KR101674122B1 - Prevention or Treatment for ischemic stroke using miR-135-5p - Google Patents

Abstract

The present invention is directed to the prevention or treatment of ischemic stroke using tissue protective miRNAs that are regulated by ischemic preconditioning.

Description

Prevention or Treatment of ischemic stroke using miR-135b-5p [0002]

The present invention is directed to the prevention or treatment of ischemic stroke using tissue protective miRNAs that are regulated by ischemic preconditioning.

Ischemic stroke is a disease in which cerebral blood flow is blocked due to stenosis or occlusion of the cerebral blood vessels due to thrombosis or embolism. If the blood supply is not re-supplied in a short period of time, the supply of nutrients such as oxygen and glucose, Resulting in permanent damage to nerve cells.

Currently, drugs used for the treatment and prevention of ischemic stroke are hypertension remedy, cerebral blood flow improvement agent, and hyperlipemia treatment agent. However, it is known that only the thrombolytic agent can reduce the brain damage caused by ischemia after the disease. However, thrombolytic agents are limited in their use due to the side effects of bleeding, so less than 10% of all patients are eligible for such treatment. Therefore, it is urgent to develop other therapeutic agents that can reduce brain damage caused by ischemic stroke.

Recent studies have shown that pre-treatment of repeated ischemia over a short period of time can paradoxically demonstrate a protective effect against ischemia. Such endogenous protective actions are called "ischemic tolerance" and ischemic tolerance Induced stimulation was named "ischemic preconditioning (IP)." According to these studies, exposure of the brain to subthreshold ischemic stimulation to an extent that does not damage neurons, prior to induction of ischemic stroke in animal models, may protect the brain from subsequent ischemic stroke . For example, ischemic preconditioning reduces the size of infarcts, reduces apoptosis, a characteristic feature of reperfusion injury after ischemia, facilitates restoration of function after reperfusion, and prevents arrhythmias (Dirnagli, Simon RP, Hallenbeck JM Trends Neurosci 2003; 26: 248-254).

In this regard, studies on the protein and signal pathways involved in ischemic preconditioning are under way, but the mechanism of how ischemic preconditioning induces ischemic tolerance is uncertain, and studies to elucidate this mechanism suggest that treatment with ischemic stroke It is expected to provide important targets for drug development for prevention.

In the present invention, tissue protective miRNAs regulated by ischemic preconditioning have been identified, and they can be utilized as a therapeutic agent for enhancing the protective effect of brain tissue, thus completing the present invention.

Accordingly, it is an object of the present invention to provide an ischemic brain stem comprising an oligonucleotide complementarily binding to one or more miRNAs selected from the group consisting of miR-33-5p, miR-135b-5p and miR-551b- Or a pharmaceutically acceptable salt thereof.

It is another object of the present invention to provide an effective amount of an oligonucleotide complementarily binding to one or more miRNAs selected from the group consisting of miR-33-5p, miR-135b-5p and miR-551b- A method for treating ischemic stroke.

In the present invention, the miRNA was extracted from the cortex of the brain after sacrifice in an ischemic precursor-induced experimental animal, and the expression level of the cortical miRNA of the control group without the ischemic preconditioning Were compared. As a result, miR-33-5p, miR-135b-5p, and miR-551b-3p, which specifically express expression decreased by ischemic preconditioning, were found (FIGS.

In addition, inhibitors of miR-33-5p, miR-135b-5p and miR-551b-3p were introduced into N2a cells (murine neuroblastoma cells) The ratio of mRNA expression of Bax (pro-apoptotic Bcl2 family) and Bcl2 (anti-apoptotic Bcl2 family) decreased to 19-27%. When the activity of these miRNAs decreased, the brain cells were protected from apoptosis (Fig. 5).

Thus, miR-33-5p, miR-135b-5p and miR-551b-3p, which are regulated by ischemic preconditioning, are each associated with brain tissue protection and are used as targets to improve the protective effect of brain tissue Can be utilized.

Accordingly, in one aspect, the present invention provides a method for producing a miRNA comprising complementarily binding oligonucleotides to one or more miRNAs selected from the group consisting of miR-33-5p, miR-135b-5p and miR-551b-3p , And pharmaceutical compositions for preventing or treating ischemic stroke.

In another aspect, the present invention provides a method for producing an miRNA comprising the steps of: (a) contacting an effective amount of an oligonucleotide complementarily binding to one or more miRNAs selected from the group consisting of miR-33-5p, miR-135b-5p and miR- To a method for treating ischemic stroke.

Hereinafter, the present invention will be described in more detail.

MicroRNAs (or miRNAs) are non-coding RNAs that regulate a variety of gene expression and inhibit the translation of mRNA or induce the degradation of mRNA, thereby suppressing the expression of the gene at the post-transcriptional stage. Post-transcriptional regulation is used as a powerful regulatory method in the process of precise and precise gene expression, such as intracellular signal transduction. Since about half of the gene expression changes due to external stimuli are regulated at the post-transcriptional stage, Has been reported to play an important role in the regulation of the expression of the entire gene. In some studies, miRNAs have been shown to play an important role in controlling many biological processes such as cell differentiation, proliferation, apoptosis, development, immunity, metabolism and stem cell maintenance. In addition, miRNAs generally have very high specificity and efficacy by simultaneously controlling the expression of a plurality of target genes rather than one.

In the present invention, miR-33-5p, miR-135b-5p and miR-551b-3p are derived from animals including humans such as monkeys, pigs, horses, cows, sheep, dogs, cats, mice, And those derived from humans are preferable.

In the present invention, the nucleic acid molecules constituting miR-33-5p, miR-135b-5p and miR-551b-3p may each have a nucleotide length of 18 to 100 nt. In a preferred embodiment, the nucleic acid molecule may be in the form of a mature miRNA having a length of 19 to 25 nt, more preferably 21, 22 or 23 nt, and in another preferred embodiment, a precursor miRNA of 50 to 100 nt, more preferably 65 to 95 nt Lt; / RTI >

Sequence information of the miR-33-5p, miR-135b-5p and miR-551b-3p nucleic acid molecules in the mature miRNA or precursor miRNA form is available from NIH GenBank and miRBASE (http: mirbase.org/) and the like. More specifically, the nucleotide sequence of the mature form of miR-33-5p is registered with the gene registry number MIMAT0000667 (SEQ ID NO: 1) and the precursor form is registered with MI0000707 (SEQ ID NO: 2). The nucleotide sequence of the mature form of miR-135b-5p is registered with the gene registry number MIMAT0000612 (SEQ ID NO: 3), and the precursor form is registered with MI0000646 (SEQ ID NO: 4). The nucleotide sequence of the mature form of miR-551b-3p is registered with the gene registry number MIMAT0003890 (SEQ ID NO: 5) and the precursor form with the MI0004131 (SEQ ID NO: 6).

In addition, the miRNAs used in the present invention functionally equivalent to the miRNA nucleic acid molecule even when a functional equivalent of the nucleic acid molecule constituting the miRNA, for example, a part of the base sequence of the miRNA nucleic acid molecule is modified by deletion, substitution or insertion This is a concept that includes variants that can be made. For example, the miRNA of the present invention may exhibit at least 80% homology with the nucleotide sequence of each corresponding SEQ ID NO, and preferably at least 90%, more preferably at least 95%. Such homology can be readily determined by comparing the sequence of the nucleotide with the corresponding portion of the target gene using computer algorithms well known in the art, for example Align or BLAST algorithms.

In addition, miRNA nucleic acid molecules used in the present invention may exist in single stranded or double stranded form. Although mature miRNA molecules are predominantly single stranded, precursor miRNA molecules may contain a partial self-complementary structure (e.g., a stem-loop structure) that can form double strands. In addition, the nucleic acid molecule of the present invention may be in the form of RNA, peptide nucleic acids (PNA), or locked nucleic acid (LNA).

The nucleic acid molecule of the present invention can be isolated or prepared using standard molecular biology techniques, such as chemical synthesis methods or recombinant methods, or commercially available.

In the present invention, the expression of miR-33-5p, miR-135b-5p and / or miR-551b-3p is specifically reduced by ischemic preconditioning. Thus, by reducing the expression or activity of miR-33-5p, miR-135b-5p and / or miR-551b-3p, the condition of ischemic tolerance by ischemic preconditioning can be mimicked.

Examples of a substance that decreases the expression or activity of miR-33-5p, miR-135b-5p, and / or miR-551b-3p include miR-33-5p, miR- Lt; RTI ID = 0.0 > 3p. ≪ / RTI > The oligonucleotides may complementarily bind miR-33-5p, miR-135b-5p and / or miR-551b-3p to reduce the expression of these miRNAs or inhibit their function.

As used herein, the term "complementary" means that the oligonucleotide is sufficiently complementary to hybridize selectively to an miRNA target under a given hybridization or annealing condition, preferably under physiological conditions, and is substantially complementary, And perfectly complementary, and preferably means completely complementary.

In the present invention, the oligonucleotide may be a DNA or RNA molecule, and more preferably an RNA molecule. The oligonucleotides may also include modifications, for example, 2'-O-modified oligonucleotides, phosphorothioate-backbone deoxyribonucleotides, PNA (peptide nucleic acid) have.

In the present invention, the oligonucleotide complementarily binding to the miRNA may be an antisense oligonucleotide, an antagomir, an siRNA (small interfering RNA), a shRNA (short hairpin RNA), an RNA plasmid, For example.

In one embodiment, the antisense oligonucleotides complementarily bind to single-stranded fragments of miRNA molecules in a cell to decrease the miRNA's protease efficiency, thereby inhibiting their expression or activity and inhibiting their function. As described above, miRNA molecules may exist in single stranded or double stranded form. Although mature miRNA molecules are predominantly single stranded, precursor miRNA molecules are partially self-complementary (e.g., stem- and loop-structure) structures that can form double-stranded portions. Thus, the antisense oligonucleotides of the invention may be complementary to a single strand fragment of a precursor miRNA molecule, or complementary to a mature miRNA molecule.

The antisense oligonucleotides are capable of inhibiting the expression or activity of a target miRNA when introduced into a cell and are preferably resistant to an endogenous nuclease such as exonuclease and endonuclease, Stable, optionally modified oligonucleotide.

In addition, the antisense oligonucleotides may contain a complementary sequence to the target miRNA, preferably the miRNA, seed sequence. In general, miRNA seed sequences are very important sequences for target recognition and are conserved for a variety of species. Thus, in one embodiment, the antisense oligonucleotides of the invention may have a sequence complementary to the sequence of miR-33-5p, miR-135b-5p and / or miR-551b-3p, Can inhibit miR-33-5p, miR-135b-5p, and / or miR-551b-3p.

In another embodiment, an antagonist refers to an oligonucleotide designed to silence intrinsic miRNAs, which may have a sequence that is at least partially, preferably completely complementary, to the target miRNA, Gt; < / RTI > The antagonist may comprise one or more modified nucleotides (e.g., 2'-O-methyl-sugar modifications). In addition, the antagonist may comprise one or more phosphorothioate linkages and may have a partial or complete phosphorothioate backbone. In addition, in order to improve in vivo delivery and stability, the antagonist may be conjugated to cholesterol or other sites at its 3'-end. Suitable antagonists may have a length of 7-50 nucleotides, preferably 10-40 nucleotides, more preferably 15-30 nucleotides, and most preferably 20-25 nucleotides, to inhibit the target miRNA.

In another embodiment, the siRNA refers to a short double-stranded RNA that specifically acts on miRNAs and can induce RNAi (RNA interference) phenomenon through cleavage of miRNA molecules. In the present invention, the siRNA is composed of an antisense RNA strand comprising a sense RNA strand containing a sequence homologous to a part or all of the nucleic acid sequence of the target miRNA and a sequence complementary thereto, Sequence. ≪ / RTI >

In another embodiment, the shRNA is designed to overcome the disadvantages of high cost biosynthetic cost of siRNA, short time maintenance of RNA interference effect due to low cell transfection efficiency, and the like. From the promoter of RNA polymerase III, adenovirus, And a plasmid expression vector system. The shRNA is converted into an siRNA having a correct structure by the siRNA processing enzyme (Dicer or Rnase III) present in the cell, and the silencing of the target miRNA is performed .

In another embodiment, the RNA aptamer refers to a nucleic acid ligand molecule that binds to and has an antagonistic effect on miRNA by its ability to adopt a particular three-dimensional conformation. Typically, the aptamer may be a small nucleic acid of 15-50 bases in length that folds into a defined secondary and tertiary structure, such as a stem-loop structure. It is preferred that the aptamer binds to the target molecule at a kd of less than 10 ^-6 , 10 ^-8 , 10 ^-10 , or 10 ^-12 . The aptamer can bind to a target molecule with a very high specificity. The aptamer may also be composed of a mixture of multiple ribonucleotide units, deoxyribonucleotide units, or two types of nucleotide residues. The aptamer may further comprise one or more modified base, sugar or phosphate backbone units.

In another embodiment, a ribozyme refers to a nucleic acid molecule capable of catalyzing a chemical reaction either intramolecularly or between molecules. The ribozymes that can be used in the present invention include nuclease based on ribozymes found in natural systems, or any of the different types of ribozymes that catalyze nucleic acid polymerase type reactions, such as the Hammerhead lysozyme, Hairpin Libojaim and tetrahimena libojaim. Also, although not found in natural systems, ribozymes that are engineered to catalyze specific reactions in vivo are available. The ribozyme can cleave the RNA or DNA substrate, and preferably can cleave the RNA substrate. The ribozyme typically cleaves the nucleic acid substrate through recognition and binding and subsequent cleavage of the target substrate, which is based on base pair interaction, and can thus be characterized by target specific cleavage.

Alternatively, oligonucleotides that complementarily bind miRNAs in the present invention can be prepared by methods known in the art to increase their affinity for their targets and provide resistance to mismatch of the target sequence Modified oligomer mimetics, such as peptide nucleic acids (PNAs) and fixed nucleic acids (LNAs). The LNA is a modified ribonucleotide with an additional bridge between the 2 'to 4' carbons of the ribose sugar moiety and has a locked form whereby the oligonucleotide with the LNA has improved thermal stability. In addition, the PNA may comprise a peptide-based backbone instead of a sugar-phosphate backbone.

In addition, the oligonucleotides of the present invention can be modified by methods known in the art to increase stability and increase resistance to nuclease degradation. But are not limited to, for example, modification of the oligonucleotide backbone, modification of the sugar moiety, or modification of the base. Examples of backbone modifications include backbone modifications such as phosphorothioate, morpholino or phosphonocarboxylate linkages. Examples of sugar variants include those wherein the 2 'position of the ribose moiety is an -O-lower alkyl group comprising 1-6 saturated or unsaturated carbon atoms, or -O-aryl containing 2-6 carbon atoms, -O-substituted < / RTI > The -O-alkyl, aryl or allyl group may be unsubstituted or substituted with an amino or halo group (e.g., halo, hydroxy, trifluoromethylcyano, nitroacyl acyloxy, alkoxy, carboxy, carbalkoxyl or amino group) . In addition, the oligonucleotides of the present invention may have 2 ' -O-alkylated ribonucleotides at the 3 ', 5 ' or 3 ' and 5 ' ends and at least 4 or 5 consecutive nucleotides may be so modified . Examples of 2'-O-alkylated groups include 2'-OC _{1 -4} alkyl oligonucleotides, preferably 2'-O-methyl, 2'-O-ethyl, Butyl, but is not limited thereto.

In the present invention, an oligonucleotide that binds complementarily to an miRNA may be contained in an expression vector or may be provided in a form introduced into a cell.

In one embodiment, an oligonucleotide that binds complementarily to the miRNA may be provided in an expression vector for intracellular delivery. Oligonucleotides that complementarily bind miRNA in the present invention can be introduced into cells using various transformation techniques such as a complex of nucleic acid and DEAE-dextran, a complex of nucleic acid and nucleoprotein, a complex of nucleic acid and lipid, and the like , And can be provided in a form contained in a carrier that enables efficient introduction into the cell for this purpose. Preferably, the carrier is a vector, and both viral vectors and non-viral vectors are usable. As the viral vector, for example, lentivirus, retrovirus, adenovirus, herpes virus and avipox virus vector can be used. However, But is not limited thereto.

The expression vector may further include a selection marker to facilitate selection of the transformed cells. Markers conferring selectable phenotypes such as, for example, drug resistance, resistance to nutritional requirements, cytotoxic agents or expression of surface proteins, such as green fluorescent protein, puromycin, neomycin, hygromycin, Histidine dehydrogenase (hisD) and guanine phosphoribosyltransferase (Gpt).

In another embodiment, an oligonucleotide that binds complementarily to a miRNA in the present invention may be provided in a form introduced into the cell. These cells are capable of expressing high levels of oligonucleotides that bind complementarily to miRNA nucleic acid molecules or miRNAs and by implanting these cells into brain tissue, brain tissue can be protected from damage by ischemic stroke.

Examples of the method of introducing into cell include G-fectin, Mirus TrasIT-TKO lipophilic reagent, lipofectin, lipofectamine, cellfectin, cationic phospholipid nanoparticles, cationic polymer, cationic micelle, cationic May be introduced into a cell together with a delivery reagent containing an emulsion or liposome, or may be conjugated with a biocompatible polymer such as polyethylene glycol to increase intracellular absorption.

The pharmaceutical composition of the present invention may further comprise a pharmaceutically acceptable carrier and may be formulated together with a carrier. The pharmaceutically acceptable carrier means a carrier or diluent which does not irritate the organism and does not interfere with the biological activity and properties of the administered compound. Examples of the pharmaceutical carrier which is acceptable for the composition to be formulated into a liquid solution include sterilized and sterile water suitable for the living body such as saline, sterilized water, Ringer's solution, buffered saline, albumin injection solution, dextrose solution, maltodextrin solution, glycerol, One or more of these components may be mixed and used. If necessary, other conventional additives such as an antioxidant, a buffer, and a bacteriostatic agent may be added. Can also be formulated in the form of solutions or suspensions (e. G., Microparticles, liposomes, or integrated with cells).

The pharmaceutical composition of the present invention can be applied to any formulation containing it as an active ingredient, and can be manufactured and administered as an oral or parenteral formulation. Administration refers to the introduction of a pharmaceutical composition of the present invention to a patient in any suitable manner and includes the transfer of nucleic acid molecules by viral or non-viral techniques or the transplantation of cells expressing nucleic acid molecules. The route of administration of the composition of the present invention may be administered through various routes of oral or parenteral administration, as long as it can reach the target tissues, preferably topically to the brain tissue.

The compositions and methods of treatment of the present invention are applicable to any animal in which ischemic stroke can occur, and the animal includes human and primate as well as cattle such as cows, pigs, sheep, horses, dogs and cats.

It will be apparent to those skilled in the art that the appropriate total daily usage of the compositions of the present invention may be determined by treatment within the scope of sound medical judgment. The specific therapeutically effective amount for a particular patient will depend upon a variety of factors, including the type and extent of the response to be achieved, the specific composition, including whether or not other agents are used, the age, weight, general health status, sex and diet, The route of administration and the fraction of the composition, the duration of the treatment, and the amount of radiation to be irradiated, and the similar factors well known in the medical arts. For example, it may be used at a dose of 0.001 占 퐂 / kg to 100 mg / kg (body weight) per day, but is not limited thereto. The effective amount of the pharmaceutical composition suitable for the purpose of the present invention is preferably determined in consideration of the above-mentioned matters.

In accordance with the present invention, therapeutic agents that enhance the protective effect of brain tissue can be developed using tissue protective miRNAs that are regulated by ischemic preconditioning.

FIG. 1 shows miRNA microarray results of five miRNAs expressing 1.5 times or more specific expression change by ischemic preconditioning.
FIG. 2 shows the results of qRT-PCR showing changes in miR-33-5p expression by ischemic preconditioning. MCAO means middle cerebral artery occlusion, and IP means ischemic preconditioning.
FIG. 3 shows the results of qRT-PCR showing changes in miR-135b-5p expression by ischemic preconditioning. MCAO means middle cerebral artery occlusion, and IP means ischemic preconditioning.
Fig. 4 shows the results of qRT-PCR showing changes in miR-551b-3p expression by ischemic preconditioning. MCAO means middle cerebral artery occlusion, and IP means ischemic preconditioning.
FIG. 5 shows the results of measurement of changes in Bax / Bcl2 mRNA ratio by extracting total RNA after transfection of inhibitors against miR-33a-5p, miR-135b-5p and miR-551b- .

Hereinafter, the present invention will be described in detail with reference to examples. However, the following examples are illustrative of the present invention, and the present invention is not limited by the following examples.

Example  1. Ischemic preconditioning

A 10-week-old male mouse (C57BL6) (12 mice, Orient Bio) underwent isoflurane 1.2% inhalation anesthesia and the skin of the right parietal area was incised, 2 mm posteriorly from the bregma and 5 mm to the right, parietal Bone flow was measured by attaching a fiber optic probe of a laser Doppler flowmeter to the computer. The skin of the anterior neck of the mouse was incised, and the bilateral common carotid artery was exposed. The bilateral common carotid arteries occlusion was performed for 3 minutes at 5-minute intervals. The skin was sutured and restored in anesthesia. .

Example  2. Ischemic Brainpunch  Judo

The optical fiber measurement device was attached to the mouse (ischemic preconditioning group) and the ischemic preconditioning control group (control group), which had passed 24 hours after the ischemic preconditioning, in the same manner as in Example 1 under suction inhalation. A 6-0 nylon suture with silicone end coated on the internal carotid artery arteriotomy was obtained by dissecting and closing the external carotid artery after skin incision of the anterior cervix. And allowed to advance to the origin of the middle cerebral artery, which is a point where the brain was reduced by more than 85% of normal cerebral blood flow, and then maintained for 30 minutes. The nylon suture was then slowly removed and perfused again. Reperfusion was confirmed to be more than 80% and restored in anesthesia after skin suture.

Example  3. In brain tissue Total RNA  extraction

At 3 hours or 6 hours after induction of ischemic stroke, mice were euthanized immediately after ischemic preconditioning and control mice and brain was extracted and ipsilateral cortex was isolated. Total RNA, including small RNA, was extracted from the cortex using the mirVana miRNA isolation kit (Ambion). The extraction method was carried out according to the manufacturer's manual. Extracted total RNA was quantified by spectrophotometer and RNA status was confirmed by electrophoresis on 1% agarose gel.

Example  4. miRNA Microarray

Using an Agilent mouse 8 x 60K miRNA microarray, miRNA microarrays were performed.

Expression values of each miRNA obtained after scanning were subjected to log2 transformation and finally used for statistical analysis. The Bayesian t test (Limma: Linear Models for Microarray Data, Gordon K. Smyth.) Method was used to identify differential expression miRNAs (DEmiR) in both groups. Finally, DEmiR was selected as the miRNA with a p value <0.05 and an absolute value of log2 (fold change) greater than 0.585.

Example  5. miRNA  For confirmation of expression Quantitative real - time  PCR ( qRT - PCR )

To confirm the expression of each miRNA, complementary DNA was obtained using TaqMan MiRNA reverse kit (Applied Biosystems). In detail, 0.3 μl of 100 mM dNTPs, 3.0 μl of MultiScribe ™ Reverse Transcription, 1.5 μl of 10 × RT buffer, and 0.19 μl of RNase inhibitor were added to the plate, and the total volume of the plate was adjusted to 7.0 μl by adding water free of nucleic acid hydrolase Then, 5.0 μl of RNA and 3.0 μl of RT primer pool were added and mixed. After cooling for 30 minutes at 16 ° C, 30 minutes at 42 ° C, and 5 minutes at 85 ° C, the mixture was cooled to 4 ° C.

A total of 10.0 μl was prepared by mixing 0.5 μl of 20 × TaqMan MiRNA Assays, and 0.665 μl of cDNA and 5.0 μl of TaqMan 2X Universal PCR Master Mix using TaqMan MiRNA Assays (Applied Biosystems). The resulting mixture was incubated in an ABI 7500 fast sequence detection system (Applied Biosystems) 10 minutes at 95 ° C, 15 seconds at 60 ° C, and 60 seconds at 40 ° C. The expression of snoRNA 202 was used as an internal control and the expression of each miRNA was corrected for the expression level of snoRNA 202 using the ΔΔC _T method.

The TaqMan MiRNA Assays used to investigate the expression of each miRNA are as follows: miR-135b-5p (Part Number P / N 4427975 Assay ID TM002261, RT002261), miR-551b-3p ID No. TM001535, RT001535), miR-33-5p (Part Number P / N 4427975 Assay ID TM465396, RT465396) and snoRNA 202 (Part Number P / N 4427975 Assay ID 001232).

Example  6. miRNA inhibitor  Cell introduction and Total RNA  extraction

N2a cells (Neuro-2a, murine neuroblastoma cells) were plated in 2 × 10 ⁵ cells on a 6-well plate, and after 24 hours, they were transfected with media not containing antibiotics and transfected with miRNA inhibitors. The miRNA inhibitor is miR-33a-5p inhibitor (manufacturer ambion, product name mirVana® miRNA inhibitor (Part number: 4464084), catalog number MH12410), miR-135b-5p inhibitor ), Catalog number MH13044) and miR-551b-3p inhibitor (manufacturer ambion, product name mirVana® miRNA inhibitor (Part number: 4464084), catalog number MH11653). The miRNA inhibitor to be introduced into cells was mixed with Opti-MEM to a final concentration of 50 nM. Lipofectamine 2000, a transfection reagent, was also added to Opti-MEM at a defined rate and allowed to react at room temperature for 5 minutes. At the end of the reaction time, the cells were mixed with the inhibitor and reacted at room temperature for 20 minutes before the cells were treated.

At 24 hours after transfection, the cells were collected and total RNA was isolated using RNeasy Plus Mini kit (Qiagen). The extraction method was carried out according to the manufacturer's manual. Extracted total RNA was quantified by spectrophotometer and RNA status was confirmed by electrophoresis on 1% agarose gel.

Example  7. Bax Wow Bcl2  For confirmation of expression Quantitative real - time PCR  ( qRT - PCR )

Bax (pro-apoptotic Bcl2 family) promotes apoptosis and Bcl2 (anti-apoptotic Bcl2 family) inhibits apoptosis. Bcl-2 and Bax form a homodimer or a heterodimer to control cell death. Therefore, the inhibition of cell death can be confirmed by measuring the expression ratio of Bax / Bcl2 mRNA. Therefore, cDNA was synthesized with extracted total RNA and qRT-PCR was performed on Bax and Bcl2.

Superscript II reverse transcriptase (Invitrogen) was used for cDNA synthesis. 1 μg of total RNA and 50 ng of oligo dT were denatured at 70 ° C for 10 min. Then, 4 μl of 5 × RT buffer, 2 μl of 0.1 mM DTT, 4 μl of 2.5 mM dNTP mixture, 200 units of Superscript II reverse transcriptase, units were reacted at 25 ° C for 10 minutes, 42 ° C for 50 minutes, and 95 ° C for 5 minutes. The cDNA was synthesized and diluted 1: 4 to obtain 2 μl and used as a template for qRT-PCR. For the qRT-PCR, 20 μl of the reaction mixture containing 2 μl of cDNA, 10 μl of SYBR Premix EX Taq (Takara Bio), 0.4 μl of Rox reference dye (50 ×, Takara Bio) and 200 nM of each gene was amplified by ABI 7500fast sequence detection system (Applied Biosystems) at 95 ° C for 30 seconds, and then amplified by repeating 40 cycles (95 ° C for 3 seconds, 60 ° C for 30 seconds). The specificity of the PCR product was confirmed by reacting at 95 ° C for 15 seconds, at 60 ° C for 1 minute, and at 95 ° C for 15 seconds. Gapdh expression was used as an internal control, and the expression of Bax and Bcl2 genes was expressed as Gapdh expression level ΔΔC_T Method.

The primers of the genes used in the experiments are as follows:

mBax forward: 5'-TGA AGA CAG GGG CCT TTT TG-3 '(SEQ ID NO: 7)

mBax reverse: 5'-AAT TCG CCG GAG ACA CTC G-3 '(SEQ ID NO: 8)

mBcl2 forward: 5'-GTC GCT ACC GTC GTG ACT TC-3 '(SEQ ID NO: 9)

mBcl2 reverse: 5'-CAG ACA TGC ACC TAC CCA GC-3 '(SEQ ID NO: 10)

mGapdh forward: 5'-AAT GTG TCC GTC GTG GAT CT-3 '(SEQ ID NO: 11)

mGapdh reverse: 5'-GGT CCT CAG TGT AGC CCA AG-3 '(SEQ ID NO: 12)

Example  8. Statistical processing

The experimental results were expressed as mean ± standard deviation (n = 3) and statistical processing was performed using GraphPad Prism5 software. One-way ANOVA was used to compare the Bax / Bcl2 mRNA ratio with the negative inhibitors in each group. Post-test was performed with Bonferroni's multiple comparison test when the p-value was less than 0.05. A p-value less than 0.05 after each statistical treatment was considered to be statistically significant and marked with *.

Experiment result

1. Ischemic preconditioning, which specifically changes the expression of brain tissue miRNA  Selection

MiRNAs were extracted from the cortex of the brain after ischemic stroke, and the expression patterns of the brain cortical miRNAs in the control group without ischemic preconditioning were compared using an miRNA microarray Respectively. As a result, three kinds of miRNA-related miRNAs (miR-33-5p, miR-135b-5p, miR-551b-3p) having specifically decreased miRNA expression by 1.5 times or more were selected by ischemic preconditioning 1).

2. Ischemic preconditioning miRNA  Confirmation of expression control

When ischemic preconditioning was performed prior to exposure to ischemic stroke, qRT-PCR confirmed that the expression of miR-33-5p specifically decreased 3.3-fold compared to the control group without the pretreatment (Fig. 2).

In addition, when ischemic preconditioning was performed prior to exposure to ischemic stroke, the expression of miR-135b-5p was specifically reduced about 2.0-fold compared to the control group without the pretreatment using qRT-PCR (Fig. 3).

In addition, when ischemic preconditioning was performed prior to exposure to ischemic stroke, the expression of miR-551b-3p was specifically reduced by about 2.0-fold compared to the control group without the pretreatment using qRT-PCR (Fig. 4).

3. Controlled by ischemic preconditioning miRNA Of cell protection activity

The cytoprotective effects of miR-33a-5p, miR-135b-5p and miR-551b-3p, which specifically express miRNAs by ischemic preconditioning, were examined using an in vitro cell system. Each miRNA inhibitor was transfected into N2a cells to reduce the expression of each miRNA and the expression ratios of Bax (pro-apoptotic Bcl2 family) and Bcl2 (anti-apoptotic Bcl2 family) mRNA were calculated.

As a result, when the activity of miR-33a-5p, miR-135b-5p and miR-551b-3p was treated to decrease the activity of each miRNA, the expression ratio of Bax / Bcl2 mRNA was 19 ~ 27% (Fig. 5). When the activity of these miRNAs is decreased, the neuronal cells are protected from apoptosis (Fig. 5).

<110> Ewha University - Industry Collaboration Foundation <120> Prevention or Treatment for ischemic stroke using miR-135b-5p <130> DPP20151857KR <150> KR10-2014-0093256 <151> 2014-07-23 <160> 12 <170> Kopatentin 1.71 <210> 1 <211> 21 <212> RNA <213> Homo sapiens, mature miR-33-5p <400> 1 gugcauugua guugcauugc a 21 <210> 2 <211> 69 <212> RNA <213> Homo sapiens, miR-33-5p precursor <400> 2 cuguggugca uuguaguugc auugcauguu cuggcaauac cugugcaaug uuuccacagu 60 gcaucacgg 69 <210> 3 <211> 23 <212> RNA <213> Homo sapiens, mature miR-135b-5p <400> 3 uauggcuuuu cauuccuaug uga 23 <210> 4 <211> 97 <212> RNA <213> Homo sapiens, miR-135b-5p precursor <400> 4 cgcucugcug uggccuaugg cuuuucauuc cuaugugauu gcugcuccga acucauguag 60 ggcuaaaagc caugggcuac agugaggggc aagcucc 97 <210> 5 <211> 21 <212> RNA <213> Homo sapiens, mature miR-551b-3p <400> 5 gcgacccaua cuugguuuca g 21 <210> 6 <211> 98 <212> RNA <213> Homo sapiens, miR-551b-3p precursor <400> 6 gugcucuugu ggcccaugaa aucaagcuug ggugagaccu ggugcagaac aggaaggcga 60 cccauacuug guuucagugg cugcaagaau gacugcau 98 <210> 7 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> mBax forward primer <400> 7 tgaagacagg ggcctttttg 20 <210> 8 <211> 19 <212> DNA <213> Artificial Sequence <220> <223> mBax reverse primer <400> 8 aattcgccgg agacactcg 19 <210> 9 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> mBcl2 forward primer <400> 9 gtcgctaccg tcgtgacttc 20 <210> 10 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> mBcl2 reverse primer <400> 10 cagacatgca cctacccagc 20 <210> 11 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> mGapdh forward primer <400> 11 aatgtgtccg tcgtggatct 20 <210> 12 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> mGapdh reverse primer <400> 12 ggtcctcagt gtagcccaag 20

Claims (5)

A pharmaceutical composition for the prevention of ischemic stroke caused by neuronal cell damage comprising an oligonucleotide complementarily binding to a precursor of miR-135b-5p or miR-135b-5p,
The miR-135b-5p comprises the nucleotide sequence of SEQ ID NO: 3, the miR-135b-5p precursor comprises SEQ ID NO: 4,
The precursors of miR-135b-5p and miR-135b-5 are characterized in that their expression is regulated by ischemic preconditioning,
Wherein said composition protects neuronal cells.
The oligonucleotide complementary to the miR-135b-5p or miR-135b-5p precursor is selected from the group consisting of an antisense oligonucleotide, an antagomir, an siRNA (small interfering RNA), a short hairpin RNA), RNA &lt; / RTI &gt;
The oligonucleotide complementary to the miR-135b-5p or miR-135b-5p precursor may be DNA, RNA, a 2'-O-modified oligonucleotide, a phosphorothioate-backbone deoxyribonucleotide , PNA (peptide nucleic acid) or LNA (locked nucleic acid).
The composition according to claim 1, wherein the oligonucleotide that binds complementarily to the miR-135b-5p or miR-135b-5p precursor is contained in an expression vector or introduced into a cell.
A composition for protecting a brain cell comprising an oligonucleotide complementarily binding to a precursor of miR-135b-5p or miR-135b-5p,
The miR-135b-5p comprises the nucleotide sequence of SEQ ID NO: 3, the miR-135b-5p precursor comprises SEQ ID NO: 4,
Wherein the precursors of miR-135b-5p and miR-135b-5 are expressed by ischemic preconditioning.

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