9a). This may be a result of miR-1-BS inducing myocyte differentiation faster in proliferation culture conditions. This differentiation of muscle cells can be molecularly confirmed by markers the amount of expression of genes that increase expression during differentiation, so that the amount of sk-actin and Myogenin expression as a primer specific for the messenger RNA, RT (Reverse transcription) -PCR was performed to detect the b-actin messenger RNA with no difference in expression level even during differentiation (Fig. 9b). As a result, it was confirmed that the amount of transcription of sk-actin and Myogenin increased when both miR-1 (1) and miR-1-BS (1BS) were expressed. In the case of Myogenin, the amount of miR-1-BS was increased by miR-1-BS. increased more than miR-1

miR-155 is a microRNA that inhibits myocyte differentiation (Seok H et. Al, JBC, 286 (41): 35339-46-2011), and miR-155-BS is effective in the same way as described above for miR-1. Observation was made through differentiation of skeletal muscle cells (C2C12) (FIG. 9C). As a result, after expressing the control group NT, miR-155, miR-155-BS, no morphological differences of cells were observed in the growth culture conditions (GM), but the deficient culture conditions (inducing differentiation of muscle cells) In DM), the control group is differentiated and suppressed when miR-155 is expressed, but when the miR-155-BS is introduced, the differentiation disappears. The immunostaining experiment through the myosin 2 heavy chain protein It was confirmed through (Fig. 9c). In particular, when the C2C12 cells expressing miR-155-BS were examined in detail, it was observed that miR-155-BS induced cell death. In contrast, no cell death was observed in myocytes to which miR-155 was delivered (FIG. 9C, bottom). In order to investigate this in detail, C2C12 cells expressing the corresponding microRNAs were grown in proliferation culture conditions for 48 hours, and then cell death was examined by applying the same flow cytometry as the method of Example 3 (FIG. 9D). As a result, it was confirmed that the experimental group delivered miR-155-BS increased by about 1.5-2.5 times more apoptosis than the control group (Fig. 9d, e).

Through the above examples, miR-1-BS promotes the differentiation of skeletal muscle cells to induce coarse muscle fiber differentiation, miR-155-BS induces the death of skeletal muscle cells, and this function is the existing muscle We found that miR-1's ability to contract cells and miR-155's ability to inhibit muscle cell differentiation were different.

Example 10 Discovery of an Irregular Target Site That Allows Wobble Sequences at the Initiation Site of a MicroRNA in an Ago HITS CLIP Assay

As a method to analyze the target of the microRNA at the transcript level, an experimental method called Ago HITS CLIP has been developed and widely used (Nature, 2009, 460 (7254): 479-86). Ago HITS CLIP experiments induce a covalent bond between RNA and agon nut protein in the cell by irradiating UV to a cell or tissue sample, and use the antibody that specifically recognizes the agon nut in the RNA-agon nut complex thus produced. After separation by immunoprecipitation, RNA is analyzed by next-generation sequencing. The sequencing data thus obtained can not only identify the target mRNA of the microRNA through bioinformatics but also accurately analyze the binding position and sequence (Nature, 2009, 460 (7254): 479-86). . Ago HITS-CLIP was first applied to the cerebral cortical tissues of mice to map the targets of microRNAs in the brain tissues. These multiple Ago HITS-CLIP assays have revealed the location of microRNA binding in multiple tissues, particularly similar to the initiation of microRNA binding in these target sequences, but other forms of non-normal binding targets in the target mRNA sequence. Many were found (Nat Struct Mol Biol. 2012 Feb 12; 19 (3): 321-7).

It has been observed that some of the known nonnormal binding laws exist as wobble through the G: U nucleotide sequence in addition to the known base sequences. Wobble nucleotide sequences are found in certain RNAs, unlike conventional nucleotide sequences, and it has been suggested that the well-known G: U wobble can also be arranged in non-normal targets of microRNAs. However, the binding was weak and not frequently observed. Especially, in case of G: A wobble, it was possible to form a nucleotide sequence in specific RNA, but it was not investigated at all. However, in the case of the microRNA initiation sequence, generally weak G: A nucleotide sequence may be relatively important because the free energy of the nucleotide sequence is structurally stabilized by the agon nut protein. Paying attention to the following analysis.

First, data from Ago HITS-CLIP on gray matter of post-brain tissue to determine whether a wobble nucleotide sequence including G: A is present in microRNA targets in human brain tissue (Boudreau RL et al, Neuron, Vol. 81 (2), 2014, 294-305) (FIG. 10A). The analysis was based on the method of developing Ago HITS-CLIP (Nature, 2009, 460 (7254): 479-86), from the sequenced FASTQ file to the Bowtie2 program. The sequencing results were also arranged in human microRNA sequences, and 20 kinds of microRNAs (top20 miRNA: FIG. 10B) that frequently bind agon nut proteins in the brain tissues were also analyzed. Finally, the canonical target site having a complementary sequence from the second to the eighth end of the corresponding sequence was examined for the top20 miRNA thus identified (FIG. 10b). At this time, it was observed that a large number of normal initiation target sites were found at the microRNA binding sites (FIG. 3B, median value 1292.5 sites, margin of error +/− 706.62). Subsequently, it was examined in the Ago HITS-CLIP data whether there was an irregular target site capable of binding to G: U or G: A wobble nucleotide sequences in the starting nucleotide sequence of such microRNA. At this time, as a control for the analysis, it was analyzed using a target sequence that cannot complementarily arrange bases due to the same base sequence as the microRNA seed base sequence.

As a result of the analysis, both the targets with G: A wobble (median 1119.5, error range +/- 526.48) and the targets with G: U wobble (median 995 site, error range +/- 523.22) were both control (median 891 Site, margin of error +/- 410.71), and particularly showed that the microRNA target site with G: A wobble is present about 1.1 times more than G: U wobble (FIG. 10b). MiR-124, which is specifically expressed in brain tissue, has two Gs at the end and 4th and 5th from the 5 'end, thereby enabling G: U and G: A wobble sequences. 10c). Therefore, the G: A, G: U wobble nucleotide sequence by G at this specific position was analyzed (Fig. 10d), the most common initiation target site (seed) of miR-124 was found (1,992). However, it was found that the fourth made G: U wobble (1,542), and then the G: A wobble (1,542). In addition, there were many target sites consisting of G: U wobbles (1,800) and G: A wobbles (1,198) in number 5 G. All the sites examined were more than the number of control groups (647). It could be confirmed (Fig. 10d). Thus, when a microRNA recognizes a target mRNA through its nucleotide sequence, the microRNA allows not only a regular nucleotide sequence through the nucleotide but also irregularly G: U or G: A wobble nucleotide sequence, and through this binding It was found that it can bind to the target mRNA. Target recognition of microRNAs through these G: A wobble bases is a novel result that has not been reported, and thus it can be seen that it is recognized as an irregular target in several microRNAs as observed in the above examples.

Through the above embodiments, there is a case in which the irregular target recognition of the microRNA allows the G: A wobble sequence in the initiation sequence, through which many microRNAs bind to the messenger RNA of the target gene and inhibit the expression of the gene. It is believed to be.

Example 11 Development of mIRNA-GU Recognizing Non-regular G: A Initiation Array Site of MicroRNA and Confirmation of miR-124-G5U Enhancing Neuronal Cell Death Upon Application of miR-124

In Example 10, it was found that the irregular target recognition of the microRNA may allow a G: A wobble sequence to the initiation sequence, which may be referred to as an irregular G: A initiation sequence site, and may be complementarily arranged. The miRNA-GU sequence of the novel RNA interference inducing substance was invented as follows. Applying this sequencing technique to miR-124, miR-124 has an initiation sequence of 5'-AAGGCAC-3 'and the non-normal GA sequence initiation targets are G: A at base 4 and G at base 5: Wobble nucleotide sequence is possible, and thus, when the G: A wobble sequence is replaced with the sequence of miRNA-GU, which is a form of replacing the existing complementary nucleotide sequence, miR-124-G4U (5'-AAUGCAC-3 '), Can be changed to miR-124-G5U (5'-AAGUCAC-3 '). The modified miR-124-G4U and miR-124-G5U combine the irregular target sites that miR-124 weakly binds and recognizes in the form of G: A wobble arrays in a complementary arrangement. Can display the function of an irregular G: A initiation array target.

Therefore, in order to examine the biological function of the miRNA irregular GA sequence initiation target, an experiment was performed by applying the irregular GA sequence initiation target to miR-124. To this end, NT (described as N2a in FIG. 11B), miR-124, miR-124-G4U, and miR-124-G5U were previously introduced into the neuroblastoma N2a as a control group according to the same method as in the previous example. Observation was performed while incubating for 72 hours (FIG. 11B). As a result, when miR-124 is expressed, a differentiation phenomenon occurs in which long nerve branch processes are formed, but in the experimental group to which miR-124-G4U and miR-124-G5U are delivered, no differentiation occurs (FIG. 11b). Since the neurodifferentiation function of the existing miR-124 was not observed when it was changed to miRNA-GU, a change in the degree of cell death was observed in the Sh-sy-5y cell line, a human neuroblastoma. Flow cytometry at this time was similar to the method used in Example 9, using a Muse Annexin V and Dead Cell Assay Kit (millipore's company), was performed through the Muse 'Cell Analyzer (Milipore). As a result, the expression of miR-124-G-5U was confirmed to increase cell death more than two times compared to the control group (Fig. 11c), which was subdivided into early apoptosis or late apoptosis. In the analysis group, the miR-124-G-5U group was significantly increased in both early and late apoptosis groups compared to the miR-124 group. (FIG. 11D). In the experimental group to which miR-124-G-4U was delivered, late apoptosis was significantly suppressed compared to the experimental group to which miR-124 was delivered (FIG. 11 c, d). Could be (FIG. 11 b).

Based on this, miR-124-regulated GAR initiation target inhibition by miR-124-GU was confirmed that the ability of neuronal differentiation induced by miR-124 disappears, but instead exhibits other biological function, cell death control function. .

Example 12 Promoting Cell Division of Neuroblasts Induced by miR-124-G4U

In the previous example, miR-124-G4U loses neuronal cell differentiation capacity and does not show any function even in cell death, and thus cell division was examined. In other words, control NT, miR-124, miR-124-G4U, miR-124-G5U were introduced into N2a, a neuroblastoma cell, respectively, in the same manner as in the previous example, and then flow cytometry for cell division proliferation phenomenon. Was carried out (FIG. 12A). In this case, the cells were treated using the Muse Ki67 Proliferation Kit (millipore, Inc.) according to the experimental method provided by the manufacturer, and analyzed by cell division using the Muse Cell Analyzer (Milipore). This method quantitatively analyzes the number of cells with increased cell division proliferation by measuring the number of cells stained with Ki67. When miR-124 is expressed in N2a cells, the cells differentiated as compared to the control NT, resulting in a decrease in the intensity of Ki67 staining. However, the miR-124-G4U-delivered experimental group had slightly increased Ki67-stained cells from 61% to 65% compared to the control group (NT). In comparison, miR-14-G5U was no difference compared to the control group.

In addition, flow cytometry was performed using the sh-sy-5y cell line, a human neuroblastoma, to investigate the effect of miR-124-G4U on the cell cycle (FIG. 12B). In this case, the function of miR-124-G4U and miR-124-G5U was analyzed by Muse Cell Analyzer (Milipore) using Muse ^TM Cell Cycle Kit (Catalog No. MCH100106, Milipore). . As a result, cell cycle arrest was observed in which the number of cell cycles in miR-124 increased in G0 / G1, but miR-124-G4U and miR-124-G5U were miR-124-induced cells. The cleavage arrest phenomenon disappeared and the cell cycle was observed to rotate as in the control group.

Through the above examples, it was found that miR-124-G4U has a function of increasing cell division proliferation of neuroblasts among RNA interference-inducing modified sequences that inhibit miR-124 irregular GA array initiation targets.

Example 13 Confirmation Using a Fluorescent Protein Reporter that miR-1 Can Inhibit Expression of an Irregular GA Array Initiation Target Gene in a Cardiomyocyte Cell Line

In the above example, after discovering that a microRNA can irregularly bind to a target mRNA by allowing a G: A wobble sequence in the initiation sequence, and that it may have new functions, the binding actually occurs in the target gene. Reporter experiments were conducted to determine if inhibition could occur at the cellular level. At this time, the experiment was conducted on miR-1, which is known to be expressed in muscle cells such as cardiomyocytes, h9c2, in particular, focusing on the G present in the 7th from the 5 'end of miR-1, G: A wobble nucleotide sequence was performed (FIG. 13). Gene suppression by microRNA was used to construct and use a fluorescent protein expression reporter system to more accurately measure at the individual cell level (FIG. 13). Fluorescent protein expression reporter system is composed of two colors of fluorescent protein expressed in one vector, in which SV40 promoter expresses green fluorescent protein (GFP) and its 3'UTR (3'untranslated region) By arranging several target sites of microRNA in the region, it is possible to measure the gene suppression effect by microRNA through the intensity of green fluorescent protein, while at the same time red fluorescent protein (RFP) is a TK promoter The green fluorescence signal was normalized to the intensity of the green fluorescent signal (FIG. 13A, above).

In this fluorescent protein expression reporter vector, the irregular target site is complementary to the bases from the 5 'end of the miR-1 to the 2nd to 8th bases, and the G: A wobble sequence to the 7th base (7G). After the reporter was prepared by inserting (A-site) (GFP-7G: A site), it was experimentally confirmed whether it was inhibited by miR-1. In this experiment, 500ng GFP-7G: A site reporter vector and 25uM of miR-1 were co-transfected into cardiomyocyte H9c2 using lipofectamine 2000 (Invitrogen) reagent according to the protocol provided by the company. After 24 hours, each fluorescence signal was measured by flow cytometry through Attune NxT of Life Technologu. As a result, miR-1 expression of the 7G: A site of miR-1 was very efficiently inhibited (FIG. 13A, middle), compared with cells into which control nucleic acid (cont; NT) was introduced (FIG. 13A, left). Confirmed. In other words, according to the expression level of the two fluorescent proteins in the cardiomyocyte cell line h9c2 in four parts (Q5-1, Q5-2, Q5-3, Q5-4), the control nucleic acid in the case of miR-1 introduced cells, the decrease in the case where the introduction of more than ³ of the 10 century, red fluorescent protein and 10 ^3rd century the cellular distribution of the visible green fluorescent protein 59.51% (control:: Q5-3) 41.80% (Q5-3 miR-1) from 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 without the introduction of miR-1. This can be expected to be inhibited via G; A wobble nucleotide sequence with reporter target mRNA by miR-1 already expressed in h9c2 cells. To confirm this point, miRIDIAN microRNA Hairpin inhibitor (miR-1 inhibitor, FIG. 13A, right), which can inhibit the expression of miR-1 in h9c2 cells, was purchased from Dharmacon and transfected into cells at a concentration of 50 uM. It was. These results, the distribution of the visible (Fig. 13a, left side of the figure) miR-1 inhibitor one case (Fig. 13a, right figure), ten ^3rd century red fluorescent protein and green fluorescent protein of 10 ³ intensity of introducing than in control cells Increased by 81.56% (miR-1 inhibitor: Q5-3), it was confirmed that miR-1 present in the cell can inhibit gene expression through the 7G: A site of miR-1 (Fig. 13a) .

In addition, the control group (GFP-no site) without the microRNA target site in the fluorescent protein expression reporter and GFP-7G: A site, which is a reporter containing the 7G: A site of miR-1, were introduced into H9c2 cells to compare their activities. As a positive control, it was confirmed by quantitative comparison with the case where miR-1 was introduced together (FIG. 13B). In this analysis, the red fluorescent protein (RFP) value measured by the flow cytometer was divided into sections, and the green fluorescent protein (GFP) value was averaged and converted into a logarithmic ratio, where the green fluorescence of the control GFP-no site was obtained. The protein level was set to 1 (relative log GFP) and calculated relative. At this time, when there is a site that is complementary base sequence through the G: A wobble (GFP-7G: A site) for mR-1 and G base No. 7, especially red fluorescent protein of 1.5-3 value In the section showing the expression (log RFP), it was confirmed that the expression of the green fluorescent protein (relative log GFP) through the G: A wobble (relative log GFP) is about 10% lower than 1. This inhibition was observed to be the same as the pattern of suppressing the expression of green fluorescent protein (relative log GFP) by 40% -10%, especially in the expression of red fluorescent protein (log RFP) in the 1.5-4 section (log RFP). .

In order to confirm that the results observed in the above examples are not affected by the difference in the intensity of the different promoters used in the fluorescent protein reporter and the fluorescence activity of the two different fluorescent proteins, the two fluorescent proteins are interchanged. P.UTA.3.0 (Lemus-Diaz N et al, Scientific Reports, (7), 2017), a fluorescent protein reporter, was purchased from Addgene (plasmid # 82447) and used. The reporter experiment was performed by inserting the miR-1-7G; A sequence five times in the 3 'UTR portion of the red fluorescent protein (RFP) controlled by the SV40 promoter in the p.UTA.3.0 vector and then reporting The vector (RFP-7G: A site) was constructed and the red fluorescent protein expression level was standardized using the green fluorescent protein (GFP) value expressed by the Tk promoter as a standard reflecting the degree of introduction into the cell. Modified and used (FIG. 13C, above). As a result, as in the previous example, the 7G; A site of miR-1 was inhibited by miR-1 expressed in h9c2, and the control group of the reporter without the miRNA target site (RFP-no site) and 7G of miR-1; The results of the reporter containing the A site (RFP-7G: A site) were compared and confirmed. At the same time, the positive control group observed the same phenomenon as the previous example in co-transfection with miR-1 (FIG. 4C-D).

From the above, it can be concluded that the non-normal target of the microRNA found through the Ago HITS-CLIP analysis can not only bind through the G: A wobble sequence in the initiation sequence of the microRNA, but also inhibit the expression of the target gene. Confirmed. Thus, in view of this, if the target site through the G; A wobble array can be sufficiently combined with the microRNA, and if this point can be induced historically through miRNA-GU, it may be necessary to It is thought that only the biological function of the microRNA can be utilized.

Example 14 Confirmation of Cardiomyocyte-induced Hypertrophy-inducing Function by Regulating Irregular GA Array Initiation Target Gene for miR-1 G Bases 2, 3, and 7

 This non-regular gene suppression phenomenon is miR-1 of the cardiomyocytes described in the previous example to confirm the possibility that the G: A wobble sequence can act as an atypical target of the microRNA, and may regulate biological function through miRNA-GU. The experiment was carried out mainly.

In order to investigate whether an irregular G: A wobble target has a specific biological function, miR-1 should not recognize a regular target but only the non-normal G: A wobble array target site. The miRNA-GU prepared by substituting the G with U such that the G in the starting region is arranged with A instead of C was used in the experiment. In this experiment, primary rat myocardial cells (primary rat neonatal cardiomyocyte culture) were cultured from rat heart tissue to perform physiologically similar conditions. At this time, the culture of cardiomyocytes was isolated from cardiac tissues of rats at day 1 after birth by enzymatic reaction (Cellutron neomyt kit), and cardiomyocytes were 10% FBS (fetal bovine serum), 5% HS. brdU was added to a medium prepared by mixing Dulbecco's modified Eagle's medium (Invitrogen) and M199 (WellGene) medium at 4: 1 supplemented with horse serum, 100 U / ml penicillin, and 100 μg / ml steptomycin. Cultures were distinguished from other cells having a cell cycle present in the tissue.

The primary cultured cardiomyocytes thus obtained were first identified whether myocardial hypertrophy is induced (FIG. 14a). Cardiomyocytes have the characteristic of stopping the cell cycle and increasing the size of myocardial cells, which is called myocardial hypertrophy. Myocardial hypertrophy is a cardiac disease model system that has played a role in compensating myocardial cell function, is also known as an intermediate pathology of heart disease, and is well known for changes in cell morphology and gene expression profiles. In a cellular model of myocardial hypertrophy, cardiomyocytes can be induced with an adrenal receptor agonist such as phenylephrine (PE) (Molkentin, JD et al, 1998, Cell 93 (2), 215-228). Therefore, the cultured cardiomyocytes were treated with 100 uM of phenylephrine to induce myocardial hypertrophy, thereby morphologically confirming that the size of the cardiomyocytes was increased compared to the control group (FIG. 6A, rCMC) which had not been treated with anything. (FIG. 14A, + PE). In addition, when miR-1 was introduced into primary cardiomyocytes, the size of the cardiomyocytes was observed to be reduced (FIG. 14A, upper right row). This is consistent with well-known myocardial hypertrophy 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 Gs at the initiation region, and thus miR-1 is substituted with U in order to recognize only irregular G: A wobble array target sites without recognition of regular targets. The 2nd position (miR-1-G2U), the 3rd position (miR-1-G3U), the 7th position based on the position of G ' Synthesis was carried out by applying to the position (miR-1-G7U). At this time, the substitution base sequence of miR-1 used in FIG. 14 is as follows (miR-1-G2U: 5'p-UUGAAUGUAAAGAAGUAUGUAU-3 '; miR-1-G3U: 5'p-UGUAAUGUAAAGAAGUAUGUAU-3'; miR -1-G7U: 5'p-UGGAAUUUAAAGAAGUAUGUAU-3 ') It was synthesized as a guide strand, and the carrier strand was designed by the conventional miR-1, chemically synthesized by Bioneer, separated by HPLC, and provided by the company. According to one method, a duplex of a guide strand and a transport strand was prepared and used. Transfection of the corresponding microRNA into the primary cardiomyocyte line was performed at a concentration of 50 uM using RNAimax (Invitrogen) reagent.

As a result, when miR-1-G2U or miR-1-G3U or miR-1-G7U were introduced into primary cardiomyocyte culture, the morphology of the cardiomyocytes was similar to that of the cells that induced myocardial hypertrophy (FIG. 14A, + PE). It could be observed that the larger (Fig. 14a). At this time, in order to quantitatively analyze each cell size, more than 100 cells were quantified through the Image J program (NIH), and thus miR-1 substituents synthesized to recognize only the G: A wobble array site of miR-1 ( miR-1-G2U, miR-1-G3U, miR-1-G7U) were analyzed to be 1.5 to 1.8 times larger than the control (NT) nucleic acid. This is similar to the myocardial cell size of the phenylephrine (PE) drug-induced myocardial cell hypertrophy model, miR-1 itself has been shown to reduce the cell size in the form of cardiomyocytes, thereby reducing the regular target inhibitory effect It was observed to exhibit a different function in cell morphology than target inhibition via G; A wobble (FIG. 14B). In addition, in order to confirm the myocardial hypertrophy observed in this example at the molecular level, GAPDH was tested for the expression of the ATL (atrial naturiuretic peptide) gene, which is known to increase its expression as a marker, through qPCR (Quantitative Polymerase Chain Reaction) experiment. Compared to the measured value was measured as a relative value (Fig. 14c). As a result, when miR-1 substituents (miR-1-G2U, miR-1-G3U, miR-1-G7U) synthesized to recognize only the G: A wobble array site of miR-1 were introduced into cardiomyocytes, In all, it was confirmed that the ANP expression was significantly increased by 1.2 to 1.5 times compared to the control group (NT) through four repeated experiments. This increase in ANP expression is less than that of the phenylephrine (PE) drug-treated cardiomyocyte group compared to the control group by about two times the size of the control group, but significantly reduced ANP expression when the original miR-1 was introduced. Contrary to the phenomenon, miR-1 exhibits a completely different biological function through the G: A wobble array site.

From the above, it can be finally seen that the non-normal target inhibited by the G: A wobble configuration in the microRNA initiation region may have a biological function different from the conventional normal target recognition. These findings indicate that the function through the regular target of the microRNA is different from the function through the non-normal target, which suggests that if only the G: A wobble target of the microRNA can be suppressed, then the G: A wobble target can be suppressed. Only biological functions that occur by can be selectively controlled.

Example 15 Induction of Cardiac Hypertrophy Through miR-133-G4U Expression in Myocardial Cell Line (H9c2)

For non-regular target phenomena where the G: A wobble sequence acts on the end of the microRNA, it additionally functions in cardiomyocytes in addition to miR-1, as discussed in the previous example, to confirm its biological function in cardiomyocytes. The miRNA-GU was applied to miR-133, which is known to be. miR-133 regulates myocyte development and disease pathology and is known to have 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 identified miR-133-G4U (5'-UUUUGUCCCCUUCAACCAGCUG-3 '), which is an interference-inducing nucleic acid that specifically inhibits target recognition through the fourth G of the 5' end of the irregular GA array target site of miR-133. ) Was synthesized by Bioneer as in the above example, and delivered to the cardiomyocyte line h9c2 using RNAiMAX reagent (Invitrogen) as a duplex of 50 nM concentration to observe the change in cell morphology (FIG. 15A). As a result, expression of miR-133-G4U increased the size of H9c2 cells compared to the control group (NT) (FIG. 15A), and quantitatively analyzed the size of 100 or more cells through the Image J (NIH) program. , It was confirmed that about 2 times increased significantly (Fig. 15b).

 Thus, the deterioration in the expression of miR-133's irregular GA sequence initiation target caused by miR-133-G4U induces cardiac hypertrophy, which is inferred as a new function different from the normal initiation target inhibition function of miR-133 that inhibits cardiac hypertrophy. can do.

Example 16 Inhibition of Expression of the Irregular GA Sequence Initiation Site Gene by miR-122 in Liver Cancer Cells Using Luciferase Reporter

In the previous example 10, the discovery that miRNAs irregularly bind to the G: A wobble sequence initiation site led to the invention of miRNA-GU that specifically recognizes only these irregular target bindings, which miR-124, miR- When applied to 1, miR-133, it was observed in Examples 11, 12, 14, 14 that the effect is different from the existing function. In addition, in order to confirm whether it is possible to actually inhibit the expression of a target gene having a non-regular G: A wobble sequence initiation site in the microRNA, it confirmed the function in myocardial tissue cell line targeting miR-1 in Example 13 It was. In addition, the use of a luciferase reporter system to determine whether the miR-122 specifically expressed in liver tissue and liver cancer cells can inhibit the expression of the non-regular G: A wobble sequence initiation target gene (FIG. 16).

MiR-122 that expresses and functions specifically in liver cancer or liver tissues has a 2, 3, or 5, terminal basis based on its initiation sequence (5 'initiation base 1-8 bases: 5'- UGG AGU GU-3') The fifth, fifth, and seventh G's have a G: A wobble array. Therefore, first of all, in order to measure the inhibition efficiency of the corresponding target site that the second G can be recognized irregularly through the G: A wobble configuration, the IC50 (Inhibitory concentration 50) method was performed in the same manner as in Example 2. ) Was measured and compared with the normal initiation site (Fig. 16a).

In order to detect the inhibition of expression of a non-regular GA sequence initiation target capable of GA base arrangement with the G base of miR-122, the luciferase reporter vector for performing the experiment was subjected to a nonnormalization of the G base of miRNA-122. The GA sequence initiation site (2G: A) sequence (5 'base of bases 1-9: 5'-CAC ACU CAA-3') is 3 'of the Renilla luciferase gene in the psi-check2 (Promega) vector. The UTR (3'untranslated region) was prepared by introducing five consecutive arrays. At the same time, the luciferase reporter vector for the normal initiation site (bases 1-9 of the 5 'initiation: 5'-CAC ACU CCA-3') was also identically produced.

With the luciferase reporter vector thus constructed, the target gene having a normalized seed site and a non-normal GA arraying site (2G: A site) through the second G at the 5 'end at various concentrations of miR-122 was obtained. The inhibition of expression was measured by the change in the activity of the corresponding luciferase reporter and the value of IC50 (inhibitory concentration 50) showed that miR-122 was about 0.5 nM for the normal initiation target site, and the irregular GA array initiation site (2G: A site) was measured at about 7 nM, and miR-122 inhibited the expression of genes having a non-regular GA sequence initiation site (2G: A site), and the efficiency was lower than that of normal initiation site. (FIG. 16A). In addition, when the concentration at which gene expression inhibition was started was examined with a luciferase reporter in a non-normal GA array initiation target, it was confirmed that luciferase enzyme activity was inhibited at a concentration condition of 1.5 nM or more (FIG. 16A). This showed that the miR-122 inhibited the expression of the normal initiation target site by about two times the inhibition efficiency.

This experiment synthesizes duplexes of miR-122 by the same method used in the above examples, and uses the Lipofectamine 2000 reagent with the corresponding psi-check2 vector (50 ng) at various concentrations (0, 1, 5, 10, 25 nM). (Invitrogen) was co-transfected into approximately 10,000 hepatocellular carcinoma cell lines (Huh7, KCLB: 60104) according to the manufacturer's protocol, and incubated in 96 wells. Lucifer was performed in the same manner as in Example 2. Laze activity was measured and performed.

After confirming that miR-122 inhibits the expression of the non-regular GA sequence initiation site gene, the Huh7 cell line, which is a hepatic cancer cell with miR-122, is used to confirm that it is equally regulated by miR-122 present in the cell. Luciferase reporter experiments were performed on the irregular GA arraying initiation site (2G: A) caused by the 2nd G and the 3rd G on the 5 'end (Fig. 16b). In order to proceed with these experiments, the luciferase reporter vector (luc-3G: A site) for the irregular GA array initiation site (3G: A), which is caused by the third G at the 5 'end, the second and third at the 5' end. Luciferase reporter vector for the nonnormal GA sequence initiation site (luc-2G: A, 3G: A site) caused by the first G, a luciferase reporter vector (luc that measures completely complementary to the sequence of the entire miR-122) -PM site) was constructed in the same manner as described above and with the luciferase reporter vector (luc-2G: A site) for the non-normal GA array initiation site (2G: A) caused by the second G at the 5 'end. The experiment was performed by measuring the activity of luciferase after introduction into the liver cancer cell line together (FIG. 16B).

As a result, when the luc-PM site vector was introduced into the cells in Huh7, which is known to have miR-122, the activity was reduced by about 50% compared to the control in which the luciferase vector itself was introduced. It was confirmed that 122 exists, and at the same time miR-122 is the third irregular GA array initiation target reporter (Luc-3G: A), the second and third irregular GA array initiation target reporter (Luc-2G: A, 3G: A) It was confirmed that the activity of. In addition, to determine whether this inhibitory effect was induced by miR-122, the expression inhibitor of miR-122 (hsa-miR-122-5p inhibitor, IH-300591-06-0010) was purchased from Damacon Corporation and treated with 50 nM. At this time, it was confirmed that all such suppression phenomenon disappeared.

Through the above examples, miR-122 was found to be able to inhibit the expression of the 2nd non-regular GA sequence initiation target on the 5 'end, although weaker than the normal initiation site, and also naturally in Huh7, a liver cancer cell. Existing miR-122 inhibited the expression of the 3rd non-regular GA sequence initiation target gene on the 5 'end and the 2,3 non-regular GA sequence initiation target gene. In particular, it was confirmed that the inhibitory effect made in Huh7, a hepatic cancer cell, is controlled by miR-122 by disappearing by expression inhibitor treatment of miR-122 (FIG. 16B). Thus, it can be seen that miR-122 has a regulatory function of inhibiting the expression of the non-regular GA sequence initiation target gene.

Example 17 Confirmation of Cell Migration Inhibition of Hepatocellular Carcinoma Cells by Inhibiting Expression of Specific Irregular GA Arrangement Initiation Target Gene of miR-122

In order to examine the hepatocellular intracellular function of miR-122's irregular GA sequence initiation target, after applying miRNA-GU which complementarily recognizes and inhibits the irregular GA sequence initiation target site, it is introduced into hepG2, a liver cancer cell, Wound healing assay was performed to examine how the ability of cell migration, which is important for cancer metastasis, to change among liver cancer cell properties.

The wound healing assay in hepG2, a hepatocellular carcinoma cell line, was first complementarily recognized by the second irregular GA array initiation target, the third irregular GA array initiation target, and the second and third irregular GA array initiation targets at the 5 'end of miR-122. MiRNA-GU was synthesized into the corresponding sequence and introduced into hepG2 cells by the same method as in the above example, and after 24 hours of incubation, scratches were applied to the cell layer using a 1000ul tip. The cells were cultured for up to 48 hours, and the movement of cells in each experimental group was observed by comparison with the introduction of NT-6pi as a control group. 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 the cell migration in which the formed flaw was almost filled during the cell culture for 48 hours. However, in the experimental group delivered miR-122-G2U and miR-122-G2,3U siRNA, this cell migration was inhibited, and this inhibition was the strongest in the experimental group delivered miR-122-G2,3U siRNA. (FIG. 17A). As a result of quantitative measurement, it was confirmed that the miR-122-G2U, miR-122-G2, 3U siRNA was inhibited 2-3-fold cell migration compared to the control (Fig. 17b). Correspondingly, the quantitative analysis was carried out by observing the cell migration of miR-122 in the form of cells, and then analyzed the observation results by using the Image J program (NIH). Additionally, as a result of experiments on miR-122-G3U siRNA-induced cell migration, cell migration inhibition function could not be observed by miR-122-G3U (Fig. 17C-D).

Based on this, inhibition of irregular GA sequence initiation target of miR-122 inhibits the migration of hepatocellular carcinoma cells, which is the GA sequence of base 2 acts preferentially, and additionally, when GA sequence of base 3 is additionally added, It can be seen that the enhancement of migration inhibition function is induced.

Example 18 Confirmation of Increase in Cell Cycle Arrest Due to Regulation of Non-regular GA Arrangement Initiation Target by miR-122-G2,3U

Cell migration is closely related to cell division, especially in cancer cells (Cancer research, 2004, 64 (22): 8420-8427). Therefore, flow cytometry was performed in the same manner as in Example 12 to determine whether inhibition of hepatic cancer cell migration induced by miR-122-G2,3U is related to cell cycle regulation. At this time, NT-6pi, miR-122, miR-122-G2, miR-122-3U, miR-122-G2, 3U were delivered to HepG2, a liver cancer cell line, and the cell cycle of each cell was measured. In the G2,3U-delivered experimental group, the percentage of cells with G0 / G1 distribution increased from an average of 52% of the control group (NP-6pi) to 68%, and the cell distribution of the G2 / M phase was significantly lower. (FIG. 18). However, miR-122, miR-122-G2U, miR-122-G3U did not observe a significant difference compared to the control. At this time, the cell cycle assay was performed by transferring the synthesized siRNA duplex to HepG at a concentration of 50 nM, incubating for 24 hours, and using a Muse Cell Cycle Kit (Catalog No. MCH100106, Milipore) according to a protocol provided by the company. Experiments were performed and measured with a Muse Cell Analyzer (Milipore).

Therefore, it was confirmed that the cancer cell function controlled by miR-122-G2,3U lowers cell division (G2 / M) and induces cell cycle arrest (G0 / G1). The results can be interpreted that miR-122 recognizes the irregular GA sequence initiation site by simultaneously G: A wobble arrangement of both the second G and the third G based on the 5 'end, thereby preventing the progression of the liver cancer cell cycle.

Example 19 Observation of Induction of Cell Cycle Steady State by Regulation of Non-regular GA Array Initiation Targets by miR-122-G2,3U and miR-122-G2,7U

The function of miR-122 on the irregular GA array initiation target was found in Example 17 that miR-122-G2,3U inhibits the migration of hepatocytes, in particular the expression of miR-122-G2,3U. Inhibition of the cell migration induced by the effect of maximizing the G 3 base when the regulatory effect of the G base is added to the biological function of the regulation of the non-regular GA sequence initiation of G base 2 (Fig. 17). Therefore, in order to determine whether the G bases 2, 3, and 7 of miR-122 alter the biological function of non-regular GA sequence initiation target inhibition, miR-122-G7U, miR-122-G3,7U, miR-122 Experiments were conducted to further confirm cell cycle regulation function of -G2,7U siRNA.

At this time, hepG2, a liver cancer cell line, was used in the same manner, and prepared in the same miRNA-GU duplex as in the previous Example 18, and transfected into cells at a concentration of 50 nM. However, in this cell cycle observation, after 24 hours of incubation, nocodazole was treated at 100ng / ml for 16 hours to synchronize HepG2 cells with G2 / M (dividing ready / dividing), and then how many The amount of cells in cell cycle arrest at G0 / G1 was measured with a Muse Cell Analyzer (Milipore) flow cytometer as cells stopped at G2 / M (FIG. 19).

As a result, miR-122-G2U showed no difference in cell count in G0 / G1, which is cell cycle stationary, compared to NT-6pi, but miR-122-G2,3U siRNA, miR-122-G2,7U. When regulating the non-regular GA array initiation target site attached to the second G, it was confirmed that the ratio of cell cycle stationary state (G0 / G1) was increased (FIG. 19A). But miR-122-G3, 7U did not show a significant increase (Fig. 19a). In the quantitative analysis, the experimental group delivered miR-122-G2,3U siRNA increased the rate of cell cycle arrest (G0 / G1) more than about 2 times compared to the control group, followed by miR-122-G2, In the 7U experimental group, it was observed that the ratio of the cell cycle stationary state (G0 / G1) was about 1.5 times higher (FIG. 19B).

Based on this, it was confirmed that the cell cycle arrest state can be increased through the regulation of the irregular GA sequence initiation target by miR-122-G2,3U siRNA and miR-122-G2,7U siRNA.

 Example 20 Observation of Induction of Cell Cycle Steady State Induced by Regulation of Irregular GA Arrangement Initiation Targets by let-7a-G2U and let-7a-G2,4U

A representative microRNA that acts as a tumor suppressor is the let-7 family. It is reported that the function of regulating the developmental process of the pretty little nematode (Nature, 2000, 403 (6772): 901-906), then suppressed expression in various cancer cells (Cancer Res., 2004, 64 (11): 3753- 3756) and anticancer function through control of tumorigenesis (Cell, 2005, 120 (5): 635-647; Genes Dev. 2007, 21 (9): 1025-1030), It has been studied as an important gene with the possibility of cancer diagnosis and anticancer drug development. In this regard, the present inventors conducted an experiment to investigate the biological function induced by the inhibition of let-7's non-normal GA array initiation target.

The starting sequence from the 1st to 9th 5 'terminal of let-7a is 5'-UGA GGU AGU-3', and the G which is capable of G: A wobble sequence is 2nd, 4th, 5th, 8th Exists in The present inventors pay attention to the second and fourth G among them, modified them with miRNA-GU, synthesized them, and introduced them into HepG2 cells, which are liver cancer cell lines, and then, how the cell cycle was affected in Example 19 was carried out. The experiment was carried out in the same manner. The base sequence for let-7 synthesized at this time 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 GUA GGU UGU AGU G UU-3 '; let-7a-G2,4U: 5'- U UAU GUA GUA GGU UGU AUA G UU -3 ').

As a result, let-7a did not show any difference compared to the control group NT-6pi, but let-7a-G2U and let-7a-G2,4U increased the cell cycle arrest state at G0 / G1, let-7-G4U could be observed to increase the amount of cells in G2 / M rather quickly to cycle the cell cycle (FIG. 20A). Quantification of this in 4 replicates showed that in the let-7a-G2U experimental group, the distribution of G1 / G0 cells increased about 1.5 times compared to the control group (Nt-6pi), and the distribution of G2 / M cells decreased. Was observed (Figure 20b). Through this, inhibition of expression of let-7a's second non-regular GA sequence initiation target was confirmed to induce cell cycle arrest of HepG2 cells. In the case of G base 4 at the beginning of let-7a, the single GA sequence (let-7a-G4U) proceeds rapidly to the G2 / M state while reducing the induction of cell cycle arrest of HepG2. Sol drug treatment resulted in high cell numbers. In addition, when 2 and 4 simultaneously arranged GA (let-7a-G2,4U), HepG2 induced cell cycle arrest. This cell cycle arrest was not observed in HepG2 cells to which let-7a was delivered (FIG. 20 b). Thus, inhibition of the expression of let-7a at the 2nd and 2nd non-regular GA sequence initiation targets induces the cell cycle arrest of HepG2 cells, which is dependent on the function that let-7a has in HepG2 cell regulation performed by the present inventors. Confirms a completely different function.

Through the above embodiment, let-7 serves to promote cancer cell cycle arrest by inhibiting the gene of the non-regular GA initiation sequence through G at the 5 ′ end 2, more preferably at the 5 ′ end 2 It can be seen that the effect is greatest when G and G number 4 work together in a G; A wobble arrangement. In addition, the non-regular GA initiation site through let-7's 5 ′ end G is likely to promote cancer cell proliferation, leading to cancer cell proliferation.

Example 21 Observation of Reverse Differentiation Promoting Induced by MiR-302a-4GU and miR-372-4GU Induced Target Inhibition of Non-regular GA Arrangement by OCT4 Promoter Reporter

Differentiated cells can regain their differentiation ability by artificial expression of several transcription factors. Finally, the cells that acquire omnipotent differentiation capacity such as embryonic cells are called induced pluripotent stem cells. This technique introduces four factors (Oct3 / 4, Sox2, c-Myc, Klf4) into mouse embryonic fibroblasts (MEM), and induces pluripotent stem cells (iPS) with differentiation diversity like stem cells. cell: inducible pluripotent stem cells) have been reported (Cell, 2006, 126 (4): 663-676). Similarly, it has been reported that induced pluripotent stem cells can be produced by delivery of four factors (OCT4, SOX2, NANOG, and LIN28) to human somatic cells (Science, 2007, 318 (5858): 1917-1920). This field is highly applicable in that it can generate regeneration potential through any cell including human cells through dedifferentiation process, and after the first discovery, induced pluripotent stem cell generation. Efforts have been made to increase efficiency. MicroRNA-induced pluripotent stem cells (mirPS), reported as part of the method, are also effective in inducing pluripotency. MicroRNAs such as miR-302a and miR-372 can be used alone or in four factors (Oct3 / 4, Sox2, c-Myc, Klf4) has been reported to function to reverse differentiate cells (RNA, 2008 14 (10): 2115-2124; Nat Biotechnol. 2011, 29 (5): 443-448). Therefore, the present inventors conducted an experiment to determine whether inhibition of the expression of miR-302a and miR-372 atypical GA sequence initiation targets can promote the process of dedifferentiating cells.

First, in order to monitor pluripotency induction, a plasmid containing an Oct4 promoter reported to be activated in stem cells and a green fluorescent protein expressed depending thereon was purchased (Addgene # 21319) (FIG. 21 a). When the Oct4 expression reporter vector (pOct4: GFP) was introduced into the differentiated cervical cancer cell HeLa, the green fluorescent protein was not expressed, but when the cell was reversed to obtain differentiation ability, the green fluorescent protein was expressed in the Oct4 expression reporter. Will be. Experiments were performed with miR-302a and miR-372, which are microRNAs known to cause reverse differentiation with such Oct4 expression reporter vectors. The 2nd to 8th sequences based on the 5 'end of the miR-302a and miR-372 start sequences are "AA GUG CU", and thus, the irregular GA sequence starts through the 4th and 6th G based on the 5' end. Do. However, the inventors carried out the experiment by synthesizing miR-302a-G4U and miR-372-G4U by paying attention to the fourth G.

Firstly, the experiment was performed by delivering Oct4 expression reporter vector (pOct4: GFP) to HeLa cells using Lipofectamine 2000 (Invitrogen) reagent according to the manufacturer's protocol, followed by guide strands according to human miR-302a and miR-372 sequences, Carrier strands were synthesized in Bioneer as in Example, prepared as duplexes, and delivered sequentially at 50 nM concentration using RNAimax (Invitrogen) reagent. In addition, the preparation of non-regular GA sequence initiation target specific siRNA of miR-302a and miR-372 was prepared in the same manner as in the above example by substituting U for G base in the end, and delivered to the cells at a concentration of 50 nM. The base sequence of the nucleic acid used therein is as follows (miR-302a: 5'- UAAGUGCUUCCAUGUUUUGGUGA-3 '; miR-302a-4GU: 5'- UAAUUGCUU CCAUGUUUUGGUGA-3'; miR-302a transport strand: 5'- ACUUAAACGUGGAUGUACUUGCU- 3 ′; miR-372: 5′-AAAGUGCUGCG ACAUUUGAGCGU-3 ′; miR-372-G4U: 5′-AAAUUGC UGCGACA UUUGAGCGU-3 ′, miR-372 Transport Strand: 5′-CCUCAAAUGUGGAGCACUAUUCU-3 ′). The HeLa cells incorporating the reporter vector and RNA 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 steptomycin for 14 days. In the transfection, cells were cultured in a complete medium without antibiotics.

 As a result, the experimental group to which miR-302a or miR-372 were delivered alone showed colony cell growth compared to the control group, but green fluorescent protein expression indicating the activity of Oct4 promoter was not observed until 14 days of culture. Did. However, in the experimental group in which miR-302a and miR-372 non-regular GA sequence initiation target inhibitory siRNA were introduced, not only cluster growth but also green fluorescent protein expression indicating the activity of Oct4 promoter was observed (FIG. 21 bc, red arrow). This resulted in a stronger and larger green fluorescent protein expressing cell population when miR-302a and miR-372 and their respective GA sequence initiation target inhibitory siRNAs were delivered to cells at the same time (FIG. 21 bc). Based on this, it can be seen that inhibition of miR-302a and miR-372 non-regular GA sequence initiation targets can promote differentiation of cells and acquisition of differentiation capacity through Oct4 expression.

Through the above embodiments, miR-372-G4U and miR-302a-G4U express only existing miR-372 and miR-302a alone to promote cell dedifferentiation more efficiently than causing cell dedifferentiation. It can be seen that miR-372-G4U and miR-302a-G4U can be used as a material to promote the reverse differentiation of cells.

[Example 22] Confirmation of the phenomenon that the 2'OMe modification at the 6th end of the 5 'end did not suppress the irregular nuclear ridge target of the RNA interference derivative.

In the above embodiment, the nonnuclear nucleus target exhibits a novel biological function that is completely different from the normal initiation target, and thus invents miRNA-BS, an RNA interference-inducing nucleic acid that only regulates the inhibition of expression of the nonnuclear nucleus target, and confirms its function. It was. However, in the case of miRNA-BS, the initiation sequence was changed to complementarily arrange the irregular nuclear ridge site of the existing miRNA, but the new initiation site was confirmed through the changed initiation sequence. Therefore, it has been found that an RNA interference inducing nucleic acid such as miRNA-BS recognizes only a normal starting sequence and a technology that does not recognize an irregular nuclear ridge site. Thus, the inventors of the present invention is important to the base of the position 6 position 5 'end in order to make a non-normal nuclear fusion, the degree of irregular nuclear fusion occurs depending on the sequence intensity of the base sequence in the 6 position Note that it can be, in order to reduce the intensity of the nucleotide sequence at position 6, the following experiment was carried out. That is, a methyl group (2 'OMe) is put in the 2' position of the ribosil ring of the nucleotide 6 of miR-124, and the modified interference inducing nucleic acid (miR-124-6me) is a non-normal nuclear fusion target of miR-124. The inhibition function was observed by the luciferase reporter experiment. 2'OMe modified miR-124 was prepared through synthesis (Bionia), and the luciferase reporter experiment was carried out in the same manner as in the previous Example 2 and the gene inhibition efficiency was measured by IC50 (FIG. 22). . The luciferase reporter vector used at this time was used for the normal initiation site of the miR-124 (Luc-seed) and the irregular nucleation site (Luc-nucleation bulge).

As a result, the inhibitory effect of the non-normal bump target gene by miR-124 shown in Example 2 was completely eliminated, whereas the inhibition of gene expression through the normal initiation site resulted in 2'OMe being 6th nucleotide with an IC50 value of 0.3 nM. It was observed that even when added, still shows a good inhibitory effect (FIG. 22).

According to the above embodiment, when the 2'OMe modification is applied to the 6th 5 'end of the nucleic acid inducing RNA interference, the expression suppression effect of the normal initiation target gene induced by the RNA interference inducing nucleic acid is maintained, Inhibition of the expression of the irregular nuclear target gene was confirmed to disappear. Thus, the 2'OMe modifications applied at the 6th end of the 5 'end may be applied to miRNA-BS to maintain the original intention of specifically inhibiting only the nonnuclear nucleus site of the miRNA, while newly emerging nonnuclear nucleus bonds may By eliminating it completely, the side effects can be minimized.

<110> Korea University <120> RNA interference inducing nucleic acids suppressing noncanonical          targets of microRNA and use <130> MP19-161 <150> KR 10-2018-0063055 <151> 2018-05-31 <160> 102 <170> KoPatentIn 3.0 <210> 1 <211> 6 <212> RNA <213> Artificial Sequence <220> <223> 5 '2 ~ 7 of miR-124-BS <400> 1 aaggcc 6 <210> 2 <211> 6 <212> RNA <213> Artificial Sequence <220> <223> 5 '2 ~ 7 of miR-122-BS <400> 2 aaggcc 6 <210> 3 <211> 6 <212> RNA <213> Artificial Sequence <220> <223> 5 '2 ~ 7 of miR-155-BS <400> 3 uaaugg 6 <210> 4 <211> 6 <212> RNA <213> Artificial Sequence <220> <223> 5 '2 ~ 7 of miR-1-BS <400> 4 ggaauu 6 <210> 5 <211> 21 <212> RNA <213> Artificial Sequence <220> <223> miR-124-BS <400> 5 uaaggccacg cggugaaugc c 21 <210> 6 <211> 21 <212> RNA <213> Artificial Sequence <220> <223> miR-122-BS <400> 6 uggaguugug acaauggugu u 21 <210> 7 <211> 22 <212> RNA <213> Artificial Sequence <220> <223> miR-155-BS <400> 7 uuaaugggcu aaucgugaua gg 22 <210> 8 <211> 21 <212> RNA <213> Artificial Sequence <220> <223> miR-1-BS <400> 8 uggaauugua aagaaguaug u 21 <210> 9 <211> 9 <212> RNA <213> Artificial Sequence <220> <223> 5 '1 ~ 9 of miR-124-G4U <400> 9 uaaugcacg 9 <210> 10 <211> 9 <212> RNA <213> Artificial Sequence <220> <223> 5 '1 ~ 9 of miR-124-G5U <400> 10 uaagucacg 9 <210> 11 <211> 9 <212> RNA <213> Artificial Sequence <220> <223> 5 '1 ~ 9 of miR-124-G4,5U <400> 11 uaauucacg 9 <210> 12 <211> 9 <212> RNA <213> Artificial Sequence <220> <223> 5 '1 ~ 9 of miR-1-G2U <400> 12 uugaaugua 9 <210> 13 <211> 9 <212> RNA <213> Artificial Sequence <220> <223> 5 '1 ~ 9 of miR-1-G3U <400> 13 uguaaugua 9 <210> 14 <211> 9 <212> RNA <213> Artificial Sequence <220> <223> 5 '1 ~ 9 of miR-1-G7U <400> 14 uggaauuua 9 <210> 15 <211> 9 <212> RNA <213> Artificial Sequence <220> <223> 5 '1 ~ 9 of miR-1-G2,3U <400> 15 uuuaaugua 9 <210> 16 <211> 9 <212> RNA <213> Artificial Sequence <220> <223> 5 '1 ~ 9 of miR-1-G3,7U <400> 16 uguaauuua 9 <210> 17 <211> 9 <212> RNA <213> Artificial Sequence <220> <223> 5 '1 ~ 9 of miR-1-G2,7U <400> 17 uugaauuua 9 <210> 18 <211> 9 <212> RNA <213> Artificial Sequence <220> <223> 5 '1 ~ 9 of miR-1-G2,3,7U <400> 18 uuuaauuua 9 <210> 19 <211> 9 <212> RNA <213> Artificial Sequence <220> <223> 5 '1 ~ 9 of miR-122-G2U <400> 19 uugagugug 9 <210> 20 <211> 9 <212> RNA <213> Artificial Sequence <220> <223> 5 '1 ~ 9 of miR-122-G3U <400> 20 uguagugug 9 <210> 21 <211> 9 <212> RNA <213> Artificial Sequence <220> <223> 5 '1 ~ 9 of miR-122-G5U <400> 21 uggauugug 9 <210> 22 <211> 9 <212> RNA <213> Artificial Sequence <220> <223> 5 '1 ~ 9 of miR-122-G7U <400> 22 uggaguuug 9 <210> 23 <211> 9 <212> RNA <213> Artificial Sequence <220> <223> 5 '1 ~ 9 of miR-122-G9U <400> 23 uggaguguu 9 <210> 24 <211> 9 <212> RNA <213> Artificial Sequence <220> <223> 5 '1 ~ 9 of miR-122-G2,3U <400> 24 uuuagugug 9 <210> 25 <211> 9 <212> RNA <213> Artificial Sequence <220> <223> 5 '1 ~ 9 of miR-122-G2,5U <400> 25 uugauugug 9 <210> 26 <211> 9 <212> RNA <213> Artificial Sequence <220> <223> 5 '1 ~ 9 of miR-122-G2,7U <400> 26 uugaguuug 9 <210> 27 <211> 9 <212> RNA <213> Artificial Sequence <220> <223> 5 '1 ~ 9 of miR-122-G2,9U <400> 27 uugaguguu 9 <210> 28 <211> 9 <212> RNA <213> Artificial Sequence <220> <223> 5 '1 ~ 9 of miR-122-G3,5U <400> 28 uguauugug 9 <210> 29 <211> 9 <212> RNA <213> Artificial Sequence <220> <223> 5 '1 ~ 9 of miR-122-G3,7U <400> 29 uguaguuug 9 <210> 30 <211> 9 <212> RNA <213> Artificial Sequence <220> <223> 5 '1 ~ 9 of miR-122-G3,9U <400> 30 uguaguguu 9 <210> 31 <211> 9 <212> RNA <213> Artificial Sequence <220> <223> 5 '1 ~ 9 of miR-122-G5,7U <400> 31 uggauuuug 9 <210> 32 <211> 9 <212> RNA <213> Artificial Sequence <220> <223> 5 '1 ~ 9 of miR-122-G5,9U <400> 32 uggauuguu 9 <210> 33 <211> 9 <212> RNA <213> Artificial Sequence <220> <223> 5 '1 ~ 9 of miR-122-G7,9U <400> 33 uggaguuuu 9 <210> 34 <211> 9 <212> RNA <213> Artificial Sequence <220> <223> 5 '1 ~ 9 of miR-133-G4U <400> 34 uuuuguccc 9 <210> 35 <211> 9 <212> RNA <213> Artificial Sequence <220> <223> 5 '1 ~ 9 of miR-133-G5U <400> 35 uuuguuccc 9 <210> 36 <211> 9 <212> RNA <213> Artificial Sequence <220> <223> 5 '1 ~ 9 of miR-124-G4,5U <400> 36 uuuuuuccc 9 <210> 37 <211> 9 <212> RNA <213> Artificial Sequence <220> <223> 5 '1 ~ 9 of let-7-G2U <400> 37 uuagguagu 9 <210> 38 <211> 9 <212> RNA <213> Artificial Sequence <220> <223> 5 '1 ~ 9 of let-7-G4U <400> 38 ugauguagu 9 <210> 39 <211> 9 <212> RNA <213> Artificial Sequence <220> <223> 5 '1 ~ 9 of let-7-G5U <400> 39 ugaguuagu 9 <210> 40 <211> 9 <212> RNA <213> Artificial Sequence <220> <223> 5 '1 ~ 9 of let-7-G8U <400> 40 ugagguauu 9 <210> 41 <211> 9 <212> RNA <213> Artificial Sequence <220> <223> 5 '1 ~ 9 of let-7-G2,4U <400> 41 uuauguagu 9 <210> 42 <211> 9 <212> RNA <213> Artificial Sequence <220> <223> 5 '1 ~ 9 of let-7-G2,5U <400> 42 uuaguuagu 9 <210> 43 <211> 9 <212> RNA <213> Artificial Sequence <220> <223> 5 '1 ~ 9 of let-7-G2,8U <400> 43 uuagguauu 9 <210> 44 <211> 9 <212> RNA <213> Artificial Sequence <220> <223> 5 '1 ~ 9 of let-7-G4,5U <400> 44 ugauuuagu 9 <210> 45 <211> 9 <212> RNA <213> Artificial Sequence <220> <223> 5 '1 ~ 9 of let-7-G4,8U <400> 45 ugauguauu 9 <210> 46 <211> 9 <212> RNA <213> Artificial Sequence <220> <223> 5 '1 ~ 9 of let-7-G5,8U <400> 46 ugaguuauu 9 <210> 47 <211> 9 <212> RNA <213> Artificial Sequence <220> <223> 5 '1 ~ 9 of miR-302a-G4U <400> 47 uaauugcuu 9 <210> 48 <211> 9 <212> RNA <213> Artificial Sequence <220> <223> 5 '1 ~ 9 of miR-302a-G6U <400> 48 uaaguucuu 9 <210> 49 <211> 9 <212> RNA <213> Artificial Sequence <220> <223> 5 '1 ~ 9 of miR-302a-G4,6U <400> 49 uaauuucuu 9 <210> 50 <211> 9 <212> RNA <213> Artificial Sequence <220> <223> 5 '1 ~ 9 of miR-372-G4U <400> 50 aaauugcug 9 <210> 51 <211> 9 <212> RNA <213> Artificial Sequence <220> <223> 5 '1 ~ 9 of miR-372-G6U <400> 51 aaaguucug 9 <210> 52 <211> 9 <212> RNA <213> Artificial Sequence <220> <223> 5 '1 ~ 9 of miR-372-G9U <400> 52 aaagugcuu 9 <210> 53 <211> 9 <212> RNA <213> Artificial Sequence <220> <223> 5 '1 ~ 9 of miR-372-G4,6U <400> 53 aaauuucug 9 <210> 54 <211> 9 <212> RNA <213> Artificial Sequence <220> <223> 5 '1 ~ 9 of miR-372-G4,9U <400> 54 aaauugcuu 9 <210> 55 <211> 9 <212> RNA <213> Artificial Sequence <220> <223> 5 '1 ~ 9 of miR-372-G6,9U <400> 55 aaaguucuu 9 <210> 56 <211> 22 <212> RNA <213> Artificial Sequence <220> <223> miR-124-G4U <400> 56 uaaugcacgc ggugaaugcc aa 22 <210> 57 <211> 21 <212> RNA <213> Artificial Sequence <220> <223> miR-124-G5U <400> 57 uaagucacgc ggugaaugcc a 21 <210> 58 <211> 22 <212> RNA <213> Artificial Sequence <220> <223> miR-124-G4,5U <400> 58 uaauucacgc ggugaaugcc aa 22 <210> 59 <211> 22 <212> RNA <213> Artificial Sequence <220> <223> miR-1-G2U <400> 59 uugaauguaa agaaguaugu au 22 <210> 60 <211> 22 <212> RNA <213> Artificial Sequence <220> <223> miR-1-G3U <400> 60 uguaauguaa agaaguaugu au 22 <210> 61 <211> 22 <212> RNA <213> Artificial Sequence <220> <223> miR-1-G7U <400> 61 uggaauuuaa agaaguaugu au 22 <210> 62 <211> 22 <212> RNA <213> Artificial Sequence <220> <223> miR-1-G2,3U <400> 62 uuuaauguaa agaaguaugu au 22 <210> 63 <211> 22 <212> RNA <213> Artificial Sequence <220> <223> miR-1-G3,7U <400> 63 uguaauuuaa agaaguaugu au 22 <210> 64 <211> 22 <212> RNA <213> Artificial Sequence <220> <223> miR-1-G2,7U <400> 64 uugaauuuaa agaaguaugu au 22 <210> 65 <211> 22 <212> RNA <213> Artificial Sequence <220> <223> miR-1-G2,3,7U <400> 65 uuuaauuuaa agaaguaugu au 22 <210> 66 <211> 22 <212> RNA <213> Artificial Sequence <220> <223> miR-122-G2U <400> 66 uugaguguga caaugguguu ug 22 <210> 67 <211> 22 <212> RNA <213> Artificial Sequence <220> <223> miR-122-G3U <400> 67 uguaguguga caaugguguu ug 22 <210> 68 <211> 22 <212> RNA <213> Artificial Sequence <220> <223> miR-122-G5U <400> 68 uggauuguga caaugguguu ug 22 <210> 69 <211> 22 <212> RNA <213> Artificial Sequence <220> <223> miR-122-G7U <400> 69 uggaguuuga caaugguguu ug 22 <210> 70 <211> 22 <212> RNA <213> Artificial Sequence <220> <223> miR-122-G9U <400> 70 uggaguguua caaugguguu ug 22 <210> 71 <211> 22 <212> RNA <213> Artificial Sequence <220> <223> miR-122-G2,3U <400> 71 uuuaguguga caaugguguu ug 22 <210> 72 <211> 22 <212> RNA <213> Artificial Sequence <220> <223> miR-122-G2,5U <400> 72 uugauuguga caaugguguu ug 22 <210> 73 <211> 22 <212> RNA <213> Artificial Sequence <220> <223> miR-122-G2,7U <400> 73 uugaguuuga caaugguguu ug 22 <210> 74 <211> 22 <212> RNA <213> Artificial Sequence <220> <223> miR-122-G2,9U <400> 74 uugaguguua caaugguguu ug 22 <210> 75 <211> 22 <212> RNA <213> Artificial Sequence <220> <223> miR-122-G3,5U <400> 75 uguauuguga caaugguguu ug 22 <210> 76 <211> 22 <212> RNA <213> Artificial Sequence <220> <223> miR-122-G3,7U <400> 76 uguaguuuga caaugguguu ug 22 <210> 77 <211> 22 <212> RNA <213> Artificial Sequence <220> <223> miR-122-G3,9U <400> 77 uguaguguua caaugguguu ug 22 <210> 78 <211> 22 <212> RNA <213> Artificial Sequence <220> <223> miR-122-G5,7U <400> 78 uggauuuuga caaugguguu ug 22 <210> 79 <211> 22 <212> RNA <213> Artificial Sequence <220> <223> miR-122-G5,9U <400> 79 uggauuguua caaugguguu ug 22 <210> 80 <211> 22 <212> RNA <213> Artificial Sequence <220> <223> miR-122-G7,9U <400> 80 uggaguuuua caaugguguu ug 22 <210> 81 <211> 22 <212> RNA <213> Artificial Sequence <220> <223> miR-133-G4U <400> 81 uuuugucccc uucaaccagc ug 22 <210> 82 <211> 22 <212> RNA <213> Artificial Sequence <220> <223> miR-133-G5U <400> 82 uuuguucccc uucaaccagc ug 22 <210> 83 <211> 22 <212> RNA <213> Artificial Sequence <220> <223> miR-124-G4,5U <400> 83 uuuuuucccc uucaaccagc ug 22 <210> 84 <211> 22 <212> RNA <213> Artificial Sequence <220> <223> let-7-G2U <400> 84 uuagguagua gguuguauag uu 22 <210> 85 <211> 22 <212> RNA <213> Artificial Sequence <220> <223> let-7-G4U <400> 85 ugauguagua gguuguauag uu 22 <210> 86 <211> 22 <212> RNA <213> Artificial Sequence <220> <223> let-7-G5U <400> 86 ugaguuagua gguuguauag uu 22 <210> 87 <211> 22 <212> RNA <213> Artificial Sequence <220> <223> let-7-G8U <400> 87 ugagguauua gguuguauag uu 22 <210> 88 <211> 22 <212> RNA <213> Artificial Sequence <220> <223> let-7-G2,4U <400> 88 uuauguagua gguuguauag uu 22 <210> 89 <211> 22 <212> RNA <213> Artificial Sequence <220> <223> let-7-G2,5U <400> 89 uuaguuagua gguuguauag uu 22 <210> 90 <211> 22 <212> RNA <213> Artificial Sequence <220> <223> let-7-G2,8U <400> 90 uuagguauua gguuguauag uu 22 <210> 91 <211> 22 <212> RNA <213> Artificial Sequence <220> <223> let-7-G4,5U <400> 91 ugauuuagua gguuguauag uu 22 <210> 92 <211> 22 <212> RNA <213> Artificial Sequence <220> <223> let-7-G4,8U <400> 92 ugauguauua gguuguauag uu 22 <210> 93 <211> 22 <212> RNA <213> Artificial Sequence <220> <223> let-7-G5,8U <400> 93 ugaguuauua gguuguauag uu 22 <210> 94 <211> 23 <212> RNA <213> Artificial Sequence <220> <223> miR-302a-G4U <400> 94 uaauugcuuc cauguuuugg uga 23 <210> 95 <211> 23 <212> RNA <213> Artificial Sequence <220> <223> miR-302a-G6U <400> 95 uaaguucuuc cauguuuugg uga 23 <210> 96 <211> 23 <212> RNA <213> Artificial Sequence <220> <223> miR-302a-G4,6U <400> 96 uaauuucuuc cauguuuugg uga 23 <210> 97 <211> 23 <212> RNA <213> Artificial Sequence <220> <223> miR-372-G4U <400> 97 aaauugcugc gacauuugag cgu 23 <210> 98 <211> 23 <212> RNA <213> Artificial Sequence <220> <223> miR-372-G6U <400> 98 aaaguucugc gacauuugag cgu 23 <210> 99 <211> 23 <212> RNA <213> Artificial Sequence <220> <223> miR-372-G9U <400> 99 aaagugcuuc gacauuugag cgu 23 <210> 100 <211> 23 <212> RNA <213> Artificial Sequence <220> <223> miR-372-G4,6U <400> 100 aaauuucugc gacauuugag cgu 23 <210> 101 <211> 23 <212> RNA <213> Artificial Sequence <220> <223> miR-372-G4,9U <400> 101 aaauugcuuc gacauuugag cgu 23 <210> 102 <211> 23 <212> RNA <213> Artificial Sequence <220> <223> miR-372-G6,9U <400> 102 aaaguucuuc gacauuugag cgu 23

Claims (12)

An RNA interference inducing nucleic acid in which at least one of the double strands of a nucleic acid inducing RNA interference is modified, wherein some sequences of a particular microRNA are modified to inhibit the noncanonical target gene of the microRNA,
The RNA interference-inducing nucleic acid comprises four to five base sequences from the 5 'end of a particular microRNA, the sixth and seventh bases are identical, and the sixth and seventh bases are the sixth of a particular microRNA. A base having a base sequence complementary to that of the base, and a base sequenceable to the sixth base of the specific microRNA include all complementary bases including G: A, G: U wobble sequences Characterized in that, or
The RNA interference-inducing nucleic acid has a guanine base of the base sequence between the 5 'end and the ninth base of a specific microRNA having a modified base sequence substituted with at least one or more of uracil or adenine (Uracil) or adenine (Adenine) Characterized by
RNA interference induced nucleic acid.
The RNA interference inducing nucleic acid according to claim 1, wherein the RNA interference inducing nucleic acid selectively inhibits the irregular target gene of the microRNA and does not inhibit the regular target gene of the microRNA.
According to claim 1, wherein the specific microRNA is at least one selected from the group consisting of miR-124, miR-155, miR-122, miR-1, let-7, miR-133, miR-302a and miR-372 RNA interference inducing nucleic acid.
The RNA interference inducing nucleic acid according to claim 1, wherein the RNA interference inducing nucleic acid has a second to seventh base sequence at the 5 'end.
RNA interference-inducing nucleic acid (miR-124-BS) which shows the base sequence of 5'-AA GGC C-3 'as an RNA interference-inducing nucleic acid that inhibits an irregular target gene of miR-124;
RNA interference-inducing nucleic acid (miR-122-BS) which shows the base sequence of 5'-GG AGU U-3 'as an RNA interference-inducing nucleic acid that inhibits an irregular target gene of miR-122;
RNA interference-inducing nucleic acid (miR-155-BS) which shows the base sequence of 5'-UA AUG G-3 'as an RNA interference-inducing nucleic acid that inhibits an irregular target gene of miR-155; or
RNA interference-inducing nucleic acid (miR-1-BS) which shows the base sequence of 5'-GG AAU U-3 'as an RNA interference-inducing nucleic acid that inhibits an irregular target gene of miR-1.
The RNA interference inducing nucleic acid according to claim 4, wherein the base sequence of the RNA interference inducing nucleic acid is represented by any one or more of the following:
RNA interference-inducing nucleic acid (miR-124-BS) which shows the base sequence of 5'-UAA GGC CAC GCG GUG AAU GCC-3 'as an RNA interference-inducing nucleic acid that inhibits an irregular target gene of miR-124;
RNA interference-inducing nucleic acid (miR-122-BS) which shows the base sequence of 5'-UGG AGU UGU GAC AAU GGU GUU-3 'as an RNA interference-inducing nucleic acid that inhibits an irregular target gene of miR-122;
RNA interference-inducing nucleic acid (miR-155-BS) which shows the base sequence of 5'-UUA AUG GGC UAA U CGU GAU AGG-3 'as an RNA interference-inducing nucleic acid that inhibits an irregular target gene of miR-155;
RNA interference-inducing nucleic acid (miR-1-BS) showing the base sequence of 5'-UGG AAU UGU AAA GAA GUA UGU-3 'as an RNA interference-inducing nucleic acid that inhibits an irregular target gene of miR-1.
The RNA interference inducing nucleic acid according to claim 1, wherein the RNA interference inducing nucleic acid has a nucleotide sequence between the 5 'end and the ninth base in any one or more of the following:
As RNA Interference Inducing Nucleic Acids That Inhibit the Irregular Target Gene of miR-124
5'-UAA UGC ACG-3 '(miR-124-G4U), 5'-UAA GUC ACG-3' (miR-124-G5U) or 5'-UAA UUC ACG-3 '(miR-124-G4, RNA interference inducing nucleic acid comprising the base sequence of 5U);
As RNA Interference Inducing Nucleic Acids That Inhibit the Irregular Target Gene of miR-1
5'-UUG AAU GUA-3 '(miR-1-G2U), 5'-UGU 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 RNA interference inducing nucleic acids comprising the nucleotide sequence of -1-G2,7U) or 5'-UUU AAU UUA-3 '(miR-1-G2,3,7U);
RNA Interference-Inducing Nucleic Acids That Inhibit the Irregular Target Gene of miR-122
5'-UUG AGU GUG-3 '(miR-122-G2U), 5'-UGU 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 an RNA interference inducing nucleic acid comprising the nucleotide sequence of 5'-UGG AGU UUU-3 (miR-122-G7,9U);
As RNA Interference Inducing Nucleic Acids That Inhibit the Minormal Target Gene of miR-133
5'-UUU UGU CCC-3 '(miR-133-G4U), 5'-UUU GUU CCC-3' (miR-133-G5U) or 5'-UUU UUU CCC-3 '(miR-133-G4, RNA interference inducing nucleic acid comprising the base sequence of 5U);
As RNA Interference Inducing Nucleic Acids That Inhibit the Irregular Target Gene of let-7
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 An RNA interference inducing nucleic acid comprising the nucleotide sequence of -3 '(let-7-G4,8U), or 5'-UGA GUU AUU-3'(let-7-G5,8U);
RNA Interference-Inducing Nucleic Acids That Inhibit the Irregular Target Gene of miR-302a
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 , RNA interference inducing nucleic acid comprising the base sequence of 6U); or
RNA Interference Inducing Nucleic Acids That Inhibit MiR-372 Irregular Target Genes
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 '( RNA interference inducing nucleic acid comprising the nucleotide sequence of miR-372-G6,9U).
The RNA interference inducing nucleic acid according to claim 6, wherein the base sequence of the RNA interference inducing nucleic acid is represented by any one or more of the following:
As RNA Interference Inducing Nucleic Acids That Inhibit the Irregular Target Gene of miR-124
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 RNA interference inducing nucleic acids showing the nucleotide sequence of CGG UGA AUG CCA A-3 '(miR-124-G4,5U);
As RNA Interference Inducing Nucleic Acids That Inhibit the Irregular Target Gene of miR-1
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 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'-UUU AAU UUA AAG AAG UAU GUA U- RNA interference inducing nucleic acid showing a nucleotide sequence of 3 '(miR-1-G2,3,7U);
RNA interference inducing nucleic acids that inhibit the non-regular target gene of miR-122 as 5'-UUG AGU GUG ACA AUG GUG UUU G-3 '(miR-122-G2U), 5'-UGU 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 RNA interference inducing nucleic acid showing the nucleotide sequence of (miR-122-G7,9U);
RNA interference inducing nucleic acids that inhibit the non-regular target gene of miR-133 as 5'-UUU UGU CCC CUU CAA CCA GCU G -3 '(miR-133-G4U), 5'-UUU GUU CCC CUU CAA CCA GCU G-3 an RNA interference inducing nucleic acid comprising a nucleotide sequence of '(miR-133-G5U) or 5'-UUU UUU CCC CUU CAA CCA GCU G-3'(miR-133-G4,5U); RNA interference-inducing nucleic acids that inhibit the irregular target gene of let-7 are 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 Base sequence of '-UGA UGU 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) RNA interference inducing nucleic acid;
RNA interference-inducing nucleic acids that inhibit the irregular target gene of miR-302a as 5'-UAA UUG CUU CCA UGU UUU GGU GA-3 '(miR-302a-G4U), 5'-UAA GUU CUU CCA UGU UUU GGU GA-3 RNA interference inducing nucleic acids showing the base sequence of a '(miR-302a-G6U), or a 5'-UAA UUU CUU CCA UGU UUU GGU GA-3'(miR-302a-G4,6U); or
RNA interference-inducing nucleic acids that inhibit the non-regular target gene of miR-372 as 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 CUU CGA CAU UUG AGC GU-3' (miR-372-G6 , 9U) RNA interference inducing nucleic acid.
A composition for inhibiting the expression of an irregular target gene of a microRNA comprising the RNA interference inducing nucleic acid of any one of claims 1 to 7.
A composition for regulating cell cycle, differentiation, dedifferentiation, morphology, migration, division, proliferation or death, comprising the RNA interference inducing nucleic acid of any one of claims 1 to 7.
The composition of claim 9, wherein the composition is one or more of the following:
a composition for inducing cancer cell death comprising an RNA interference inducing nucleic acid that inhibits an irregular target gene of miR-124;
a composition for inducing neurites branching differentiation comprising an RNA interference inducing nucleic acid that inhibits an irregular target gene of miR-124;
a composition for inducing cell cycle arrest of liver cancer cells comprising an RNA interference inducing nucleic acid that inhibits an irregular target gene of miR-122;
a composition for promoting myocyte differentiation or promoting muscle fibrosis, including an RNA interference-inducing nucleic acid that suppresses an irregular target gene of miR-1;
a composition for inducing myocyte death, including an RNA interference-inducing nucleic acid that inhibits an irregular target gene of miR-155;
a composition for promoting cell death of neuroblastoma, including RNA interference-inducing nucleic acid that inhibits an irregular target gene of miR-124;
a composition for promoting cell division proliferation of neuroblastoma, including an RNA interference-inducing nucleic acid that inhibits an irregular target gene of miR-124;
a composition for inducing myocardial hypertrophy comprising an RNA interference inducing nucleic acid that inhibits an irregular target gene of miR-1;
a composition for inducing myocardial hypertrophy comprising an RNA interference inducing nucleic acid that inhibits an irregular target gene of miR-133;
a composition for inducing cell cycle arrest of cancer cells, including an RNA interference inducing nucleic acid that inhibits an irregular target gene of let-7;
a composition for inducing cell cycle progression activity of hepatocytes comprising an RNA interference inducing nucleic acid that inhibits an irregular target gene of let-7;
a composition for promoting reverse differentiation comprising an RNA interference inducing nucleic acid that inhibits an irregular target gene of miR-302a;
a composition for promoting reverse differentiation comprising an RNA interference inducing nucleic acid that inhibits an irregular target gene of miR-372; or
A composition for inhibiting cell migration of liver cancer cells comprising an RNA interference inducing nucleic acid that inhibits an irregular target gene of miR-122.
In one or more single strands of a double strand of nucleic acid inducing RNA interference, some sequences of a particular microRNA are modified to express the noncanonical target gene of the microRNA, including the following steps: Method for preparing RNA interference inducing nucleic acid to inhibit:
It contains four base sequences from second to fifth from the 5 'end of a particular microRNA, wherein the sixth and seventh bases are identical and the sixth and seventh bases can be aligned with the sixth base of a particular microRNA. A base having complementary base sequences to the base and capable of being aligned with the sixth base of the specific microRNA includes RNA complementary nucleic acids such that all complementary bases, including G: A and G: U wobble sequences, are included. Constructing, or
RNA interference-inducing nucleic acids are constructed such that the guanine base in the base sequence between the 5 'end and the ninth base of a specific microRNA has a modified base sequence substituted with at least one of uracil or adenine. Steps.
Transfecting the RNA interference inducing nucleic acid of any one of claims 1 to 7 to a subject cell;
Treating the test substance with the subject cell; And
Including the step of confirming the expression level or expression of the irregular target gene of the microRNA inhibited by the RNA interference-inducing nucleic acid in the target cell,
A method for screening test substances for controlling cell cycle, differentiation, retrodifferentiation, morphology, migration, division, proliferation or death.

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