KR20200047207A - Recombinant vector for producing animal model with cardiac hypertrophy, transformed model with cardiac hypertrophy by the recombinant vector and a method of production thereof - Google Patents

Abstract

The present invention relates to a recombinant vector for producing a cardiomegaly animal model, a cardiomegaly animal model transformed by the recombinant vector, and a method for producing the same. When the recombinant vector according to the present invention is specifically expressed in mouse myocardial cells, only an irregular G:A wobble base sequence target site of a microRNA is recognized and suppressed, and thus a biological function of increasing the size of the myocardial cells is exhibited and cardiomegaly is induced. According to the present invention, it is expected that an animal model induced with cardiomegaly is produced to be usefully used in the study of the progression, pathology, and physiological mechanisms of heart diseases, including cardiomegaly, at an individual level.

Description

Recombinant vector for producing animal model with cardiac hypertrophy, transformed model with cardiac hypertrophy by the recombinant vector and a method of production thereof }

The present invention relates to a recombinant vector for producing an animal model of cardiomegaly, an animal model of cardiomegaly transformed with the recombinant vector, and a method of manufacturing the same.

MicroRNA (miRNA) is a small RNA composed of ~ 21 bases, and functions to induce RNA interference that suppresses gene expression in the step after transcription of the target gene. MicroRNAs recognize hundreds of target mRNAs and inhibit their expression through nucleotide sequence of the seed region (positions 1-8) located mainly at the 5 'end, thereby controlling various biological phenomena.

In particular, when the microRNA binds to the target mRNA, at least 6 contiguous nucleotide sequences, i.e., 2nd to 7th, mainly through the seed region between the 2nd and 8th nucleotides at the 5 'end. It is known to use a nucleotide sequence or a nucleotide sequence between the 3rd and 8th nucleotides, which is called canonical target recognition, and recently, Ago, a technique for sequencing mRNA positions binding to microRNAs As HITS-CLIP was developed, it became known that there were many irregular target recognitions that deviated from these rules in addition to regular target recognition.

On the other hand, heart disease is one of the top three death diseases in Korean adults, including cancer, and various pathological stresses in the onset process begin to cause cardiac hypertrophy by changing the size of heart tissue. Hypertrophy is characterized by increased myocardial cell size and increased protein synthesis rate. Cardiac hypertrophy gradually induces myocardial fibrosis and heart failure, ultimately resulting in cardiac dysfunction (or heart failure). Particularly, in the case of heart failure, since the mortality rate is very high, especially around 50%, studies are being conducted to ultimately prevent myocardial hypertrophy or develop a treatment method, so it is necessary to establish a disease model for this.

A well-known method for inducing such hypertrophy in animal and cell models is a method of treating an adrenergic receptor agonist such as phenylephrine (PE) or isoproterenol (ISO). However, this treatment has a difficulty in repeatedly experimenting to inject into the body of a mouse at the same interval every time for at least 1 month, or inserting a pump that constantly delivers drugs into a subject through surgical surgery. In addition, there is a difference between individuals in the symptoms of cardiac hypertrophy caused by drugs, and the degree of enlargement of the heart has a problem that may be insufficient depending on the situation.

Cardiac hypertrophy is also known to be regulated by microRNA like many other diseases. In particular, miR-1 not only plays a very important role in the development of myocardial cells, but also induces a cardiac atrophy that causes the heart to shrink. It was reported. In addition, miR-133 expressed like miR-1 is also known to have a function of suppressing myocardial hypertrophy.

Therefore, in order to understand the phenomenon of hypertrophy and develop a treatment for it, a microRNA model of hypertrophy was established at the animal individual level and utilized.

Korean Registered Patent No. 1481007

The present inventors identified a G: A wobble seed site, a non-canonical target recognition form of miR-1, a microRNA known to be specifically expressed in muscle cells including the heart, as a regular seeding site (seed). As a result of expressing the recombinant vector containing the modified base sequence specifically to the mouse myocardial cells to recognize it as a site), we observed the phenomenon of myocardial cell enlargement, and attempted to construct an animal model of cardiomegaly disease using this.

Therefore, the problem to be solved by the present invention is that the present invention is represented by the nucleotide sequence of SEQ ID NO: 1 (5'-UGGAAUGUAAAGAAGUAUGUAU -3 '), based on the 5' end of the miR-1 gene inducing RNA interference. Eurasil from Guanine (G) containing at least one nucleotide of 2, 3 or 7 of the nucleotide sequence of SEQ ID NO: 1, including a total of six consecutive polynucleotides starting from any one of the nucleotides. Recombinant vector for producing an animal model of cardiomegaly, characterized by expressing an RNA interference-inducing nucleic acid containing a polynucleotide substituted with Uracil; U), an animal model of a cardiomegaly transformed with the recombinant vector, and a method for manufacturing the same Is to provide.

At this time, preferably, the sequence from the 2nd to the 7th based on the 5 'end of the RNA interference-inducing nucleic acid binding to the Argonaute protein to induce RNA interference is UGAAUG, GUAAUG, GGAAUU, GAAUGU, UAAUGU, GAAUUU, the sequence is miR-1 represented by the nucleotide sequence of SEQ ID NO: 1 shows the effect of binding to and inhibiting only the target recognized by the G: A wobble sequence in the cardiac tissue of a myocardial infarction patient, thereby exhibiting the effect of being an animal individual It can be used as an animal model of heart disease that develops in and causes heart disease occurring in humans.

However, the technical problem to be achieved by the present invention is not limited to the above-mentioned problems, and other problems not mentioned can be clearly understood by those skilled in the art from the following description. There will be.

In order to achieve the above object, the present invention includes a total of six consecutive polynucleotides starting from any one of nucleotides 2 to 3 of the 5 'end of the gene represented by the nucleotide sequence of SEQ ID NO: 1 , RNA interference-inducing nucleic acid comprising a polynucleotide in which one or more of nucleotides 2, 3, or 7 of SEQ ID NO: 1 is substituted with uracil (U) in guanine (G) It provides a recombinant vector for producing an animal model of cardiomegaly, characterized in that.

In one embodiment of the invention, the polynucleotide may be selected from the following groups of polynucleotides:

A polynucleotide represented by the nucleotide sequence of SEQ ID NO: 2 (5'-UGAAUG-3 '); A polynucleotide represented by the nucleotide sequence of SEQ ID NO: 3 (5'-GUAAUG-3 '); A polynucleotide represented by the nucleotide sequence of SEQ ID NO: 4 (5'-GGAAUU-3 '); A polynucleotide represented by the nucleotide sequence of SEQ ID NO: 5 (5'-UAAUGU-3 '); Or a polynucleotide represented by the nucleotide sequence of SEQ ID NO: 6 (5'-GAAUUU-3 ').

As another embodiment of the present invention, the recombinant vector may include a base sequence substituted with adenine (A) in front of the first nucleotide at the 5 'end of the polynucleotide, and is not limited thereto, and may bind to an agonist protein. If possible, the first nucleotide at the 5 'end can be A, U, C, G all nucleotide sequences.

In another embodiment of the present invention, the recombinant vector can express any one of the following groups of RNA interference-inducing nucleic acids:

RNA interference-inducing nucleic acid represented by the nucleotide sequence of SEQ ID NO: 7 (5'-UUG AAU GUA AAG AAG UAU GUA U-3 ');

RNA interference-inducing nucleic acid represented by the nucleotide sequence of SEQ ID NO: 8 (5'- UGU AAU GUA AAG AAG UAU GUA U-3 ');

RNA interference-inducing nucleic acid represented by the nucleotide sequence of SEQ ID NO: 9 (5'-UGG AAU UUA AAG AAG UAU GUA U-3 ');

RNA interference-inducing nucleic acid represented by the nucleotide sequence of SEQ ID NO: 10 (5'-AUG AAU GAC UGC CGG UUG UUA UG-3 ');

RNA interference-inducing nucleic acid represented by the nucleotide sequence of SEQ ID NO: 11 (5'-AGU AAU GAC UGC CGG UUG UUA UG-3 ');

RNA interference-inducing nucleic acid represented by the nucleotide sequence of SEQ ID NO: 12 (5'-AGG AAU UAC UGC CGG UUG UUA UG-3 ');

RNA interference-inducing nucleic acid represented by the nucleotide sequence of SEQ ID NO: 13 (5'-AUA AUG UAC UGC CGG UUG UUA UG-3 '); or

RNA interference-inducing nucleic acid represented by the nucleotide sequence of SEQ ID NO: 14 (5'-AGA AUU UAC UGC CGG UUG UUA UG-3 ').

As another embodiment of the present invention, the gene represented by the nucleotide sequence of SEQ ID NO: 1 may be miR-1.

As another embodiment of the present invention, the polynucleotide may be a polynucleotide that recognizes a target site where a G: A wobble arrangement occurs.

As another embodiment of the present invention, the recombinant vector is an alphaMHC (αMHC) promoter, a beta MHC (β MHC) promoter, a Nkx2.5 promoter, a cardiac troponin T (TNNT2) promoter, a muscle creatine kinase ( A promoter selected from the group consisting of muscle creatine kinase (MCK) promoter, human α-skeletal actin (HAS) promoter, and murine stem cell virus (MSCV) promoter is operably linked You can.

As another embodiment of the present invention, the cardiac hypertrophy is cardiac hypertrophy, heart failure, cardiac fibrosis, mitral valve insufficiency, aortic valve regurgitation, dilated cardiomyopathy, ischemic heart disease, ventricular septal defect, tricuspid valve It may be occlusive insufficiency, pulmonary valve insufficiency, pulmonary arterial hypertension, right ventricular myocardial infarction, cardiomyopathy involving the right ventricle, atrial septal defect, atrial fibrillation, hypertrophic cardiomyopathy or invasive cardiomyopathy.

As another embodiment of the present invention, the animal may be a mammal except human.

As another embodiment of the present invention, the animal may be a mouse.

In addition, the present invention provides a cardiac hypertrophy animal model characterized by being transformed with the recombinant vector.

In addition, the present invention (a) starting from any one of nucleotides 2 to 3 of the 5 'end of the gene represented by the nucleotide sequence of SEQ ID NO: 1 includes a total of six consecutive nucleotides, the SEQ ID NO: 1 Symbiosis by operably connecting DNA expressing a polynucleotide in which one or more of nucleotides 2, 3, or 7 of nucleotides are substituted with uracil (U) in guanine (G) Preparing a recombinant vector for animal model production;

(b) introducing the recombinant vector into an animal fertilized egg; And

(c) implanting the fertilized egg into a surrogate mother, and generating a fertilized egg to obtain a transformed animal model.

In addition, the present invention comprises the steps of (a) treating a candidate substance to the cardiomyocytes of the cardiac hypertrophy animal model;

(b) a total of six consecutive polynucleotides starting from any one of nucleotides 2 to 3 based on the 5 'end of the gene represented by the nucleotide sequence of SEQ ID NO: 1 in the myocardial cell of the cardiac hypertrophy animal model, , G: A wobble of a polynucleotide in which one or more of nucleotides 2, 3, or 7 of SEQ ID NO: 1 is substituted with uracil (U) in guanine (G) Analyzing the binding frequency with the array target site; And

(c) when the binding to the G: A wobble (wobble) array target site is reduced compared to an animal model of cardiomegaly that did not process the candidate substance, the method comprising selecting the candidate substance as a therapeutic agent for cardiomegaly, It provides a method of screening for a treatment for hypertrophy.

When the recombinant vector according to the present invention is specifically expressed in mouse myocardial cells, the biological function of increasing the size of the myocardial cells appears by recognizing and suppressing only the non-regular G: A wobble nucleotide target site of microRNA. Since this leads to cardiac hypertrophy, it is expected that an animal model in which cardiomegaly is induced may be useful for the progress and pathology of cardiac diseases including cardiomegaly at the individual level, as well as for the study of physiological mechanisms.

1A to 1D are bioinformatical analysis results of Ago HITS-CLIP, which is sequence data for a microRNA binding to an agonist protein in human brain tissue and its target mRNA position, as well as a normal target (seed) target , It is a diagram showing the result of confirming that the non-regular G: A wobble array (G: A wobble) starting target naturally exists.
Figures 2a and 2b are human RNA myocardial infarction (Cardiomyopathy) miR- expressing heart tissue specifically through Ago HITS-CLIP, a sequence mRNA data for the target mRNA position and microRNA binding to agonist protein in the heart tissue of patients As a result of bioinformatical analysis of the target binding of 1, the results of confirming the presence of normal G: A wobble (G: A wobble) binding as well as normal initiation (seed) binding are present in patients with myocardial infarction. It is a drawing.
3A to 3D show that miR-1 functions to suppress the expression of a non-regular G: A wobble sequence initiation target gene. Fluorescence-activated cell sorting (FACS) in a fluorescent protein reporter makes the cardiomyocyte cell line (H9c2). It is a figure showing the results confirmed in.
4A to 4D show the 2, 3, or 7 guanine base from the 5 'end corresponding to miR-1 to recognize only the non-regular G: A wobble sequence initiation target gene of miR-1. It is a diagram showing the result of confirming through the luciferase reporter experiment that only the non-regular G: A wobble sequence initiation target gene is inhibited by expressing it by substituting with.
5A to 5C are expressed by substituting 2, 3, or 7th guanine base from the 5 'end of miR-1 with uracil, and expressing 2, 3, or 5 from the 5' end of miR-1. This is a diagram showing the results of confirming the function of the target gene that triggers the irregular G: A wobble sequence for the 7th guanine base through observation of cell morphology and molecular biological characteristics of the myocardial hypertrophy-inducing effect.
6A to 6C show the minimum nucleotide sequence showing the regulatory function of an irregular G: A wobble sequence initiation target gene for 2, 3, or 7 guanine bases from the 5 'end of miR-1, 5 of miR-1 'The 2nd to 7th sequence from the terminal, or the 3rd to 8th sequence is defined, and RNA interference-inducing substances containing only the nucleotide sequence are introduced into the cardiomyocyte cell line (H9c2) to show the myocardial hypertrophy-inducing effect. It is a diagram showing the result confirmed through morphological observation.
Figures 7a to 7d is a micro-substitution of the 7th guanine from the 5 'end of miR-1 with uracil to control only the irregular G: A wobble sequence initiation target for the 7th guanine base from the 5' end of miR-1 It is a diagram showing the manufacturing process of a transgenic mouse expressing RNA sequence specifically for myocardial cells.
8A to 8D show myocardial hypertrophy in an individual of a transgenic mouse expressing a microRNA sequence in which the 7th guanine from the 5 'end of miR-1 is specifically substituted with uracil for myocardial cell specificity, and monitor the survival of the individual. It is a diagram showing one result.
9A to 9C show myocardial hypertrophy of transgenic mouse lines generated while maintaining an individual of a transgenic mouse expressing a microRNA sequence in which the 7th guanine from the 5 'end of miR-1 specifically substituted with uracil for myocardial cell specificity It is a diagram showing the results of observing.

The present invention can recognize a G: A wobble seeding site (G: A wobble seed site), which is a form of irregular target recognition of microRNAs found by the present inventors through Ago HITS-CLIP analysis, as a regular seed site. It is based on the technology to modify the sequence so as to develop a technology to efficiently suppress only the non-regular target gene of the microRNA through this, and it was confirmed that the effect of the technology is to selectively show only the non-regular target inhibition, which is a function of the microRNA. In particular, when the technique was applied to miR-1, a microRNA known to be expressed mainly in muscle cells including the heart, a phenomenon in which myocardial cells were enlarged was observed.

Hereinafter, the present invention will be described in detail.

The present invention includes a total of six consecutive polynucleotides starting from any one of nucleotides 2 to 3 of the 5 'end of the gene represented by the nucleotide sequence of SEQ ID NO: 1, and among the nucleotide sequences of SEQ ID NO: 1 Cardiac hypertrophy animal, characterized in that at least one of the nucleotides 2, 3 or 7 expresses an RNA interference-inducing nucleic acid comprising a polynucleotide substituted by guanine (G) to uracil (U) A recombinant vector for model construction is provided.

In the present invention, the polynucleotide is a polynucleotide represented by the nucleotide sequence of SEQ ID NO: 2 (5'-UGAAUG-3 '); A polynucleotide represented by the nucleotide sequence of SEQ ID NO: 3 (5'-GUAAUG-3 '); A polynucleotide represented by the nucleotide sequence of SEQ ID NO: 4 (5'-GGAAUU-3 '); A polynucleotide represented by the nucleotide sequence of SEQ ID NO: 5 (5'-UAAUGU-3 '); Or it may be selected from polynucleotides represented by the nucleotide sequence of SEQ ID NO: 6 (5'-GAAUUU-3 '), but is not limited thereto.

In the present invention, the recombinant vector can efficiently load agonists by including a base sequence substituted with adenine (A) in front of the first nucleotide of the 5 'end of the polynucleotide, but is not limited thereto. If it is possible to bind to the protein, the first nucleotide at the 5 'end can be A, U, C, G all nucleotide sequences.

In the present invention, the RNA interference-inducing nucleic acid is microRNA, small interfering RNA (siRNA), short hairpin RNA (shRNA), DsiRNA, lsiRNA, ss-siRNA, piRNA, endo-siRNA, And it may be selected from the group consisting of asiRNA, but if the nucleic acid inducing RNA interference is not limited to the above type.

In the present invention, the recombinant vector is an RNA interference-inducing nucleic acid represented by the nucleotide sequence of SEQ ID NO: 7 (5'-UUG AAU GUA AAG AAG UAU GUA U-3 ');

RNA interference-inducing nucleic acid represented by the nucleotide sequence of SEQ ID NO: 8 (5'- UGU AAU GUA AAG AAG UAU GUA U-3 ');

RNA interference-inducing nucleic acid represented by the nucleotide sequence of SEQ ID NO: 9 (5'-UGG AAU UUA AAG AAG UAU GUA U-3 ');

RNA interference-inducing nucleic acid represented by the nucleotide sequence of SEQ ID NO: 10 (5'-AUG AAU GAC UGC CGG UUG UUA UG-3 ');

RNA interference-inducing nucleic acid represented by the nucleotide sequence of SEQ ID NO: 11 (5'-AGU AAU GAC UGC CGG UUG UUA UG-3 ');

RNA interference-inducing nucleic acid represented by the nucleotide sequence of SEQ ID NO: 12 (5'-AGG AAU UAC UGC CGG UUG UUA UG-3 ');

RNA interference-inducing nucleic acid represented by the nucleotide sequence of SEQ ID NO: 13 (5'-AUA AUG UAC UGC CGG UUG UUA UG-3 '); or

RNA interference-inducing nucleic acid represented by the nucleotide sequence of SEQ ID NO: 14 (5'-AGA AUU UAC UGC CGG UUG UUA UG-3 ') can be expressed, but is not limited thereto.

In the present invention, the gene represented by the nucleotide sequence of SEQ ID NO: 1 may be miR-1.

In the present invention, the polynucleotide may be a polynucleotide that recognizes a target site where a G: A wobble arrangement occurs.

The term "nucleotide" in the present invention includes nitrogen-containing heterocyclic bases, sugars and one or more phosphate groups. These are the monomer units of the nucleic acid sequence. In RNA, sugar is ribose, and in DNA it is deoxyribose, ie, a sugar that lacks the hydroxyl group present in ribose. The nitrogen-containing heterocyclic base can be a purine or pyrimidine base. Purine bases include adenine (A) and guanine (G), and modified derivatives or analogs thereof. Pyrimidine bases include cytosine (C), thymine (T) and uracil (U), and their modified derivatives or analogs. In the present invention, the sugar of the nucleotide is ribose, and the base includes adenine (A), guanine (G), cytosine (C), or uracil (U).

In the recombinant vector expressing the RNA interference-inducing nucleic acid of the present invention, the recombinant vector may include DNA, and the DNA includes a polynucleotide complementary to the polynucleotide of the RNA interference-inducing nucleic acid of the present invention. For example, if the base of the RNA interference-inducing nucleic acid is adenine (A), the thymine (T) base, if the base of the RNA interference-inducing nucleic acid is guanine (G), the cytosine (C) base, the base of the RNA interference-inducing nucleic acid is cytosine In the case of (C), the base of the guanine (G) base and the RNA interference-inducing nucleic acid may include the adenine (A) base when the base is uracil (U).

In the present invention, for example, the recombinant vector is an RNA interference-inducing nucleic acid represented by the nucleotide sequence of SEQ ID NO: 7 (5'-UUG AAU GUA AAG AAG UAU GUA U-3 ');

RNA interference-inducing nucleic acid represented by the nucleotide sequence of SEQ ID NO: 8 (5'- UGU AAU GUA AAG AAG UAU GUA U-3 ');

RNA interference-inducing nucleic acid represented by the nucleotide sequence of SEQ ID NO: 9 (5'-UGG AAU UUA AAG AAG UAU GUA U-3 ');

RNA interference-inducing nucleic acid represented by the nucleotide sequence of SEQ ID NO: 10 (5'-AUG AAU GAC UGC CGG UUG UUA UG-3 ');

RNA interference-inducing nucleic acid represented by the nucleotide sequence of SEQ ID NO: 11 (5'-AGU AAU GAC UGC CGG UUG UUA UG-3 ');

RNA interference-inducing nucleic acid represented by the nucleotide sequence of SEQ ID NO: 12 (5'-AGG AAU UAC UGC CGG UUG UUA UG-3 ');

RNA interference-inducing nucleic acid represented by the nucleotide sequence of SEQ ID NO: 13 (5'-AUA AUG UAC UGC CGG UUG UUA UG-3 '); or

RNA interference-inducing nucleic acid represented by the nucleotide sequence of SEQ ID NO: 14 (5'-AGA AUU UAC UGC CGG UUG UUA UG-3 ') can be expressed.

The DNA comprises SEQ ID NO: 15: 5'-A TAC ATA CTT CTT TAC ATT CAA-3 ';

SEQ ID NO: 16: 5'-A TAC ATA CTT CTT TAC ATT ACA-3 ';

SEQ ID NO: 17: 5'-A TAC ATA CTT CTT TAA ATT CCA-3 ';

SEQ ID NO: 18: 5'-CA TAA CAA CCG GCA GTC ATT CAT-3 ';

SEQ ID NO: 19: 5'-CA TAA CAA CCG GCA GTC ATT ACT-3 ';

SEQ ID NO: 20: 5'-AT TAA CAA CCG GCA GTA ATT CCT-3 ';

SEQ ID NO: 21: 5'-CA TAA CAA CCG GCA GTA CAT TAT-3 '; or

SEQ ID NO: 22: 5'-CA TAA CAA CCG GCA GTA AAT TCT-3 '.

In the present invention, the term "target site" refers to a target nucleotide sequence that binds to the second to eighth nucleotide sequences based on the 5 'end of the micro RNA.

In the present invention, "seed region" refers to a nucleotide sequence between 2 and 8 based on the 5 'end of the microRNA.

The term "wobble (wobble) arrangement" in the present invention, when the microRNA binds to the target mRNA, even if the target region of the microRNA is not exactly complementary to the seed region of the microRNA, Guanine Refers to a G: A sequence in which the base binds to an adenine base or a base sequence through a G: U sequence in which a guanine base binds to an uracil base, and in the present invention, the G: A Alternatively, the G: U wobble nucleotide sequence of the target gene recognized by the microRNA and the microRNA is A: U, G: C, C: G, U: A in addition to the complementary sequence of G: A or G: U. It has a base sequence.

For example, when the microRNA and the target gene recognized by the microRNA are in the wobble relationship of G: A, the base region of the origin of a specific microRNA: the alignment of the base of the target gene recognized by the specific microRNA is G: A, When the microRNA and the target gene recognized by the microRNA are in the wobble relationship of G: U, the base of a specific microRNA: The sequence of the base of the target gene recognized by the specific microRNA is G: U.

The term "recombinant" in the present invention refers to a cell in which a cell replicates a heterologous nucleic acid, expresses the nucleic acid, or expresses a peptide, a heterologous peptide or a protein encoded by a heterologous nucleic acid. Recombinant cells can express genes or gene segments not found in the natural form of the cells, either in sense or antisense form. Recombinant cells can also express genes found in natural cells, but these genes have been modified and reintroduced into cells by artificial means.

The term "vector" in the present invention means a DNA structure containing a DNA sequence operably linked to a suitable regulatory sequence capable of performing expression of DNA in a suitable host. Suitable regulatory sequences include promoters for performing transcription, any operator sequences for regulating transcription, sequences encoding suitable mRNA ribosomal binding sites, and sequences that control the termination of transcription and translation. The vector of the present invention is not particularly limited as long as it can be replicated in a cell, and any vector known in the art can be used, such as a plasmid, cosmid, phage particle, or viral vector.

In the present invention, the term “recombinant vector” is used as an expression vector of a target polypeptide capable of expressing the target polypeptide with high efficiency in a suitable host cell when the coding gene of the target polypeptide to be expressed is operably linked. The recombinant vector can be expressed in a host cell. The host cell may be preferably a eukaryotic cell, and appropriately selects an expression control sequence such as a promoter, terminator, enhancer, etc. according to the type of host cell, a sequence for membrane targeting or secretion, etc. And can be combined in various ways depending on the purpose.

The term "operably linked" used in the present invention refers to a gene that needs to be expressed and a control sequence thereof are functionally linked to each other to be linked in a manner that enables gene expression.

In the present invention, the term "promotor (promotor)" refers to a nucleic acid sequence that regulates gene expression as a DNA sequencing site to which transcription regulators bind, and is intended to induce overexpression of a target gene. Examples of the promoter include a Pribno box, a Tata box, and the like. For example, the recombinant vector of the present invention includes an alpha MHC (αMHC) promoter, a beta MHC (β MHC) promoter, an Nkx2.5 promoter, a cardiac troponin T (TNNT2) promoter, a muscle creatine kinase (MCK). ) A promoter selected from the group consisting of a promoter, a human α-skeletal actin (HAS) promoter, and a murine stem cell virus (MSCV) promoter is operably linked to myocardial cell specific expression It can be controlled, and according to an embodiment of the present invention, the alpha MHC (αMHC) promoter may be operably linked, but is not limited to the type of the promoter.

In the present invention, the term "myocardial cell-specific" refers to a characteristic that is not expressed in cells other than myocardial cells or is expressed to a low degree, while expressed in a very high degree in myocardial cells.

The term "expression" in the present invention refers to a process in which a polypeptide is produced from a structural gene, the process including transcription of a gene into mRNA, and translation of such mRNA into polypeptide (s). In addition, "overexpression" refers to a state in which transcription of a gene into mRNA and translation of such mRNA into polypeptide (s) are produced more than in the normal state.

In the present invention, the term "transformation" means that the genetic properties of a living organism are changed by introducing a vector form of DNA directly into a cell physicochemically or by infecting a virus with a host cell to express a foreign gene, that is, the gene in the host cell. It means that it can be introduced and expressed in a host cell.

In the present invention, the method of introducing a vector form of DNA into a cell is not particularly limited, such as nucleofection, transient transfection, cell fusion, liposome-mediated transfection. transfection), polybrene-mediated transfection, transfection with calcium phosphate (Graham, FL et al., Virology, 52: 456 (1973)), transfection with DEAE dextran, microinjection By transfection (Capecchi, MR, Cell, 22: 479 (1980)), transfection by cationic lipids (Wong, TK et al., Gene, 10:87 (1980)), electroporation (Neumann E et al., EMBO J, 1: 841 (1982), as described in Basic methods in molecular biology, Davis et al., 1986 and Molecular cloning: A laboratory manual, Davis et al., 1986, such as transduction or transfection. Method well known to those of ordinary skill in the art Depending on the field.

In the present invention, "cardiac hypertrophy" refers to a condition in which myocardial fibers are enlarged, an increase in heart weight, and a thickening of the ventricular wall, cardiac hypertrophy, heart failure, cardiac fibrosis, mitral valve insufficiency, Aortic valve insufficiency, dilated cardiomyopathy, ischemic heart disease, ventricular septal defect, tricuspid valve insufficiency, pulmonary valve failure, pulmonary arterial hypertension, right ventricular myocardial infarction, myocardial invasion of the right ventricle, atrial septal defect, atrial fibrillation, hypertrophy It may be an initial symptom of cardiomyopathy or infiltrative cardiomyopathy, but if it is a disease related to cardiac hypertrophy other than the above-mentioned diseases, the type is not limited.

The present invention also provides an animal model of cardiac hypertrophy characterized by transformation with the recombinant vector.

In the recombinant vector for constructing an animal model of cardiac hypertrophy of the present invention, the animal may be a mammal other than a human, for example, a non-human primate, mouse, dog, cat, rabbit, horse, or cow.

According to an embodiment of the present invention, the animal may be a mouse, but if it is a mammal other than a human, there is no particular limitation on its type.

In addition, the present invention (a) starting from any one of nucleotides 2 to 3 of the 5 'end of the gene represented by the nucleotide sequence of SEQ ID NO: 1 includes a total of six consecutive nucleotides, the SEQ ID NO: 1 Symbiosis by operably connecting DNA expressing a polynucleotide in which one or more of nucleotides 2, 3, or 7 of nucleotides are substituted with uracil (U) in guanine (G) Preparing a recombinant vector for animal model production;

(b) introducing the recombinant vector into an animal fertilized egg; And

(c) implanting the fertilized egg into a surrogate mother, and generating a fertilized egg to obtain a transformed animal model.

In addition, the present invention comprises the steps of (a) treating a candidate substance to the cardiomyocytes of the cardiac hypertrophy animal model;

(b) starting from any one of nucleotides 2 to 3 of the 5 'end of the gene represented by the nucleotide sequence of SEQ ID NO: 1 in the myocardial cell of the cardiac hyperplasia animal model, and including a total of 6 polynucleotides, Target G: A wobble sequence of polynucleotides in which one or more of nucleotides 2, 3, or 7 of SEQ ID NO: 1 is substituted with uracil (U) in guanine (G) Analyzing the frequency of binding with the site; And

(c) when the binding to the G: A wobble (wobble) array target site decreases compared to an animal model of cardiomegaly that does not process the candidate substance, the method comprising selecting the candidate substance as a therapeutic agent for cardiomegaly, It provides a method of screening for a treatment for hypertrophy.

In the present invention, the candidate substance is unknown to be used for screening to examine whether it affects the binding frequency of the polynucleotide to the G: A wobble array target site in cardiomyocytes of the cardiac hyperplasia animal model. Means a substance, and may include, but is not limited to, chemicals, proteins, (poly) nucleotides, antisense-RNA, siRNA (small interference RNA), or natural product extracts.

Hereinafter, preferred embodiments are provided to help understanding of the present invention. However, the following examples are only provided to more easily understand the present invention, and the contents of the present invention are not limited by the following examples.

Example  1. Through Ago HITS-CLIP analysis Wobble  (wobble) base sequence MicroRNA  Discovering irregular target sites allowed in the origin region

As a method of analyzing the target of microRNA at the transcript level, an experimental method called Ago HITS- CLIP has been developed and widely used. In the Ago HITS CLIP experiment, RNA and ago are used in cells by irradiating UV to cells or tissue samples. After inducing a covalent bond between nut proteins, the RNA-agonut complex thus generated is isolated by immunoprecipitation using an antibody that specifically recognizes the agonut, and then the isolated RNA is next-generation sequence (Next-generation) sequencing). The sequencing data thus obtained can not only identify the target mRNA of the microRNA through bioinformatics analysis, but also accurately analyze the binding position and sequence. Ago HITS-CLIP was applied to mouse cortical tissue for the first time to map the target of microRNA in brain tissue, and since then, the method has been applied to various tissues. These Ago HITS-CLIP analyzes revealed the location of microRNA binding in different tissues, especially similar to the initiation binding of microRNAs in these target sequences, but other forms of irregular binding targets in the sequence of the target mRNA. I learned that there are many.

It has been observed that some of the found non-normal binding laws exist as wobbles through the G: U nucleotide sequence in addition to the existing known nucleotide sequence. It has been suggested that the wobble nucleotide sequence is found in a specific RNA different from the existing nucleotide sequence, and the known G: U wobble can also be arranged in a non-canonical target of microRNA. However, in comparison, the binding was weak and the G: A wobble, which was rarely observed, could form a nucleotide sequence in a specific RNA, but was not investigated at all. However, in the case of the starting sequence of the microRNA, since the free energy of the nucleotide sequence is structurally stabilized by an agonist protein, the weak G: A nucleotide sequence may be relatively important.

Thus, first, to confirm whether the wobble nucleotide sequence including G: A is present in the microRNA target in human brain tissue, Ago HITS-CLIP is performed on the gray matter of the posterior brain tissue as shown in FIG. 1A. One data was analyzed. The analysis at this time was performed by arranging the RNA sequence bound with the agonut-microRNA in the sequenced FASTQ file to the human genome sequence with the Bowtie2 program according to the method when Ago HITS-CLIP was developed. At the same time, the sequencing results were arranged in human microRNA sequences, and 20 types of microRNAs (top20 miRNAs) frequently associated with agonist proteins in corresponding brain tissues were also analyzed. Finally, we looked at the canonical target site with complementary sequences from the 2nd to the 8th based on the 5 'end of the corresponding seed sequence for the top20 miRNA thus identified. As a result, as shown in Fig. 1B, it was observed that a large number of regular starting target sites were found at the microRNA binding site (median 1292.5 sites, error range +/- 706.62). Subsequently, it was examined in the Ago HITS-CLIP data whether there is an irregular target site capable of binding to the G: U or G: A wobble nucleotide sequence in the starting sequence of the microRNA. At this time, as a control for the analysis (Control), it was analyzed using a target sequence in which complementary alignment of the base is impossible due to the same nucleotide sequence as the microRNA originating (seed) nucleotide sequence.

As a result, as shown in Fig. 1B, G: A wobble allowed target (median value 1119.5 site, error range +/- 526.48) and G: U wobble allowed target (median value 995 site, error range +/- 523.22) ) All of the sites showed a higher distribution than the control group (median 891 site, error range +/- 410.71), and in particular, the microRNA target site with G: A wobble existed about 1.1 times more than the G: U wobble. Shown.

In addition, miR-124 specifically expressed in brain tissue (5'-UAA GGC ACG CGG UGA AUG CCA A-3 '; SEQ ID NO: 23), as shown in Fig. It has the 4th and 5th from the terminal, so that G: U and G: A wobble nucleotides can be formed. Therefore, as a result of distinguishing and analyzing the G: A and G: U wobble nucleotide sequences by G at a specific position, as shown in FIG. 1D, the miR-124's canonical target site (5'-GUGCCUU-3) '; SEQ ID NO: 24) was found the most (1,992). However, it was found that the fourth is the G: U wobble (1,800) (4G: U site), and the fourth is the G: A wobble (1,542). 4G: A site). In addition, the 5th G also showed G: U wobble (1,427) (5G: U site) and G: A wobble (1,196) irregular target sites (5G: A site). It was confirmed that all sites existed more than the number of controls (647).

Therefore, when the microRNA recognizes the target mRNA through the nucleotide sequence of the initiating region, it allows not only regular nucleotide sequence through the initiating region, but also irregularly G: U or G: A wobble nucleotide sequence. It has been found that it can bind to the target mRNA. Target recognition of the microRNA through the G: A wobble base is a new result that has not been reported, and it was confirmed that irregular microscopic target recognition and regulation in several microRNAs as observed above.

Example  2. Analysis of miR-1 through analysis of Ago HITS-CLIP results in cardiac tissue of patients with myocardial infarction G: A Wobble  (wobble) base sequence MicroRNA  Discovering irregular target sites allowed in the origin region

In Example 1, the presence of a wobble nucleotide sequence including G: A among microRNA targets in human brain tissue was confirmed, and it was confirmed whether such irregular G: A wobble nucleotide sequence binding was also present in the heart tissue of a patient with heart disease. In order to perform the analysis, data on Ago HITS-CLIP (Nucleic Acids Res. 2016 Sep 6; 44 (15): 7120-7131) was performed on left ventricular tissue of 6 patients with myocardial infarction (Cardiomyopathy).

First, in the previous study, as a result of Ago HITS-CLIP in the cardiac tissue of a myocardial infarction patient, it was found that the Ago protein-microRNA complex binds to 4000 mRNAs. Of the dogs, how much distribution of the non-normal binding G: U and G: A wobble base sequence binding sites is distributed, as well as the regular initiating binding site (mied), the binding site of miR-1, which is specifically expressed in cardiac tissue. I analyzed it. Analysis at this time, let-7 / 98 family, miR-27-3p, miR-143, miR-126 always show high expression without tissue specificity of the individual even transcript containing miR-1 target site In order to exclude the possibility of overlapping binding by -3p or the like, it was performed by limiting to a transcript having only the target site of miR-1 without including the target sites of micro RNAs as described above. In addition, the target site of miR-1 investigated in the analysis is defined as a site (seed site) that binds with the nucleotide sequence of 2 to 8 (7 base targets) based on the 5 'end of micro RNA. In the case of the non-regular G: A wobble nucleotide sequence target site, it was analyzed by first confining to the G: A wobble nucleotide sequence target of the starting portion between 2 and 8 based on the 5 'end. As an analytical control for target binding in the assay, 7 nucleotide combinations of 7 bases that can enter 52.5 nucleotides, which is the average of the sequencing lengths read by the sequencing analyzer, were reported to be 4000 transcripts that were reported to bind to the Ago protein 7 Substituted as a base target, a mathematically calculated value was used.

As a result, as shown in Fig. 2A, 120 normal target sites composed of only 7 bases of miR-1 were found in the heart tissue of patients with myocardial infarction. The number of non-canonical G: A wobble base sequence target sites of miR-1 present at the initiation site is 55, which is about 45% of the normal target site, and is a random 7 base target calculation value used as a control, 11.23. It was observed to be higher than the dog. This shows that the frequency of non-regular G: A wobble nucleotide sequence target binding is high even when compared to the result of analysis of one target binding of miR-124 that is not expressed at all in the heart tissue. Therefore, even in the heart tissue of patients with myocardial infarction, miR-1 It was confirmed that the binding was performed with an irregular G: A wobble base sequence.

 Subsequently, the non-regular G: A wobble base sequence target site of miR-1 was analyzed for the position of each G base. As a result, as shown in FIG. 2B, there are 21 non-regular G: A wobble base sequence targets (G2A) for the second G base, which corresponds to 38% of the analyzed non-standard G: A wobble base sequence, and the third G There are 12 non-canonical G: A wobble nucleotide targets (G3A) for the base, 22% of the non-canonical G: A wobble nucleotide sequence analyzed, and the non-canonical G: A wobble nucleotide target (G7A) site for the 7th G base. Is 22, which corresponds to 40% of the analyzed irregular G: A wobble base sequence. The non-canonical G: A wobble base binding target present at the normal foot unit value was highest for the non-canonical G: A wobble base sequence target for the 7th G base.

Through this, in the heart tissue of patients with myocardial infarction, there is an irregular G: A wobble base sequence target binding in addition to the miR-1 regular target binding, and in the case of a normal initiation position, the 7th G base allows A and wobble base sequence. The most irregular binding was observed. Through these observations, it was confirmed that the target expression control function of miR-1 can be mediated through non-canonical target binding as well as regular target binding.

Example  3. Cardiomyocyte cell line  ( H9c2 ) Using a fluorescent protein reporter miR -1 non-regular G: A  Array inception target  Confirm the effect of suppressing gene expression

After discovering that in Example 1 and 2 above, the microRNA can bind to the target mRNA by allowing the G: A wobble sequence in the initiating sequence, and whether such binding can actually cause inhibition of the target gene. Reporter experiments were performed at the cellular level. At this time, the confirmation experiment was conducted on miR-1, which is known to be highly expressed in muscle system cells, such as the cardiomyocyte cell line H9c2, and as observed in Example 2, the 7th from the 5 'end of miR-1 Focusing on the G present in, it was performed on the G: A wobble nucleotide sequence through it. In order to more accurately measure gene inhibition by microRNA, a fluorescent protein expression reporter system was constructed and used. The fluorescent protein expression reporter system was constructed so that two colors of fluorescent proteins were expressed in one vector, where the green fluorescent protein (GFP) was expressed in the SV40 promoter, and the 3 'non-global portion (3') The target sites of micro RNA were continuously arranged in the untranslated region: 3'UTR) to measure the gene suppression effect by the microRNA through the intensity of the green fluorescent protein, and at the same time, the red fluorescent protein (red fluorescent protein) : RFP) was expressed by the Tk promoter, and the green fluorescence signal was standardized and used.

A non-canonical target site that is complementary to bases 2 to 8 from the 5 'end of miR-1 in the fluorescent protein expression reporter vector constructed as described above, and base-arranges with a G: A wobble for the 7th base G ( 7G: A-site) (5'p-AAAUUCCA-3 '; SEQ ID NO: 25) was inserted to prepare a reporter (GFP-7G: A site), and it was confirmed through experiments that it was inhibited by miR-1 Did. At this time, the experiment simultaneously introduced a 500 ng GFP-7G: A site reporter vector and 25 nM miR-1 into the cardiomyocyte cell line H9c2 according to the protocol provided by the company using lipofectamine 2000 (Invitrogen) reagent (co- transfection), and each fluorescence signal was measured by flow cytometry through Attune NxT of Life Technology 24 hours later.

As a result, as shown in FIG. 3A, the control nucleic acid (cont; NT; cel-miR-67) inhibits the 7G: A site of miR-1 very efficiently (in the middle of FIG. 3A) by the expression of miR-1. duplex) was introduced and compared (left side of FIG. 3A). That is, as a result of analyzing into four parts (Q5-1, Q5-2, Q5-3, Q5-4) according to the expression level of the two fluorescent proteins in the cardiomyocyte cell line H9c2, in the case of cells introduced with miR-1, the control group reduced to: (Q5-3 miR-1) 41.80 % from: (Q5-3 control) the distribution of the cells 10 shown the ^3rd century red fluorescent protein and green fluorescent protein of the intensity of 10 ³ than if the nucleic acid is introduced into 59.51% It was observed.

From the above, it was observed in the control that the GFP-7G: A site reporter vector was inhibited to some extent in H9c2 cells without the introduction of miR-1. This can be expected to be suppressed by miR-1 already expressed in H9c2 cells through G: A wobble nucleotide sequence with the reporter target mRNA. To confirm the above, a miRIDIAN microRNA Hairpin inhibitor (miR-1 inhibitor, right of FIG. 3A) capable of inhibiting the expression of miR-1 in H9c2 cells was purchased from Dharmacon and used by transfecting the cells at a concentration of 50 nM. Did.

As a result, when the introduction of the miR-1 inhibitor than the control group, 10 ³ and 10 ^3-intensity red fluorescence protein intensity of the green fluorescent protein showing the distribution is 81.56% of the cells of the: increased (miR-1 inhibitor Q5-3) From the results, it was confirmed that miR-1 present in the cell can inhibit gene expression through the 7G: A site of miR-1.

In addition, the control (GFP-no site) without a microRNA target site in the fluorescent protein expression reporter and GFP-7G: A site, a reporter containing the 7G: A site of miR-1, were introduced into H9c2 cells to compare their activity. It was confirmed by quantitative comparison with the case where miR-1 was introduced together as a positive control. In this case, the red fluorescence protein (RFP) value measured by the flow cytometer was divided by section, and the green fluorescence protein (GFP) value at this time was averaged and converted to a log ratio, where the green fluorescence of the control GFP-no site The protein level was set to 1 (relative log GFP) and calculated relative to it.

As a result, as shown in Fig. 3B, for miR-1 and the 7th G base, there is a site that is complementary to the base through G: A wobble (GFP-7G: A site), in particular It was confirmed that the expression of the green fluorescent protein (relative log GFP) through G: A wobble was lowered by 12.5% than 1 in the section where the expression of the red fluorescent protein (log RFP) of 1.5 to 2.5 was seen. In the positive control group, it was observed that the expression of the green fluorescent protein (relative log GFP) of about 40% to 25% in the red fluorescent protein expression (log RFP) of 1.5 to 2.5 sections was the same as the pattern of inhibition. .

In order to confirm that the results observed above are not affected by the difference in the intensity of the different promoters used in the corresponding fluorescent protein reporter and the fluorescence activities of the two different fluorescent proteins, the two fluorescent proteins are exchanged with each other. Protein reporter p.UTA.3.0 was purchased from Addgene (plasmid # 82447) and used. In this case, the reporter experiment was repeated after inserting the miR-1-7G; A sequence 5 times into the 3 'UTR portion of the red fluorescent protein (RFP) controlled by the SV40 promoter in the p.UTA.3.0 vector. The reporter vector (RFP-7G: A site) was prepared and the expression value of red fluorescent protein was standardized by reflecting the degree of introduction of green fluorescent protein (GFP) expressed by the Tk promoter into cells. It was used by modification.

As a result, as shown in Fig. 3c, as described above, the 7G: A site of miR-1 is inhibited by miR-1 expressed in H9c2, and the control group of the reporter (RFP-no site) without the miRNA target site The results of the reporter (RFP-7G: A site) containing the 7G: A site of miR-1 were compared and confirmed, and at the same time, as shown in FIG. 3D, the positive control group also miR-1 in co-transfection with miR-1. The phenomenon of inhibiting the -7G: A site was observed.

From the above, it can be seen that the non-normal target of the microRNA discovered through the Ago HITS-CLIP analysis can actually bind to the microRNA's initiation sequence through the G: A wobble array, and suppress the expression of the target gene. Confirmed.

Therefore, from this point, it is possible to sufficiently bind to the microRNA using the target site through the G: A wobble sequence, and this point is via the G: A wobble sequence control specific substitution nucleic acid (miRNA-GU). It could be inferred that, if similarly inducible, only the biological function of microRNAs by non-canonical target inhibition could be utilized.

Example 4. Luciferase  Through reporter experiments miR - 1 variant MicroRNA  Irregular G: A Wobble  Array inception Suppressive effect on the site  Confirm

Based on the fact that in Examples 1, 2, and 3, miR-1 irregularly permits G: A wobble alignment in the initiation sequence, binds to the target mRNA and inhibits the expression of the target gene, only these irregular targets We wanted to investigate the potential for this other biological function. For this purpose, miR-1, which recognizes both a regular initiation target and an irregular G: A wobble sequence target, cannot be used, so artificially the second guanine base from the 5 'end of miR-1 is uracil. Substituted with miR-1-2GU (5'-UGAAUG-3 '; SEQ ID NO: 2), miR-1-3GU (5'-GUAAUG-3) with 3rd guanine base substituted with uracil '; SEQ ID NO: 3), irregular G including the sequence of miR-1-7GU (5'-GGAAUU-3'; SEQ ID NO: 4) with the 7th Guanine base replaced by Uracil: It was designed as shown in Fig. 4a so as to align only the A wobble array target.

Additionally, the following experiment was performed to confirm whether miR-1 modified microRNA of the sequence shown in FIG. 4A can actually inhibit an irregular G: A wobble alignment target. First, on the guide strand, the same sequence as miR-1 is synthesized, but RNA with 2, 3, or 7 guanine bases substituted with uracil from the 5 'end is synthesized, and a modification is applied to the carrier strand. It was prepared by synthesizing the same sequence of unsupported miR-1. This RNA was chemically synthesized through ST Pharma, Trilink Technologies or Bioneer, separated by HPLC, and prepared as a duplex of the guide strand and the carrier strand according to the method provided by the company.

The target inhibitory effect of miR-1 modified microRNA of FIG. 4A synthesized by the above method was measured by a luciferase reporter.

In other words, the 75 nM siRNA prepared in the above-described duplex is a HeLa cell using a Lipofectamine 2000 reagent (Invitrogen) using a psi-check2 vector expressing Renilla luciferase in HeLa cells (ATCC CCL-2). The results were investigated by co-transfection. At this time, in the 3'UTR (3'untranslated region) portion, complementary to the base from the 5 'end of the miR-1 to the 2nd to 8th base, while the 2nd (Fig. 4b), the 3rd (Fig. 4c), or For the 7th (FIG. 4D) base G, a reporter inserting a non-canonical target site nucleotide sequenced with a G: A wobble was used.

In addition, in the experiment, HeLa cells were cultured in Dulbecco's modified Eagle's medium (Invitrogen) supplemented with 10% fetal bovine serum (FBS), 100 U / ml penicillin, and 100 μg / ml steptomycin, and transduced in complete medium without antibiotics. Speculation was performed. 24 hours after transfection, the effect of siRNA was investigated by measuring the activity of luciferase according to the manufacturer's protocol using Promega's Dual-luciferase reporter asssay system. It was standardized and calculated by the activity of firefly luciferase in at least 3 repeated experiments using a Glomax Luminometer.

As a result of measuring the inhibitory effect on an irregular G: A wobble array target at various concentrations and examining IC ₅₀ (inhibitory concentration 50), FIG. 4B (IC ₅₀ = 0.2nM), FIG. 4C (IC ₅₀ = 2.2nM), and As shown in Figure 4d (IC ₅₀ = 4nM), when replacing the 2, 3, or 7th guanine base from the 5th end of miR-1 with uracil (miRNA-GU), the luciferase reporter It was found that the irregular G: A wobble sequence initiation site was specifically inhibited.

Example  5. miR Irregular for the 2, 3, 7th G base of -1 G: A Wobble  Confirmation of the hypertrophy-inducing effect of myocardial cells through the regulation of the sequence-targeting target gene

5-1. Preparation for the experiment

The non-regular gene suppression phenomenon identified in Example 3 is a G: A wobble sequence control specificity verified in Example 4 in which the possibility that the G: A wobble base sequence acts as a non-regular target of the microRNA and can also regulate biological functions. In order to confirm through an appropriately substituted nucleic acid (miRNA-GU), experiments were conducted mainly on miR-1 of the cardiomyocytes examined in Examples 2 and 3 above.

In order to investigate whether an irregular G: A wobble target has a specific biological function, miR-1 must recognize a non-regular G: A wobble array target site, not a regular target. The miRNA-GU produced by substituting the corresponding G with U so that the G in the initiation region was aligned with A rather than C was used in the experiment. In this experiment, in order to perform a more physiologically similar condition to the heart, a new white paper myocardial cell (neonatal cardiomyocyte) was cultured from a rat heart tissue. At this time, the culture of new white paper cardiomyocytes is performed by separating new white paper myocardial cells through an enzyme reaction from the heart tissue of rat neonate on day 1 (Cellutron's neomyt kit), and the new white paper cardiomyocytes are 10% FBS (fetal Dulbecco's modified Eagle's medium (Invitrogen) and M199 (WellGene) medium supplemented with bovine serum), 5% HS (horse serum), 100 U / ml penicillin, and 100 μg / ml steptomycin, prepared in a 4: 1 medium The brdU was added to the cells, and cultured separately from other cells having a cell cycle present in the heart tissue.

5-2. miR -1 decrease in myocardial cell size confirmed

It was preferentially confirmed whether myocardial hypertrophy is induced in the new white paper myocardial cells obtained from Example 4-1. Myocardial cells have the characteristic of stopping the cell cycle and increasing the size of myocardial cells, which is called myocardial hypertrophy. Myocardial hypertrophy has played a role in compensating for myocardial cell dysfunction, also known as an intermediate stage of pathology of heart disease, and is a heart disease model system well known for cell morphological observation and gene expression profile changes. In the cell model of myocardial hypertrophy, myocardial hypertrophy can be induced by treating adrenal receptor receptor agonists such as phenylephrine (PE) to myocardial cells.

Therefore, the cultured new white paper myocardial cells were treated with 100 μM of phenylephrine to induce myocardial hypertrophy, and as a result, the size of the new white paper myocardial cells compared to the control group (rCMC), which was not treated as shown in FIG. The increase was confirmed morphologically (+ PE). In addition, it was observed that the size of the myocardial cells decreases when miR-1 is introduced into the new white paper myocardial cells. This is consistent with the well-known myocardial hypertrophy and miR-1 function in myocardial cells.

5-3. miR Confirmation of induction effect of -1 substituent on myocardial cell hypertrophy

miR-1 contains 3 G bases in the initiation region, and thus miR- substituted with U in order to recognize only the irregular G: A wobble sequence target site without recognizing the regular target. 1 was designed, and according to the position where the G base is located, in the 2nd position (miR-1-G2U), 3rd position (miR-1-G3U), and 7th position (miR-1-G7U) based on the 5 'end The miR-1 substituent was synthesized by application. At this time, the substituted base sequence of miR-1 used is as follows (miR-1-G2U: 5'p-UUGAAUGUAAAGAAGUAUGUAU-3 ', SEQ ID NO: 7; miR-1-G3U: 5'p-UGUAAUGUAAAGAAGUAUGUAU-3' , SEQ ID NO: 8; miR-1-G7U: 5'p-UGGAAUUUAAAGAAGUAUGUAU-3 ', SEQ ID NO: 9), synthesized as a guide strand, 2, 3, 7th base from the 5' end of the substituted miR-1 The carrier strand (hsa-miR-1-5p; miRBase accession number: MIMAT0031892, 5'-ACAUACUUCUUUAUAUGCCCAU-3 '; SEQ ID NO: 26) that forms a duplex with the guide strand is not completely complementary for humans recorded in miRBase By designing the miR-1 sequence of After chemically synthesized and produced by Bioneer, it was separated by HPLC and used as a duplex of guide and carrier strands according to the method provided by the company. The transfection of the microRNA into the white paper heart muscle cell line was performed at a concentration of 50 nM using RNAimax (Invitrogen) reagent.

As a result, as shown in FIG. 5A, when the miR-1-G2U or miR-1-G3U or miR-1-G7U was introduced into the new white paper myocardial cell culture, the new white paper myocardial cell morphology induced myocardial hypertrophy. It was observed that the level increased to (+ PE). At this time, in order to quantitatively analyze each cell size, more than 100 cells were quantified through the Image J program (NIH), and synthesized to recognize only the G: A wobble array site of miR-1 as shown in FIG. 5B. The miR-1 substituents (miR-1-G2U, miR-1-G3U, miR-1-G7U) were analyzed to be about 1.5 to 1.8 times larger in size compared to when the control (NT) nucleic acid was introduced. . This is similar to the myocardial cell size of the myocardial hypertrophy model induced by the phenylephrine (PE) drug, and miR-1 itself shows the effect of reducing the cell size in the form of myocardial cells, thereby preventing its regular target suppression effect. It was observed that it exhibits different functions in cell morphology than target inhibition through G: A wobble.

In addition, in order to confirm the myocardial hypertrophy observed in this example at the molecular level, the expression of the ANP (atrial naturiuretic peptide) gene, which is known to increase its expression as a marker, is expressed through GAPDH through qPCR (Quantitative Polymerase Chain Reaction) experiment. Compared with and measured by relative value.

As a result, as shown in FIG. 5C, a miR-1 substituent (miR-1-G2U, miR-1-G3U, miR-1-G7U) synthesized to recognize only the G: A wobble sequence site of miR-1 was newly developed. When introduced into myocardial cells, it was confirmed through four repeated experiments that the ANP expression was significantly increased by 1.2 to 1.5 times compared to the control (NT). The increase in ANP expression was less than that of the phenylephrine (PE) drug-treated cardiomyocyte test group, which was about 2 times larger in size than the control group, but ANP expression was significantly reduced when the original miR-1 was introduced. In contrast to this phenomenon, it can be seen that miR-1 exhibits completely different biological functions through the G: A wobble sequence site.

Example  6. miR Irregular for the 2nd, 3rd and 7th G bases of -1 G: A Wobble  Confirmation of the hypertrophy-inducing effect of myocardial cells through a minimal sequencing function that regulates the target gene sequence

6-1. miR Irregular for the 2nd, 3rd and 7th G bases of -1 G: A Wobble  Array inception target  Definition of minimum sequencing with gene regulation

In Example 5, it was confirmed that non-regular G: A wobble sequence initiation target regulation of miR-1's 2, 3, and 7 G bases can induce hypertrophy of myocardial cells, which is completely different from the conventional normal target inhibition. Could. Subsequently, in the base sequence of the replacement nucleic acid that allows only the non-regular G: A sequence for the 2nd, 3rd, and 7th G bases of miR-1, an experiment was conducted to define the minimum base sequence that induces myocardial hypertrophy.

This is essential information for selectively modulating the biological function of the microRNA by the G: A wobble target. Therefore, the base sequence from 2 to 7 of the 5 'end known as the regular starting site of miR-1, and the 9th to 23th bases are used as a control NT (cel-miR-67-5p: 5' -CGC UCA UUC UGC CGG UUG UUA UG-3 ', has the ribonucleic acid base sequence of base sequence 27), and in the case of the 8th base, changes to A base to make it different from the miR-1 base sequence, the 1st base is miR Although different from the -1 nucleotide sequence, RNA substituted with A base was designed as a guide strand for efficient agonist loading and synthesized in Bioneer as in Example 4 above. This was named miR-1_2-7, and its base sequence was shown in FIG. 5A and was as follows (miR-1_2-7: 5'-AGG AAU GAC UGC CGG UUG UUA UG-3 '; SEQ ID NO: 28).

For ribonucleic acid miR-1_2-7, which contains only the minimum target base sequence of miR-1, ribonucleic acid was designed by replacing the 2nd, 3rd, and 7th G base positions with U bases capable of G: A alignment, respectively. , This nucleotide sequence can be defined as the minimum nucleotide sequence that enables the G: A sequence at the 2nd, 3rd and 7th G base positions of miR-1. Each substitution base sequence for the target base sequence of miR-1_2-7 is shown in FIG. 5A as follows (miR-1_2-7_G2U: 5'-AUG AAU GAC UGC CGG UUG UUA UG-3, SEQ ID NO: 10; miR -1_2-7_G3U: 5'-AGU AAU GAC UGC CGG UUG UUA UG-3 ', SEQ ID NO: 11; miR-1_2-7_G7U: 5'-AGG AAU UAC UGC CGG UUG UUA UG-3', SEQ ID NO: 12).

In addition, the minimum target nucleotide sequence of miR-1 may also consist of 6 consecutive nucleotide sequences from 3 to 8 based on the 5 'end. Therefore, the 6th nucleotide sequence from the 3rd to the 8th of miR-1 is used as a control, arranged at the 2nd to 7th position based on the 5 'end of the NT ribonucleic acid base sequence (SEQ ID NO: 27), and the 1st base and 8th base. The second base was substituted with A base to have a different base sequence from miR-1, and ribonucleic acid having NT base sequence from 9 to 23 was devised, which was miR-1_3-8 (5'-AGA AUG UAC UGC CGG UUG UUA UG -3 '; SEQ ID NO: 29).

Based on this, RNAs in which the positions of the 3rd and 7th G bases of miR-1_3-8 were replaced with U bases capable of G: A alignment were designed, and this nucleotide sequence was 3rd and 7th G of miR-1. It can be defined as another minimal nucleotide sequence that enables G: A alignment at the base position. Each substitution base sequence for the base sequence of miR-1_3-8 is shown in FIG. 6A and is as follows (miR-1_3-8_G3U: 5'-AUA AUG UAC UGC CGG UUG UUA UG-3 ', SEQ ID NO: 13; miR -1_3-8_G7U: 5'-AGA AUU UAC UGC CGG UUG UUA UG-3 ', SEQ ID NO: 14). Designed as a guide strand, chemically synthesized and produced by Bioneer as in Example 5, separated by HPLC, and prepared and used as a duplex of the guide strand and the carrier strand according to the method provided by the company. Did.

6-2. Irregular G: A Wobble  Array inception target  Confirmation of hypertrophy-inducing effect of myocardial cells through minimal sequencing with gene regulation

The transfection of the microRNA prepared in Example 6-1 into the cardiomyocyte cell line (H9c2) was performed at a concentration of 50 nM using RNAimax (Invitrogen) reagent.

As a result of observing changes in cell morphology, miR-1_2-7 and miR-1_3-8 cells containing 6 nucleotide sequences, known as the minimum target nucleotide sequence, were introduced as shown in FIG. 6B. Similarly, it was confirmed that the cell size was reduced. On the other hand, miR-1_2-7_G2U, miR-1_2-7_G3U, miR-1_2-7_G7U, miR-1_3-8_G3U, miR-1_3-8_G7U In the case of the introduced cells, hypertrophy was observed compared to the cells into which the control group NT (H9c2) was introduced. Through this, the minimum target nucleotide sequence consisting of 6 nucleotide sequences in the normal target regulation function of miR-1 could be confirmed through a change in cell morphology. Through this, it was confirmed through observation of cell hypertrophy that it showed a new function, which is completely different from the existing normal target control function.

In addition, in order to quantitatively analyze the target regulatory function of the substituted nucleic acid introduced in Example 6-1, the size of about 50 cells per experimental group was quantified through the Image J program (NIH). As a result, as shown in Figure 6c, miR-1_2-7, miR-1_3-8, miR-1 introduced cells were all reduced by about 23% compared to when the control (NT) nucleic acid was introduced. I could confirm. On the other hand, nucleic acids miR-1_2-7_G2U, miR-1_2-7_G3U, miR-1_2-7_G7U, miR-1_3-8_G3U, miR-1_3-8_G7U, which were synthesized by recognizing only the G: A wobble sequence site, were introduced. In the case of cells, it was analyzed that the size of the control (NT) nucleic acid was increased by about 2.5 times compared to the introduced cells. This is similar to the myocardial cell size change (Fig. 4b: about 1.5 times) showing the treatment of phenylephrine (PE), a drug for inducing myocardial hypertrophy, among the experimental groups performed in Example 5, miR-1_G2U, miR-1_G3U , As a result similar to the myocardial hypertrophy induced by miR-1_G7U substitution nucleic acid (Fig. 5b: about 1.5 to 1.8 times), it can be said that the minimum nucleotide sequence functioning through the G: A wobble sequence of miR-1 was defined. .

From the above, it was confirmed that the non-canonical target inhibiting through the G: A wobble sequence in the microRNA origin region may exhibit a biologically different function from the existing normal target recognition, and the minimum nucleotide sequence that performs this function Could be defined. These findings indicate that the function through the regular target of the microRNA and the function through the irregular target are different, and in view of this, if only the G: A wobble target of the microRNA can be suppressed, the G: A wobble target It has been inferred that only the biological function caused by this can be selectively controlled. In addition, selective control of G: A wobble target regulation using minimal nucleotide sequences was expected to be more effective.

Example  7. miR -1 non-regular G: A Wobble  Array inception target  Preparation of recombinant vector for image specific expression of regulatory nucleic acid and production of transgenic mouse model

7-1. Recombinant vector preparation

In Examples 5 and 6, when a nucleic acid that selectively inhibits only the 2, 3, and 7th G: A sequence development target gene regulation of miR-1 is introduced into the myocardial myocardial cell and cardiomyocyte cell line, myocardial hypertrophy occurs. It was observed that the regulation of the normal initiation target has a new function that is completely different from the induction function. Based on this, an experiment was conducted to observe the miR-1 G: A sequence development target gene regulation function at the animal individual level, and miR-1's G: A sequence development target gene regulation nucleic acid (miR-1_G7U, sequence) Regarding No. 9), a mouse was specifically expressed and transformed into myocardial cells of a mouse to prepare a mouse.

Of the various cells that make up the heart tissue of the mouse, the cells responsible for the contraction of the heart muscle are called cardiomoycytes. The specialized function of myocardial cells that contract muscles in response to beats is regulated by transcription factors that are specifically expressed in myocardial cells, and studies of cardiomyocyte specific promoters dedicated to their expression have been conducted in the heart of mice. It is well-studied according to the timing and location of development. The most important choice in the production of transgenic mice expressing miR-1's 7th G: A sequence-targeting gene regulatory nucleic acid (miR-1_G7U) specifically in myocardial cells is the myocardial cell specificity of this substituted nucleic acid. It is the choice of promoters that regulate expression. During the development of the mouse, cardiac tissue is immature from the 8th day of the embryo (E8 day), when the linear heart (cardiac tube) is completed, but develops early enough to begin contraction, so the miR-1 targets the 7th G: A array. When the expression of the gene regulatory nucleic acid (miR-1_G7U) affects the cardiac development process due to the regulation of the embryonic stage promoter, the production of transgenic mice can be difficult as well as studies of its function. Accordingly, a gene recombination vector was produced using an αMHC promoter among promoters according to various development periods. The αMHC promoter is a part of the mouse genome that has been used since the publication of Dr. Jeffrey Robinson's thesis in 1991 (Subramaniam A et al, JBC, 1991, 266 (36): 24613-20) .From the last exon portion of βMHC, αMHC It consists of a total of 5.5 kb up to the third non-coding exon of. It is known that the expression time is continuously expressed from the 1st day of life. The vector containing the αMHC promoter was purchased from Adgene at pJG / αMHC (addgene reference: 55594).

The vector gene after the third non-coding exon of αMHC was recombined with the target gene regulatory nucleic acid (miR-1_G7U) at the 7th G: A sequence of miR-1, which is shown in FIG. 7A. The target gene regulatory nucleic acid of the 7th G: A sequence of miR-1 (miR-1_G7U: miR-1_G7T afterwards) extracts ribonucleic acid from the heart tissue of the mouse and expresses it by making it cDNA through superscript III reverse transcriptase (Invitrogen) After making the transcript, chromosome 284 nucleotide sequence containing miR-1 precursor hairpin (precursor hairpin: 77 bases) at position 2 is polymerase chain-reacted (PCR) to agarose The band was confirmed by electrophoresis on the gel, and shown in FIG. 7B (No. 1 in FIG. 7B). Then, in order to replace the G base at position 7 of miR-1 with a T base, PCR was performed from position 1 to position 7 of miR-1 in the 284 base sequence to confirm the band by electrophoresis (2 in FIG. 7B). , No. 3), conduct PCR from position 7 of miR-1 to No. 284 to confirm the band by electrophoresis (No. 4, 5 in Fig. 7B), and then No. 2, 3, 4, 5 in Fig. 7B. Target gene regulatory nucleic acid from the 7th G: A sequence of miR-1 with 284 nucleotide sequence as template (5'- TGG AAT TTA AAG AAG TAT GTA T-3 '; SEQ ID NOs: 30, 5'-ATA CAT ACT TCT TTA AAT TCC A-3 '; SEQ ID NO: 17) was prepared to be included (Fig. 7b, No. 6). The thus prepared miR-1 7th G: A wobble sequence initiation target gene regulatory nucleic acid (miR-1_G7T) was recombined into the pJG / αMHC vector through the SalI / HindIII restriction enzyme site (pJG / αMHC-miR-1_G7T).

7-2. Transgenic mouse production

When constructing a transgenic mouse, the recombinant vector prepared in Example 7-1 is injected into the nuclear part of the fertilized egg of the mouse and mixed with the mouse gene, and developed into an individual through a surrogate mother. In order to check whether the mice born in this way have a recombinant vector in the genome, it is necessary to extract nucleic acids (genomicDNA) from each mouse and screen them (positive genotyping). For efficient screening, an untranslational region (UTR) of human growth hormone (UTR) contained in the recombinant vector is used, which is natural except for mice in which the recombinant vector has been transgened. This is because the mouse genome does not contain the non-global human growth hormone region. Therefore, as shown in FIG. 7A, a recombinant vector (pJG / αMHC-miR-1_G7T) was prepared, and then an expression cassette of about 6 kb containing a promoter, miR-1_G7T of 284 base size, and a non-global region of human growth hormone was added to the recombinant vector. It was cut linearly through the included restriction enzyme BamHI. About 155 nucleotide sequences are obtained by polymerase chain reaction (PCR) reaction through a primer specifically binding to the human growth hormone non-global region contained in this linear cassette to amplify the band. It was confirmed by electrophoresis on a rose gel, which is shown in Figure 7c. Through this, it was confirmed that the mouse transformed with the recombinant vector (pJG / αMHC-miR-1_G7T) can be effectively screened through identification of a specific 155 nucleotide sequence of human growth hormone poly A. .

In addition, a linear cassette of about 6 kb was cut, microinjected into the pronuclei of the fertilized egg, and then transplanted into a surrogate mouse. Subsequently, the transformed mouse was selected by polymerase chain reaction (PCR) through the screening primer, and as shown in FIG. 7D, 155 sequencing bands were found in 2 out of 12 genomes, and a total of 9 mice In the transgenic mouse (founder), a 155 nucleotide sequence specific to human growth hormone poly A was identified. Based on this, a transgenic mouse expressing miR-1_G7T nucleic acid specifically for the heart was prepared.

Example  8. Heart-specific expression miR -One_ With G7T  Of transgenic mice Cardiomegaly  Check the induction effect

Among the total of 9 transgenic mice described in Example 7, 4 44 transgenic mice (founders) corresponding to about 44% were observed to die between 56 and 73 days of age. The cause of death was unknown, but as a result of observing the heart tissue from the recovered carcass, as shown in FIG. 8A, a heart filled with the inside of the rib cage was observed (left side in FIG. 8A), and this was tibia length. When the heart weight and tibia length ratio (HW / TL) were compared by normalizing to, the heart weight and shin obtained from 10 mice in a natural age at a similar age as shown in FIGS. 8B and 8C. It was quantitatively analyzed to be about twice as large as the tibia length ratio (HW / TL).

Also, in the form of a heart, the left ventricular wall was enlarged, and specifically, in the case of the right ventricle, it was visually confirmed that it was significantly enlarged unlike the normal heart structure. It was estimated that there were pathological symptoms such as pulmonary circulation disorder and pulmonary hypertension (middle, right in FIG. 8A).

As a result of observing the survival to date for a total of 9 transgenic mice (founder), as shown in FIG. 8D, 4 died at the age of about 2 months of age and showed a survival rate of about 56%.

In addition, the mouse transformed with a vector expressing the miR-1_G7T specifically expressing cardiac tissue by inhibiting the G: A wobble array target by replacing the 7th guanine of miR-1 with uracil (Fig. 9A, left figure) It was observed that the myocardial hypertrophy was induced in both mice from two different parental entities (founder) in the next generation F1 while maintaining the generation of the corresponding transgenic mouse (Fig. 9a, right figure), HW / TL (heart Differences were also confirmed in weight / tibia length) and HW / BW (heart weight / body weight) ratio measurements (FIGS. 9B-C).

Finally, the transgenic mouse overexpressing the 7th G: A sequence initiation target gene regulatory nucleic acid (miR-1_G7T) of miR-1 specifically in the postnatal cardiac tissue through the above Example is that cardiac hypertrophy is induced at the individual level. It was observed that it could ultimately be used as a cardiac hypertrophy animal model.

The above description of the present invention is for illustration only, and a person having ordinary knowledge in the technical field to which the present invention pertains can understand that it can be easily modified into other specific forms without changing the technical spirit or essential features of the present invention. will be. Therefore, it should be understood that the embodiments described above are illustrative in all respects and not restrictive.

<110> Korea University Industry and Academy Cooperation Foundation <120> Recombinant vector for producing animal model with cardiac          hypertrophy, transformed model with cardiac hypertrophy by the          recombinant vector and a method of production thereof <130> MP18-166 <160> 30 <170> KoPatentIn 3.0 <210> 1 <211> 22 <212> RNA <213> Homo sapiens <220> <221> gene <222> (1) .. (22) <223> miR-1 <400> 1 uggaauguaa agaaguaugu au 22 <210> 2 <211> 6 <212> RNA <213> Artificial Sequence <220> <223> 2 ~ 7 of miR-1-G2U (miR-1-2GU) <400> 2 ugaaug 6 <210> 3 <211> 6 <212> RNA <213> Artificial Sequence <220> <223> 2 ~ 7 of miR-1-G3U (miR-1-3GU) <400> 3 guaaug 6 <210> 4 <211> 6 <212> RNA <213> Artificial Sequence <220> <223> 2 ~ 7 of miR-1-G7U (miR-1-7GU) <400> 4 ggaauu 6 <210> 5 <211> 6 <212> RNA <213> Artificial Sequence <220> <223> 3 ~ 8 of miR-1-G3U <400> 5 uaaugu 6 <210> 6 <211> 6 <212> RNA <213> Artificial Sequence <220> <223> 3 ~ 8 of miR-1-G7U <400> 6 gaauuu 6 <210> 7 <211> 22 <212> RNA <213> Artificial Sequence <220> <223> miR-1-G2U <400> 7 uugaauguaa agaaguaugu au 22 <210> 8 <211> 22 <212> RNA <213> Artificial Sequence <220> <223> miR-1-G3U <400> 8 uguaauguaa agaaguaugu au 22 <210> 9 <211> 22 <212> RNA <213> Artificial Sequence <220> <223> miR-1-G7U <400> 9 uggaauuuaa agaaguaugu au 22 <210> 10 <211> 23 <212> RNA <213> Artificial Sequence <220> <223> miR-1_2-7_G2U <400> 10 augaaugacu gccgguuguu aug 23 <210> 11 <211> 23 <212> RNA <213> Artificial Sequence <220> <223> miR-1_2-7_G3U <400> 11 aguaaugacu gccgguuguu aug 23 <210> 12 <211> 23 <212> RNA <213> Artificial Sequence <220> <223> miR-1_2-7_G7U <400> 12 aggaauuacu gccgguuguu aug 23 <210> 13 <211> 23 <212> RNA <213> Artificial Sequence <220> <223> miR-1_3-8_G3U <400> 13 auaauguacu gccgguuguu aug 23 <210> 14 <211> 23 <212> RNA <213> Artificial Sequence <220> <223> miR-1_3-8_G7U <400> 14 agaauuuacu gccgguuguu aug 23 <210> 15 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> miR-1-G2U reverse complement <400> 15 atacatactt ctttacattc aa 22 <210> 16 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> miR-1-G3U reverse complement <400> 16 atacatactt ctttacatta ca 22 <210> 17 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> miR-1-G7U reverse complement <400> 17 atacatactt ctttaaattc ca 22 <210> 18 <211> 23 <212> DNA <213> Artificial Sequence <220> <223> miR-1_2-7_G2U reverse complement <400> 18 cataacaacc ggcagtcatt cat 23 <210> 19 <211> 23 <212> DNA <213> Artificial Sequence <220> <223> miR-1_2-7_G3U reverse complement <400> 19 cataacaacc ggcagtcatt act 23 <210> 20 <211> 23 <212> DNA <213> Artificial Sequence <220> <223> miR-1_2-7_G7U reverse complement <400> 20 attaacaacc ggcagtaatt cct 23 <210> 21 <211> 23 <212> DNA <213> Artificial Sequence <220> <223> miR-1_3-8_G3U reverse complement <400> 21 cataacaacc ggcagtacat tat 23 <210> 22 <211> 23 <212> DNA <213> Artificial Sequence <220> <223> miR-1_3-8_G7U reverse complement <400> 22 cataacaacc ggcagtaaat tct 23 <210> 23 <211> 22 <212> RNA <213> Homo sapiens <220> <221> gene <222> (1) .. (22) <223> miR-124 <400> 23 uaaggcacgc ggugaaugcc aa 22 <210> 24 <211> 7 <212> RNA <213> Artificial Sequence <220> <223> miR-124 seed site <400> 24 gugccuu 7 <210> 25 <211> 8 <212> RNA <213> Artificial Sequence <220> <223> miR-1-7G: A site <400> 25 aaauucca 8 <210> 26 <211> 22 <212> RNA <213> Homo sapiens <220> <221> gene <222> (1) .. (22) <223> hsa-miR-1-5p <400> 26 acauacuucu uuauaugccc au 22 <210> 27 <211> 23 <212> RNA <213> Caenorhabditis elegans <220> <221> gene <222> (1) .. (23) <223> cel-miR-67-5p <400> 27 cgcucauucu gccgguuguu aug 23 <210> 28 <211> 23 <212> RNA <213> Artificial Sequence <220> <223> miR-1_2-7 <400> 28 aggaaugacu gccgguuguu aug 23 <210> 29 <211> 23 <212> RNA <213> Artificial Sequence <220> <223> miR-1_3-8 <400> 29 agaauguacu gccgguuguu aug 23 <210> 30 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> miR-1-G7T sense strand <400> 30 tggaatttaa agaagtatgt at 22

Claims (13)

It comprises a total of six consecutive polynucleotides starting from any one of nucleotides 2 to 3 based on the 5 'end of the gene represented by the nucleotide sequence of SEQ ID NO: 1, 2 of the nucleotide sequence of SEQ ID NO: 1, Characterized in that at least one of nucleotides 3 or 7 expresses RNA interference-inducing nucleic acid comprising polynucleotides substituted with uracil (U) in guanine (G) Recombinant vector.
According to claim 1,
Recombinant vector, characterized in that the polynucleotide is selected from the following group of polynucleotides:
A polynucleotide represented by the nucleotide sequence of SEQ ID NO: 2 (5'-UGAAUG-3 '); A polynucleotide represented by the nucleotide sequence of SEQ ID NO: 3 (5'-GUAAUG-3 '); A polynucleotide represented by the nucleotide sequence of SEQ ID NO: 4 (5'-GGAAUU-3 '); A polynucleotide represented by the nucleotide sequence of SEQ ID NO: 5 (5'-UAAUGU-3 '); Or a polynucleotide represented by the nucleotide sequence of SEQ ID NO: 6 (5'-GAAUUU-3 ').
According to claim 2,
The recombinant vector is characterized in that it comprises a nucleotide sequence substituted with adenine (Adenine; A) before the first nucleotide of the 5 'end of the polynucleotide, the recombinant vector.
According to claim 1,
The recombinant vector is characterized in that it expresses any one of the following groups of RNA interference-inducing nucleic acid, recombinant vector:
RNA interference-inducing nucleic acid represented by the nucleotide sequence of SEQ ID NO: 7 (5'-UUG AAU GUA AAG AAG UAU GUA U-3 ');
RNA interference-inducing nucleic acid represented by the nucleotide sequence of SEQ ID NO: 8 (5'- UGU AAU GUA AAG AAG UAU GUA U-3 ');
RNA interference-inducing nucleic acid represented by the nucleotide sequence of SEQ ID NO: 9 (5'-UGG AAU UUA AAG AAG UAU GUA U-3 ');
RNA interference-inducing nucleic acid represented by the nucleotide sequence of SEQ ID NO: 10 (5'-AUG AAU GAC UGC CGG UUG UUA UG-3 ');
RNA interference-inducing nucleic acid represented by the nucleotide sequence of SEQ ID NO: 11 (5'-AGU AAU GAC UGC CGG UUG UUA UG-3 ');
RNA interference-inducing nucleic acid represented by the nucleotide sequence of SEQ ID NO: 12 (5'-AGG AAU UAC UGC CGG UUG UUA UG-3 ');
RNA interference-inducing nucleic acid represented by the nucleotide sequence of SEQ ID NO: 13 (5'-AUA AUG UAC UGC CGG UUG UUA UG-3 '); or
RNA interference-inducing nucleic acid represented by the nucleotide sequence of SEQ ID NO: 14 (5'-AGA AUU UAC UGC CGG UUG UUA UG-3 ').
According to claim 1,
The gene represented by the nucleotide sequence of SEQ ID NO: 1, characterized in that miR-1, recombinant vector.
According to claim 1,
The polynucleotide is a recombinant vector, characterized in that the polynucleotide that recognizes the target site where the G: A wobble (wobble) arrangement occurs.
According to claim 1,
The recombinant vector is an alpha MHC (αMHC) promoter, a beta MHC (β MHC) promoter, a Nkx2.5 promoter, a cardiac troponin T (TNNT2) promoter, a muscle creatine kinase (MCK) promoter, a human A recombinant vector, characterized in that a promoter selected from the group consisting of an alpha-skeletal actin (HAS) promoter and a murine stem cell virus (MSCV) promoter is operably linked.
According to claim 1,
The cardiomegaly is cardiac hypertrophy, heart failure, cardiac fibrosis, mitral valve insufficiency, aortic valve insufficiency, dilated cardiomyopathy, ischemic heart disease, ventricular septal defect, tricuspid valve insufficiency, pulmonary valve insufficiency, pulmonary artery Recombinant vector, characterized by hypertension, right ventricular myocardial infarction, cardiomyopathy involving the right ventricle, atrial septal defect, atrial fibrillation, hypertrophic cardiomyopathy or invasive cardiomyopathy.
According to claim 1,
Recombinant vector, characterized in that the animal is a mammal other than humans.
The method of claim 9,
Recombinant vector, characterized in that the animal is a mouse.
Claim 1 to 10, characterized in that the transformed with the recombinant vector of any one of claims, cardiomegaly animal model.
(a) starting from any one of nucleotides 2 to 3 based on the 5 'end of the gene represented by the nucleotide sequence of SEQ ID NO: 1, comprising a total of 6 consecutive nucleotides, 2 of the nucleotide sequence of SEQ ID NO: 1 Recombination for the production of asymbiotic animal model by operably connecting DNA expressing a polynucleotide in which one or more of nucleotides 3, 7 or 7 is substituted with uracil (U) from guanine (G) Preparing a vector;
(b) introducing the recombinant vector into an animal fertilized egg; And
(C) a method for producing an animal model of cardiomegaly, comprising transplanting the fertilized egg into a surrogate mother and generating a fertilized egg to obtain a transgenic animal model.
(A) processing the candidate material to the myocardial cells of the cardiac hypertrophy animal model;
(b) a total of six consecutive polynucleotides starting from any one of nucleotides 2 to 3 based on the 5 'end of the gene represented by the nucleotide sequence of SEQ ID NO: 1 in the myocardial cell of the cardiac hypertrophy animal model, , G: A wobble of a polynucleotide in which one or more of nucleotides 2, 3, or 7 of SEQ ID NO: 1 is substituted with uracil (U) in guanine (G) Analyzing the binding frequency with the array target site; And
(c) when the binding to the G: A wobble (wobble) array target site decreases compared to an animal model of cardiomegaly that does not process the candidate substance, the method comprising selecting the candidate substance as a therapeutic agent for cardiomegaly, Methods for screening for hypertrophic drugs.

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