KR102361394B1 - miRNA associated with epilepsy and uses thereof - Google Patents

Abstract

Epilepsy-related miRNA and its therapeutic agent, diagnostic composition, and use in an animal model of disease, and according to a substance capable of inhibiting miRNA according to an aspect, increases the expression of KCNQ2 to increase neuronal excitability associated with epilepsy. It can be usefully used for the treatment of epilepsy because it reduces it, and it can be used for epilepsy diagnosis methods using the same as a biomarker, and has an effect that can be usefully used for disease animal models.

Description

miRNA associated with epilepsy and uses thereof

It relates to a miRNA related to epilepsy, a therapeutic agent thereof, a diagnostic composition, and use in an animal model of a disease.

Epilepsy is a chronic neurological disorder that causes seizures and convulsions by generating electricity in the brain due to irregular excitation of cranial nerve cells. Epilepsy is caused by various causes, and epidemiological studies have reported that more than 1/3 of patients have a history of pathological changes or brain damage in the brain, and the main causes are stroke, congenital anomaly, head Trauma, encephalitis, brain tumors, degenerative encephalopathy, heredity, premature infants, injuries before and after delivery, and the like.

The KCNQ2 gene encodes a subunit of a calcium channel that regulates neuronal excitability, and it is well known that a mutation in this gene induces neonatal epilepsy. Most patients with benign neonatal epilepsy show seizures within 1 week of age and show normal life patterns, but some patient groups have neonatal epileptic encephalopathy, abnormal electroencephalography (EEG), and severe seizures. ) can lead to serious intellectual disability.

On the other hand, MicroRNAs (miRNA) are small RNA molecules of 21- to 23-nucleotides, and regulate gene expression through inhibition in the fragmentation or translation stage of target mRNA. miRNAs are involved in various physiological phenomena and diseases.

Epilepsy is a common disease affecting about 65 million people worldwide (WHO, 2004), and epilepsy patients spend a lot of medical expenses due to the disease, and they suffer a lot of economic loss because they cannot perform a normal work life. In particular, about 30% of all epilepsy patients suffer from drug-resistant epilepsy, in which epilepsy seizures are not well controlled with drugs. Therefore, the development of epilepsy drugs with a new mechanism is urgently needed.

One aspect is related to epilepsy or seizures comprising a substance capable of inhibiting the activity of any one miRNA selected from the group consisting of miR-17-5p, miR-93-5p, and miR-106b-5p as an active ingredient To provide a composition for preventing or treating a disease.

Another aspect is epilepsy comprising administering to the subject a substance capable of inhibiting the activity of any one miRNA selected from the group consisting of miR-17-5p, miR-93-5p, and miR-106b-5p Or to provide a method for preventing or treating a seizure-related disease.

Another aspect is an agent capable of detecting any one miRNA selected from the group consisting of miR-17-5p, miR-93-5p, and miR-106b-5p, for example, a nucleotide sequence, a complementary sequence thereof, or It is to provide a kit for diagnosis of epilepsy or seizure-related diseases comprising a fragment of the sequence.

In another aspect, detecting the expression of any one miRNA selected from the group consisting of miR-17-5p, miR-93-5p, and miR-106b-5p in a biological sample isolated from a subject; And to provide a method for detecting a marker in order to provide information about the diagnosis of epilepsy or seizure-related disease comprising the step of comparing the expression level of the detected marker with a control.

Another aspect is the steps of contacting the candidate material with any one miRNA selected from the group consisting of miR-17-5p, miR-93-5p, and miR-106b-5p; and a candidate substance that reduced the expression level of any one miRNA selected from the group consisting of miR-17-5p, miR-93-5p, and miR-106b-5p compared to the untreated control group for epilepsy or seizure-related disease To provide a method for screening a therapeutic agent for epilepsy or seizure-related disease, comprising the step of selecting a therapeutic agent for

Another aspect provides an animal model of epilepsy or seizure-related disease, into which a vector containing any one miRNA selected from the group consisting of miR-17-5p, miR-93-5p, and miR-106b-5p is introduced will do

Another aspect includes administering to an animal other than a human any one miRNA selected from the group consisting of miR-17-5p, miR-93-5p, and miR-106b-5p or a substance that increases its expression To provide a method for preparing an animal model of epilepsy or seizure-related disease.

One aspect is related to epilepsy or seizures comprising a substance capable of inhibiting the activity of any one miRNA selected from the group consisting of miR-17-5p, miR-93-5p, and miR-106b-5p as an active ingredient It provides a pharmaceutical composition for preventing or treating a disease.

Another aspect is epilepsy comprising administering to the subject a substance capable of inhibiting the activity of any one miRNA selected from the group consisting of miR-17-5p, miR-93-5p, and miR-106b-5p or a method for preventing or treating a seizure-related disease.

As used herein, the term “miR” or “micro RNA” refers to 21 to 23 non-coding RNAs that post-transcriptionally regulate gene expression by promoting degradation of target RNAs or inhibiting their translation. The mature sequence of the miRNA used herein can be obtained from the miRNA database (http://www.mirbase.org). In general, microRNA is transcribed into a precursor of about 70-80 nt (nucleotide) in length with a hairpin structure called pre-miRNA, and then cut by the RNAse III enzyme Dicer to produce a mature form. MicroRNA forms a ribonucleo complex called miRNP to cleave a target gene through complementary binding to a target site or inhibit translation. More than 30% of human miRNA exists as a cluster, and after being transcribed into a single precursor, it undergoes a cleavage process to form a final mature miRNA.

The term “treatment” refers to or includes the alleviation, inhibition of progression or prevention of a disease, disorder or condition, or one or more symptoms thereof, and “active ingredient” or “pharmaceutically effective amount” refers to the disease, disorder or condition, or its It may mean any amount of the composition used in the practice of the invention provided herein sufficient to alleviate, inhibit the progression or prevent one or more symptoms.

The terms “administering,” “introducing” and “implanting” are used interchangeably and in one embodiment into a subject by a method or route that results in at least partial localization of the composition according to one embodiment to a desired site. It may mean the arrangement of the composition according to

As used herein, the term “substance capable of inhibiting the activity of miR-17-5p, miR-93-5p, or miR-106b-5p miR-203” refers to any substance capable of inhibiting its expression and/or activity. include For example, the substance capable of inhibiting the activity of the miR may be a nucleic acid molecule capable of binding to all or part of the nucleotide sequence of the miR. Also, for example, antagomere, antisense molecule, bovine hairpin RNA molecule (shRNA), fire-fighting RNA molecule (siRNA), seed target LNA (Locked Nucleic Acid) oligonucleotides, decoy oligonucleotides, tough decoy oligos nucleotides, aptamers, ribozymes, or antibodies that recognize DNA:RNA hybrids. Also, for example, the nucleic acid molecule can be RNA, DNA, antagomir, antisense molecule, siRNA, shRNA, 2'-O-modified oligonucleotide, phosphorothioate-backbone deoxyribonucleotide, phosphorothioate It may be a backbone ribonucleotide, a decoy oligonucleotide, a tough decoy oligonucleotide, a peptide nucleic acid (PNA) oligonucleotide, or a locked nucleic acid (LNA) oligonucleotide.

As used herein, the term "antisense oligonucleotide" encompasses nucleic acid-based molecules that have a sequence complementary to all or part of the seed sequence of a targeted miRNA, particularly a miRNA, and thus can form a duplex with the miRNA. will do Thus, the term “antisense oligonucleotide” as used herein may refer to “complementary nucleic acid-based inhibitor”.

The antisense oligonucleotide includes various molecules, for example, ribonucleic acid (RNA), deoxyribonucleic acid (DNA), antagomere, 2'-O-modified oligonucleotide, phosphorothioate-backbone deoxyribonucleotide, phosphoro thioate-backbone ribonucleotides, decoy oligonucleotides, tough decoy oligonucleotides, peptide nucleic acid (PNA) oligonucleotides, or locked nucleic acid (LNA) oligonucleotides. The ribonucleic acid includes double-stranded small interfering RNA (siRNA), short hairpin RNA (shRNA) and ribozyme.

LNA is a modified ribonucleotide and has a locked form by including an additional bridge between the 2' to 4' carbons of the ribose sugar moiety, and the oligonucleotide with LNA has improved thermal stability.

Peptide nucleic acids (PNAs) contain a peptide-based backbone instead of a sugar-phosphate backbone. The 2'-O-modified oligonucleotide may be a 2'-O-alkyl oligonucleotide, a 2'-O-C1-3 alkyl oligonucleotide, or a 2'-O-methyl oligonucleotide.

The antisense oligonucleotide includes, in a narrow sense, an antisense oligonucleotide, an antagomere and an inhibitory RNA molecule.

As used herein, the term "antagomere" is a single-stranded chemically modified oligonucleotide, used for the silence of endogenous microRNA. Antagomere contains a non-complementary sequence at the Arganoute 2 (Ago 2) cleavage site, or the base is modified with, for example, 2' methoxy group, 3' cholesterol group, phosphorothioate to inhibit Ago2 cleavage. and has a complementary sequence to the target sequence. The antagomere according to the present specification may have a sequence that is at least partially or completely complementary to miR-106b-5p. In one embodiment the antagomere may comprise one or more modifications (eg, a 2'-0-methyl-sugar modification, or a 3' cholesterol modification). In other embodiments, the antagomere comprises one or more phosphorothioate linkages and has at least partially a phosphorothioate backbone. The length of an antagomere suitable for inhibiting miR-106b-5p and the like according to the present specification may be 7-50 nucleotides, 10-40 nucleotides, 15-30 nucleotides, 15-25 nucleotides, or 16-19 nucleotides.

As used herein, the term "complementary" means that the antisense oligonucleotide is sufficiently complementary to selectively hybridize to the miR-106b-5p target under certain hybridization or annealing conditions, preferably physiological conditions, partially or partially. It may mean to encompass both substantially complementary (substantially complementary) and perfectly complementary (perfectly complementary). Substantially complementary, although not completely complementary, refers to a degree of complementarity sufficient to bind to a target sequence and produce an effect sufficient to interfere with the effect according to the present specification, that is, miR-106b-5p activity.

As used herein, the term "nucleic acid" includes polynucleotides, oligonucleotides, DNA, RNA, and analogs and derivatives thereof, and includes, for example, peptide nucleic acids (PNA) or mixtures thereof. In addition, the nucleic acid may be single or double-stranded, and may encode a molecule including a polypeptide, mRNA, microRNA, or siRNA.

In one embodiment, the substance capable of inhibiting the activity of miR-106b-5p complementarily binds to all or part of the precursor and/or mature sequence of miR-106b-5p, in particular the seed sequence, thereby inhibiting its activity. It is an antisense oligonucleotide capable of inhibiting. Inhibition of this activity is to inhibit transcription of miR-106b-5p and/or binding of miR-106b-5p to target mRNA. The antisense oligonucleotide according to the present specification may or may not include one or more of the following modifications in the nucleotides constituting it or the backbone (backbone) connecting the nucleotides. That is, in the antisense oligonucleotide, one or more nucleotides constituting the same is LNA, or the sugar of one or more nucleotides constituting the same is 2′-O-methylated, or one or more phosphothioate in the backbone thereof.

In one embodiment, the antisense oligonucleotide or nucleic acid molecule of the present specification comprises a sequence complementary to all or part of the seed sequence of miR-106b-5p. Seed sequences are conserved sequences in various species that are very important for the recognition of target molecules of miRNAs. Since miRNA binds to the target through the seed sequence, when the interaction of the seed sequence with the target is inhibited, the translation of the target mRNA can be effectively inhibited. Each of the at least one nucleotide constituting the oligonucleotide may be 2'-O-methylated, or each of the at least one nucleotide may be LNA, or at least one of the chemical bonds constituting the backbone may be phosphothioate, but the modification may not include

In another embodiment, the substance capable of inhibiting the activity of miR-106b-5p may include the oligonucleotide sequence of SEQ ID NO: 2 or SEQ ID NO: 4.

In another embodiment, the composition may be a gene therapy agent. Specifically, the substance capable of inhibiting the miR activity may be a vector containing a substance capable of inhibiting the miR activity, or a cell transformed with or containing the same.

The term “vector” refers to a means for expressing a nucleic acid molecule of interest in a host cell. Viral vectors such as, for example, plasmid vectors, cosmid vectors and bacteriophage vectors, adenoviral vectors, retroviral vectors, RNA expression vectors, and adeno-associated viral vectors are included. Vectors that can be used as the recombinant vector include plasmids often used in the art (eg, pSC101, pGV1106, pACYC177, ColE1, pKT230, pME290, pBR322, pUC8/9, pUC6, pBD9, pHC79, pIJ61, pLAFR1, pHV14. , pGEX series, pET series, and pUC19, etc.), phage or virus (eg, SV40, etc.) can be constructed.

The recombinant vector can typically be constructed as a vector for cloning or a vector for expression. The expression vector may be a conventional vector used to express a foreign protein in plants, animals, or microorganisms in the art. The recombinant vector can be constructed through various methods known in the art. The recombinant vector can be constructed using a prokaryotic cell or a eukaryotic cell as a host. For example, when the vector used is an expression vector and a prokaryotic cell is a host, a strong promoter capable of propagating transcription (eg, pLλ promoter, CMV promoter, trp promoter, lac promoter, tac promoter, T7) promoter, etc.), a ribosome binding site for initiation of translation, and transcription/translation termination sequences. In the case of a eukaryotic cell as a host, the replication origin operating in the eukaryotic cell contained in the vector includes the f1 origin of replication, the SV40 origin of replication, the pMB1 origin of replication, the adeno origin of replication, the AAV origin of replication and the BBV origin of replication. It is not limited. In addition, a promoter derived from the genome of a mammalian cell (eg, a metallotionine promoter) or a promoter derived from a mammalian virus (eg, adenovirus late promoter, vaccinia virus 7.5K promoter, SV40 promoter, cytomegalovirus promoter and tk promoter of HSV) can be used, and generally have a polyadenylation sequence as a transcription termination sequence.

The sequence of miR-106b-5p herein is derived from mammals, for example, from humans, mice, or rats. For example, the mature sequence of miR-106b-5p may include SEQ ID NO: 1, and the precursor sequence of miR-106b-5p may include SEQ ID NO: 2.

In the present specification, inhibition of miR-106b-5p activity means inhibiting or interfering with the intracellular action or function of miR-106b-5p. Typically, miR-106b-5p interacts with an mRNA molecule encoding its target KNCQ2. Direct inhibition of binding, or direct inhibition of the function of miR-106b-5p using a small molecule inhibitor, antibody or antibody fragment, or indirect modulation using an inhibitor or small interfering RNA molecule. Furthermore, interfering with or inhibiting the activity of miR-106b-5p includes directly or indirectly inhibiting the activity of a precursor sequence (eg, SEQ ID NO: 3) and a mature sequence (eg, SEQ ID NO: 1). In addition, inhibition of miR-106b-5p activity includes suppressing the transcription of miR-106b-5p and lowering its intracellular concentration.

Without being limited to a particular theory, as used herein, miR-106b-5p binds to the 3' untranslated region of the mRNA encoding the KCNQ2 gene and inhibits its expression, thereby reducing the expression of the KCNQ2 gene or protein associated with epilepsy. it may be to do Therefore, the substance capable of inhibiting the activity of miR-106b-5p may increase the expression of KCNQ2, or the target mRNA of miR-106b-5p may be the 3'-untranslated region (UTR) of KCNQ2. have.

Therefore, inhibition of the activity of miR-106b-5p of the present specification increases the expression of KCNQ2, which can be used for the treatment of epilepsy or seizure-related diseases.

The composition of the present specification is used for the treatment of epilepsy. Epilepsy is a disease that causes various neurological symptoms such as sudden loss of consciousness, convulsions, psychiatric or sensory disturbances due to lesions or functional disorders of the brain tissue, resulting in persistent recurrent unstimulated seizures. can indicate

Epilepsy includes chronic epilepsy and status epilepticus (SE) lasting more than 30 minutes once onset. When classified by seizure, epilepsy includes partial epilepsy, representing partial epilepsy attacks, and generalized epilepsy, representing generalized epilepsy attacks. And when classifying by cause, there are idiopathic, which has no specific cause other than genetic factors, symptomatic in which lesions that cause epilepsy are found on brain images such as MRI or CT, and lesions that cause epilepsy. It is presumed to be, but it includes hidden epilepsy in which no abnormal findings are found on the current brain imaging.

As used herein, the term “seizure-associated disease” includes brain-related diseases that are accompanied by seizures, and causes seizures, eg, stroke; encephalitis; hippocampal sclerosis; cerebral palsy and congenital malformations; CNS infection-related seizures; hypoxia; brain tumor; traumatic brain injury; vascular malformations; neurodegenerative diseases; metabolic diseases such as hypoglycemia, glycogen storage disease and pyruvate dehydrogenase deficiency; multiple sclerosis; autoimmune diseases such as systemic lupus erythematosus, and seizure diseases due to unknown causes. The compositions herein are used for the treatment of seizures resulting from seizure related disorders.

The composition of the present specification may include one or more pharmaceutically acceptable diluents, carriers and/or adjuvants in addition to the above-mentioned active ingredients. The pharmaceutically acceptable carrier may be used in a mixture of saline, sterile water, Ringer's solution, buffered saline, dextrose solution, maltodextrin solution, glycerol, ethanol, liposome, and one or more of these components, and, if necessary, an antioxidant , buffers, bacteriostatic agents, and other conventional additives may be added. In addition, by additionally adding diluents, dispersants, surfactants, binders and lubricants, it can be formulated into injectable formulations such as aqueous solutions, suspensions, emulsions, pills, capsules, granules or tablets, and can act specifically on target organs. A target organ-specific antibody or other ligand may be used in combination with the carrier. Furthermore, it can be preferably formulated according to each disease or component using an appropriate method in the art or a method disclosed in Remington's Pharmaceutical Science (Remington's Pharmaceutical Science (latest edition), Mack Publishing Company, Easton PA). have. For example, it can be prepared in various formulations and dosage forms such as solutions, emulsions and liposome formulations. For example, the active ingredient may be mixed with a pharmaceutically acceptable liquid and/or fine powder solid carrier or excipient to prepare tablets, capsules, gels, syrups or suppositories. In addition, the composition according to the present specification may be prepared as a suspension using an aqueous, non-aqueous or mixed medium. Aqueous suspensions may further contain substances which increase the viscosity of the suspension, such as sodium carboxymethylcellulose, sorbitol and/or dextran.

The administration method of the composition according to the present specification is not particularly limited, and a known inhibitor administration method may be applied, and parenteral administration (eg, intranasal, intravenous, subcutaneous, intraperitoneal or It can be applied topically) or administered orally. The composition of the present specification may also be delivered by various routes, for example, by infusion or bolus injection, or administered through the epidermis or mucosa (oral mucosa, anal mucosa, intestinal mucosa, etc.). The compositions herein may also be administered systemically or locally.

Furthermore, the composition of the present specification may be delivered to the brain. Specifically, the composition may be introduced into the central nerve or the peripheral nerve through an appropriate route. Routes include intraventricular or intrathecal administration. Such administration may be accomplished using a catheter connected to a reservoir. As long as the effect according to the present specification occurs, formulations for intravenous administration, subcutaneous injection, intrathecal injection, inhalation administration, or oral administration are not excluded. In one embodiment according to the present specification, it may be administered nasally.

The composition according to the present specification is administered in a pharmaceutically effective amount. As used herein, "pharmaceutically or therapeutically effective amount" means an amount sufficient to treat a disease with a reasonable benefit/risk ratio applicable to medical treatment, and the effective dose level is determined by the type, severity, The activity of the drug, the sensitivity to the drug, the time of administration, the route of administration and the rate of excretion, the duration of treatment, factors including concurrently used drugs, and other factors well known in the medical field may be determined according to factors. The composition of the present invention may be administered as an individual therapeutic agent or in combination with other therapeutic agents, may be administered sequentially or simultaneously with conventional therapeutic agents, and may be administered singly or multiple times. In consideration of all of the above factors, it is important to administer an amount that can obtain the maximum effect with a minimum amount without side effects, which can be easily determined by those skilled in the art.

The dosage varies greatly depending on the patient's weight, age, sex, health status, diet, administration time, administration method, excretion rate, and severity of disease, etc. It may vary depending on the amount of and/or the specific efficacy of the polynucleotide used. In general, it can be calculated based on the EC50 measured to be effective in in vivo animal models and in vitro, for example, it can be 0.01 μg to 1 g per 1 kg of body weight, and in unit periods of daily, weekly, monthly or yearly, It may be administered once to several times per unit period, or may be administered continuously for a long period of time using an infusion pump. The number of repeated administrations is determined in consideration of the time the drug stays in the body, the concentration of the drug in the body, and the like. According to the course of disease treatment, the composition may be administered for relapse even after treatment has been completed.

The active ingredient according to the present specification, for example, an antisense oligonucleotide may be used in the composition by itself or in the form of a pharmaceutically acceptable salt. A pharmaceutically acceptable salt is one in which undesirable toxicity is minimized while maintaining the biological activity of the polynucleotide according to the present specification. Such salts include, for example, base addition salts formed with metal cations such as zinc, calcium, bisbus, barium, magnesium, aluminum, copper, cobalt, nickel, cadmium, sodium, potassium, etc. and salts formed with organic amino acids, or ammonia, N , N- dibenzylethylene-diamine (dibenzylethylene-diamine), D- glucosamine (glucosamine), tetraethylammonium (tetraethylammonium), or ethylenediamine (ethylenediamine) derived from cations and salts formed with, but limited to this no.

In one embodiment according to the present specification, the active ingredient of the present specification, for example, an antisense oligonucleotide may be negatively charged due to the nature of the nucleotides constituting it. Due to the lipophilic nature of the cell membrane, the uptake of antisense oligonucleotides into the cell membrane may be reduced. Interference with absorption due to this polarity can be addressed via the prodrug approach described below (Crooke, RM (1998) in Crooke, ST Antisense research and Application. Springer-Verlag, Berlin, Germany, vol. 131, pp. 103-140).

Another aspect is an agent capable of detecting any one miRNA selected from the group consisting of miR-17-5p, miR-93-5p, and miR-106b-5p, for example, a nucleotide sequence, a complementary sequence thereof Or it provides a kit for diagnosis of epilepsy or seizure-related disease comprising a fragment of the sequence.

In another aspect, detecting the expression of any one miRNA selected from the group consisting of miR-17-5p, miR-93-5p, and miR-106b-5p in a biological sample isolated from a subject; And a method of detecting a marker, or a method of diagnosing epilepsy or a seizure-related disease to provide information on the diagnosis of epilepsy or a seizure-related disease, comprising comparing the expression level of the detected marker with a control group to provide.

Specifically, the present specification provides the steps of detecting the expression of miR-106b-5p and KCNQ2 from a biological sample isolated from an individual and analyzing their interaction; and comparing the interaction of miRN-106b-5p and KCNQ2 according to the expression level of miR-106b-5p and KCNQ2 with a control group. To provide information for diagnosing epilepsy or seizure disease, detecting a marker. A method for diagnosing epilepsy or seizure-related disease is provided.

As used herein, the term “diagnosis” refers to ascertaining the presence or character of a pathological condition. For the purposes of the present specification, diagnosis may mean confirming whether epilepsy or seizure-related disease occurs, or a disease prognosis.

In the present specification, an agent capable of detecting miRNA specifically recognizes miRNA and may include a sequence complementary to the sequence. The probe or primer of the present invention may have one or more mismatched nucleotide sequences as long as it is capable of selectively hybridizing to the nucleotide sequence in addition to being completely complementary.

As used herein, the term "primer pair" includes all combinations of primer pairs consisting of forward and reverse primers recognizing a target gene sequence, and specifically, a primer pair that provides analysis results with specificity and sensitivity. Since the nucleic acid sequence of the primer is a sequence that does not match the non-target sequence present in the sample, high specificity can be conferred when the primer amplifies only the target gene sequence containing the complementary primer binding site and does not cause non-specific amplification. .

As used herein, the term “probe” refers to a material capable of specifically binding to a target material to be detected in a sample, and refers to a material capable of specifically confirming the presence of a target material in the sample through the binding. do. The type of probe molecule is not limited as a material commonly used in the art, but preferably PNA (peptide nucleic acid), LNA (locked nucleic acid), peptide, polypeptide, protein, RNA or DNA. More specifically, the probe is a biomaterial derived from or similar thereto, or manufactured in vitro, and includes, for example, enzymes, proteins, antibodies, microorganisms, animal and plant cells and organs, neurons, DNA, and RNA. and DNA includes cDNA, genomic DNA, and oligonucleotides, RNA includes genomic RNA, mRNA, and oligonucleotides, and examples of proteins include antibodies, antigens, enzymes, peptides, and the like.

The kit or diagnostic method of the present specification may additionally include other components in addition to the above components. For example, when the kit or diagnostic method of the present specification is applied to a PCR amplification process, the kit or diagnostic method of the present specification may optionally include reagents necessary for PCR amplification, such as a buffer, a DNA polymerase (eg, Thermus aquaticus). (Taq), Thermus thermophilus (Tth), Thermus filiformis, Thermis flavus, Thermococcus literalis or thermostable DNA polymerase obtained from Pyrococcus furiosus (Pfu)), DNA polymerase cofactors and dNTPs. The kit of the present specification may be manufactured in a plurality of separate packaging or compartments containing the reagent components described above. In addition, the kit may be a microarray. When the kit of the present specification is a microarray, the probe may be immobilized on the solid surface of the microarray. The nucleotide sequence of the miRNA of the present invention to be referenced when preparing a primer or probe can be confirmed in miRBase, and a primer or probe can be designed with reference to this sequence.

The step of comparing in the method of the present specification may be comparing the determined expression level of the marker with the detection result for each of the markers determined in a normal control. For example, compared to a normal control, the analyzed expression level of miR-106b-5p is decreased, the expression level of KCNQ2 is increased, or the expression of KCNQ2 is regulated by the miR-106b-5p, or the miR If the interaction between -106b-5p and KCNQ2 is increased, it may include a step of determining epilepsy or a disease related to erection.

As used herein, the term "measuring the expression level of a gene or miRNA" is a process of determining the presence and expression level of mRNA of genes in a biological sample to diagnose epilepsy or a disease related to epilepsy, measuring the amount of mRNA, or It may mean measuring the expression level of Analysis methods for this include reverse transcription polymerase reaction (RT-PCR), competitive reverse transcription polymerase reaction (Competitive RT-PCR), real-time reverse transcription polymerase reaction (Real-time RT-PCR), RNase protection assay (RPA; RNase protection) assay), Northern blotting, and a DNA chip.

As used herein, the term “protein level measurement” is a process of confirming the presence and expression level of epilepsy or erection-related disease marker proteins in a biological sample for diagnosis of epilepsy or erectile dysfunction. The amount of the protein can be confirmed using an antibody that specifically binds to the marker protein, and, for example, the protein expression level itself can be measured without using the antibody. The protein level measurement or comparative analysis method includes protein chip analysis, immunoassay, ligand binding assay, MALDI-TOF (Matrix Desorption/Ionization Time of Flight Mass Spectrometry) analysis, SELDI-TOF (Sulface Enhanced Laser Desorption/Ionization Time of Flight Mass Spectrometry analysis, radioimmunoassay, radioimmunodiffusion method, Ouchteroni immunodiffusion method, rocket immunoelectrophoresis, tissue immunostaining, complement fixation assay, 2D electrophoresis analysis, liquid chromatography-mass spectrometry Mass Spectrometry, LC-MS), LC-MS/MS (liquid chromatography-Mass Spectrometry/ Mass Spectrometry), Western blot, and ELISA (enzyme linked immunosorbent assay) may be used.

As used herein, the term "biological sample" means a sample, a non-mucosal sample, tissue, cell, whole blood, serum, plasma, saliva, cerebrospinal fluid, or samples such as urine; and the like.

Furthermore, the method according to the present application may additionally use non-marker clinical information of the test subject for seizure disease when diagnosing or prognosing the epilepsy or seizure-related disease of the test subject based on the expression level of the marker. The non-marker clinical information of the test subject may include the subject's age, sex, weight, diet, body mass, underlying disease and EEG, seizure type, brain MRI, brain CT, or cerebrospinal fluid test.

The expression level of the miRNA analyzed by the kit or method of the present specification, for example, RT (reverse transcriptase)-PCR or real-time-PCR method (see: Sambrook, J. et al., Molecular Cloning. A When the expression level measured by Laboratory Manual, 3rded. Cold Spring Harbor Press (2001)) shows an expression level of 1.2 times or more, 1.5 times or more, or 2 times or more compared to the normal control, the subject from which the analysis sample was collected (subject) ) may be determined to have epilepsy or seizure disease, especially epilepsy disease, or to have a high risk of developing it. In addition, when the interaction between miRNA and mRNA of the target gene exhibits an expression level of 1.2-fold, 1.5-fold, or 2-fold or more compared to the normal control, the subject from which the analysis sample was collected is particularly susceptible to epilepsy or seizure disease. It may be determined that you have epilepsy or have a high risk of developing epilepsy. The interaction may refer to an inverse correlation in which the expression of a target gene or protein decreases or increases according to the increase or decrease of miRNA. The method for detecting RNA-RNA interaction used in the method herein is known in the art, for example, RNA Walk (Lusting et al., Nucleic Acids Res. 2010; 38(1): those described in: e5. reference) or Yeat two hybrid system (Piganeau et al., RNA 2006; 12: 177-184), and the like, and RNA: A Laboratory Manual (Cold Spring Harbor Laboratory Press 2011) may be referred to.

Another aspect includes contacting a candidate agent with miR-106b-5p; And it provides a method of screening for a therapeutic agent for epilepsy or seizure-related disease, comprising the step of selecting a candidate substance having a reduced expression level of miR-106b-5p compared to an untreated control as a therapeutic agent for epilepsy or seizure-related disease do.

In the present specification, the miR-106b-5p RNA may be provided in the form of a cell expressing it. The activity was analyzed by expression of the miR-106b-5p RNA. For example, after contacting a cell expressing miR-106b-5p according to the present application with a test substance, the change in the expression level of miR-106b-5p is compared with a control cell that is not contacted or before the contact. , especially those with a reduction can be selected as candidates. In the present specification, the expression level of miRNA may be performed using the same method as mentioned in the above diagnostic method.

In another embodiment, the miR-106b-5p RNA is provided in the form of a cell expressing it, and the activity is determined by analysis of the interaction between the miR-106b-5p RNA and the 3'-UTR of its target KCNQ2. do. For example, after contacting a cell expressing miR-106b-5p according to the present specification with a test substance, the degree of interaction between the 3'-UTR of KCNQ2 and miR-106b-5p was measured before contact or with a non-contact control cell. By comparison, those with fluctuations or reductions in the interaction are selected as candidates. The method for detecting the RNA-RNA interaction used in the method herein may be performed using the same method as mentioned in the diagnostic method.

The type of cells used in this method, the amount and type of candidate substances, etc. vary depending on the specific experimental method and type of the test substance used, and those skilled in the art will be able to select the appropriate type, amount, and/or conditions of the cells. As a result of the experiment, a substance that causes a decrease in miR-106b-5p RNA activity in the presence of the test substance as compared to a control that has not been contacted with the candidate substance is selected as a candidate substance. About 99% or less reduction, about 95% or less reduction, about 90% reduction, about 85% reduction, about 80% reduction, about 75% reduction, about 70% reduction, about 65% or less reduction, about 60% or less compared with control reduction, about 55% reduction, about 50% or less reduction, about 45% or less reduction, about 40% or less reduction, about 30% or less reduction, about 20% or less reduction, but not excluding the scope thereof.

As used herein, the term “candidate substance” refers to a substance expected to inhibit the activity of any one miRNA selected from the group consisting of miR-17-5p, miR-93-5p, and miR-106b-5p as described above. Thus, low molecular weight compounds, high molecular weight compounds, mixtures of compounds (eg, natural extracts or cell or tissue cultures), or biopharmaceuticals (eg, proteins, antibodies, peptides, DNA, RNA, antisense oligonucleotides, RNAi, aptamers) , RNAzyme and DNAzyme), or sugars and lipids, and the like. The candidate material may be obtained from a library of synthetic or natural compounds, and methods for obtaining such a library of compounds are known in the art. Synthetic compound libraries are available from Maybridge Chemical Co. (UK), Comgenex (USA), Brandon Associates (USA), Microsource (USA) and Sigma-Aldrich (USA), and natural compound libraries are available from Pan Laboratories (USA) and It can be purchased from MycoSearch (USA). Candidate substances can be obtained by various combinatorial library methods known in the art, for example, biological libraries, spatially addressable parallel solid phase or solution phase libraries, deconvolution It can be obtained by the required synthetic library method, the “1-bead 1-compound” library method, and the synthetic library method using affinity chromatography selection. Also, for example, biological agents (Biologics) may be used for screening. Biologics refer to cells or biomolecules, and biomolecules refer to proteins, nucleic acids, carbohydrates, lipids, or substances produced using cell systems in vivo and in vitro. The biomolecule may be provided alone or in combination with other biomolecules or cells. Biomolecules include, for example, polynucleotides, peptides, antibodies, or other proteins or biological organisms found in plasma.

Another aspect provides an animal model of epilepsy or seizure-related disease genetically engineered to increase the expression or activity of miR-106b-5p.

Another aspect provides a method for preparing an animal model of epilepsy or seizure-related disease, comprising administering to an animal other than human, miR-106b-5p or a substance that increases its expression.

In one embodiment, the animal model may be one introduced with a vector containing miR-106b-5p.

Also, as used herein, the term "genetic engineering" or "genetically engineered" refers to the act of introducing one or more genetic modifications into a cell or a cell made thereby. . For example, the cell may be genetically engineered to increase the expression or activity of miR-106b-5p. The increased activity may mean that the same type of activity has a higher activity compared to the intrinsic activity that a given non-genetically engineered parental cell (eg, wild-type) does not have or has. The exogenous nucleic acid molecule may be expressed in an amount sufficient to increase the activity of the miRNA mentioned in the cell compared to its parent cell. The exogenous nucleic acid molecule may be introduced into a parent cell through an expression vector. In addition, the exogenous nucleic acid molecule may be introduced into a parent cell in the form of a linear polynucleotide. In addition, the exogenous nucleic acid molecule may be expressed from an expression vector (eg, a plasmid) in a cell. In addition, the exogenous nucleic acid molecule may be expressed by being inserted into a genetic material (eg, a chromosome) in a cell for stable expression. Accordingly, in another embodiment, a recombinant vector comprising the miRNA, the miRNA polynucleotide, and a recombinant cell comprising the recombinant vector may be provided.

The vectors and cells are as described above.

According to a substance capable of inhibiting miRNA according to an aspect, it increases the expression of KCNQ2 to reduce neuronal excitability related to epilepsy, so it can be usefully used for the treatment of epilepsy, and in a method for diagnosing epilepsy using it as a biomarker Not only can it be used, but there is an effect that can be usefully used for disease animal models.

1 is a graph showing the qPCR results of miR-105b-5p for each stage of development in the hippocampal tissue of the mouse brain.
Figure 2a is a graph showing whether miR-106b-5p according to an embodiment regulates the expression of the KCNQ2 gene.
Figure 2b is a graph showing whether miR-17-5p according to an embodiment regulates the expression of the KCNQ2 gene.
Figure 2c is a graph showing whether miR-93-5p according to an embodiment regulates the expression of the KCNQ2 gene.
Figure 2d is a view showing the mRNA of miR-106b-5p according to one embodiment, while miR-106b-5p binds to the 3' UTR of mouse KCNQ2 (wt), mouse KCNQ2 (mut) having a mutation in the seed sequence; indicates that it does not bind to the 3'UTR of
Figure 3a is a graph showing changes in the expression of KCNQ2 protein in hippocampal tissue according to the age of the mouse.
Figure 3b is a graph showing the change in miR-106b-5p expression in hippocampal tissue according to the age of the mouse.
Figure 4a is a Western blotting result showing the relative expression level of KCNQ2 according to the miR-106b-5p miRic and inhibitor treatment.
Figure 4b is a graph showing the quantification of the relative expression level of KCNQ2 according to the miR-106b-5p miRic and inhibitor treatment.
Figure 5a is a Western blotting result showing the relative expression level of KCNQ2 according to the treatment of precursor (Pre) miR-106b-5p and TuD-miR-106b-5p.
Figure 5b is a graph showing the quantification of the relative expression level of KCNQ2 according to the treatment of precursor (Pre) miR-106b-5p and TuD-miR-106b-5p.
6A is a diagram illustrating a voltage change of a neuron related to neuronal excitability in a neuron according to an increase in the expression of miR-106b-5p.
6B is a diagram showing changes in action potential, maintenance potential, and AP frequency associated with neuronal excitability in neurons according to the increase in the expression of miR-106b-5p; Red: miR106b, Black: Control)
6c is a diagram showing the change in conductance related to neuronal excitability in neurons according to the increase in the expression of miR-106b-5p.
6D is a diagram showing changes in action potential firing related to neuronal excitability in neurons according to an increase in the expression of miR-106b-5p.
FIG. 6e is a diagram showing the change in oncet time (time required from the time when current is induced to the time the action potential is fired) related to neuronal excitability in neurons according to the increase in the expression of miR-106b-5p.
7A is a diagram illustrating a voltage change of a neuron related to neuronal excitability in a neuron according to a decrease in the expression of miR-106b-5p.
7B is a diagram showing changes in action potential, maintenance potential, and AP frequency related to neuronal excitability in neurons according to the decrease in miR-106b-5p expression; Red: miR106b, Black: Control)
FIG. 7c is a diagram illustrating a change in conductance related to neuronal excitability in neurons according to a decrease in miR-106b-5p expression.
Fig. 7d is a diagram showing changes in action potential firing related to neuronal excitability in neurons according to the decrease in miR-106b-5p expression.
FIG. 7e is a diagram showing the change in oncet time (the time taken from the time the current is induced to the time the action potential is fired) related to the neuronal excitability in the nerve cells according to the decrease in the expression of miR-106b-5p.

Hereinafter, it will be described in more detail through the following examples. However, these examples are for illustrative purposes only, and the scope of the present invention is not limited to these examples.

Example 1. Identification of miRNA related to epilepsy

The KCNQ2 gene encodes a subunit of a calcium channel that regulates neuronal excitability, and mutations in this gene are known to induce neonatal epilepsy. Therefore, in this example, it was determined whether a highly conserved microRNA binding site exists in the 3'UTR (590bp) of the mouse KCNQ2 gene, TargetScan (www.targetscan.org), Miranda (www.microRNA.org). and miRDB (www.mirDB.org). Afterwards, five common miR, miR-17-5p, miR-20a-5p, miR-93-5p, miR-106a-5p, and miR-106b-5p were derived through the three programs. . Thereafter, the hippocampal tissue of mouse brain expressing KCNQ2 and functionally related to epilepsy was isolated. Specifically, mouse brains at various stages after birth were first isolated, and from these, under a stereoscopic microscope, a clear banana-shaped hippocampus tissue was isolated using forceps. RNA-seq isolates RNA from the hippocampus tissue of Shingi, separates small RNAs of less than 50 nt from the gel, and attaches an adapter to prepare a strand specific cDNA library. Thereafter, single-end 51 nt sequencing was performed using the Illumina Hiseq 2000 system, and the results are shown in Table 1 below.

RPM (read per million) values for hippocampal tissue miRNAs P1 P14 P30 P60 mmu-miR-17-5p 442.60 68.29 38.82 16.01 mmu-miR-20a-5p 189.69 38.04 24.64 8.96 mmu-miR-93-5p 752.89 155.60 109.75 93.49 mmu-miR-106b-5p 441.43 110.65 75.40 56.35

Next, RNA was extracted from hippocampal tissue for qPCR using RNeasy mini kit (QIAGEN, Germany). cDNA synthesis for miRNA qpcr was reverse transcribed using Mir-X miRNA First-Strand Synthesis Kit (clontech, USA), and cDNA synthesis for mRNA was reverse transcribed using ImProm-II™ Reverse Transcriptase kit (Promega, USA). did The synthesized samples were amplified with iTaq Universal SYBR Green Supermix (Bio-Rad, USA). Amplification and detection were performed in a CFX96TM real-time PCR system (Bio-Rad, USA), and the results are shown in FIG. 1 .

1 is a graph showing the qPCR results of miR-105b-5p for each stage of development in the hippocampal tissue of the mouse brain.

As shown in FIG. 1 , it was confirmed that miR-20a-5p and miR-106a-5p did not exist in the hippocampal tissue of the mouse brain, and thus, miR-17-5p, miR-93-5p, and miR-106b It was found that the three types of miR of -5p bind to the 3'UTR of the KCNQ2 gene and regulate its expression.

Thereafter, a luciferase assay was performed to confirm the relationship between the three types of miRNAs and the expression regulation of the KCNQ2 gene. Specifically, the total 3'-UTR sequence (WT) and mutant sequence (Mut) of mouse KCNQ2 were cloned into a luciferase reporter construct (SwitchDB, USA). HEK293T cells were aliquoted in a 48-well plate at a concentration of 2.5x10 ⁴ cells/well and cultured overnight at 37° C. under 5% CO ₂ conditions. The next day, 25ng PRL-TK and 250ng of KCNQ2-3'UTR recombinant plasmid wild type or mutant, 20nM SCR mimic or mmu-miRNA-106b-5p were mixed with Lipofectamine 2000 (Invitrogen, USA) for transfection. switched. 24 hours after transduction, cells were washed with PBS, lysed using lysis buffer (Promega, USA), and luciferase activity assay was performed using Dual-Glo ^® assay kit (Promega, USA). Cell activity was measured using Multi-Mode Readers (Bio-Tek, USA), and the results are shown in FIG. 2 .

Figure 2a is a graph showing whether miR-106b-5p according to an embodiment regulates the expression of the KCNQ2 gene.

Figure 2b is a graph showing whether miR-17-5p according to an embodiment regulates the expression of the KCNQ2 gene.

Figure 2c is a graph showing whether miR-93-5p according to an embodiment regulates the expression of the KCNQ2 gene.

Figure 2d is a view showing the mRNA of miR-106b-5p according to one embodiment, while miR-106b-5p binds to the 3' UTR of mouse KCNQ2 (wt), mouse KCNQ2 (mut) having a mutation in the seed sequence; indicates that it does not bind to the 3'UTR of

As shown in Figures 2a to 2c, it can be seen that miR-106b-5p, miR-17-5p and miR-93-5p all reduced luciferase activity. These results suggest that miR-106b-5p, miR-17-5p and miR-93-5p bind to Luc-KCNQ2 WT 3' UTR and regulate KCNQ2 protein.

In addition, as shown in Fig. 2a, in the case of miR-106b-5p, it did not bind to Luc-KCNQ2 Mut3' UTR, which was mutated in the seed sequence to which the KCNQ2 gene binds, and thus did not change the luciferase activity. found out that it wasn't.

As a result, when miR-106b-5p, miR-17-5p and miR-93-5p all regulate the expression of KCNQ2, it can be seen that, in particular, miR-106b-5p more specifically regulates the expression of KCNQ2, In the following experiments, miR-106b-5p was used.

Example 2. Changes in KCNQ2 protein, an epilepsy-related gene, and the relationship between miRNA

In this example, the expression changes of miR-106b-5p and KCNQ2 proteins of Example 1 were investigated.

First, changes in the expression of miR-106b-5p and KCNQ2 proteins were analyzed in hippocampal tissues at the stage of development of mice from 1 to 6 weeks of age. Specifically, the hippocampal tissue was dissolved with RIPA buffer (Thermo, USA) and then centrifuged at 4 °C and 15,000 rpm for 15 minutes to obtain protein. After protein quantification using BCA protein assay reagent (Thermo, USA), 30 μg of protein was separated on a 10% SDS-PAGE gel. After the protein in the gel was transferred to a PVDF membrane (Millipore, USA), blocking was performed for 1 hour with a TBST solution containing 5% skim milk powder. The primary antibody (5% milk in TBST, 1:1000) was treated and reacted overnight at 4 °C, and after washing, the secondary antibody (5% milk in TBST, 1:10,000) was reacted at room temperature for 1 hour. The bound antibody was visualized on an x-ray film using an ECL chemiluminescence system, and the results are shown in FIGS. 3A and 3B .

Figure 3a is a graph showing changes in the expression of KCNQ2 protein in hippocampal tissue according to the age of the mouse.

Figure 3b is a graph showing the change in miR-106b-5p expression in hippocampal tissue according to the age of the mouse.

As shown in FIG. 3 , KCNQ2 expression was low in the first 1 and 2 weeks and gradually increased from 3 to 5 weeks, whereas miR-106b-5p was highly expressed in the first 1 and 2 weeks and continued thereafter. It was found that the expression was low. These results indicate that KCNQ2 and miR-106b-5p exhibit an inverse correlation, indicating that KCNQ2 is regulated by miR-106b-5p.

In addition, to more specifically confirm the correlation between miR-106b-5p and KCNQ2 expression, miR-106b-5p mimic (SEQ ID NO: 1), and miR-106b-5p inhibitor (SEQ ID NO: 2) A plasmid construct containing In addition, it contains the precursor miR-106b-5p (SEQ ID NO: 3) that increases miR-106b-5p, and Tough Decoy (TD) miR-106b-5p (SEQ ID NO: 4) that decreases miR-106b-5p The plasmid construct was prepared by synthesizing a DNA sequence and cloning it into a small RNA expression vector, pmiENSR, using BamHI and HindIII sites.

Thereafter, primary neurons were isolated from mouse hippocampus, and the duplex RNA mimetics and inhibitor were transformed into neurons 3 days after in vitro culture using the Ca phosphate method. As a control group, sequences that do not exist in mice or humans (SCR mimic and SCR inhibitor) were used to prevent effects on hippocampal neurons. Thereafter, Western blotting was performed in the same manner as above, and the results are shown in FIGS. 4A and 4B .

In addition, after transforming the precursor miR-106b-5p (SEQ ID NO: 3) and TD (Tough Decoy) miR-106b-5p (SEQ ID NO: 4) into cells in the same manner as above, in the same manner as above, The expression level of KCNQ2 was confirmed, and the results are shown in FIGS. 5A and 5B.

Figure 4a is a Western blotting result showing the relative expression level of KCNQ2 according to the miR-106b-5p miRic and inhibitor treatment.

Figure 4b is a graph showing the quantification of the relative expression level of KCNQ2 according to the miR-106b-5p miRic and inhibitor treatment.

Figure 5a is a Western blotting result showing the relative expression level of KCNQ2 according to the treatment of precursor (Pre) miR-106b-5p and TuD-miR-106b-5p.

Figure 5b is a graph showing the quantification of the relative expression level of KCNQ2 according to the treatment of precursor (Pre) miR-106b-5p and TuD-miR-106b-5p.

As shown in Fig. 4, when hippocampal neurons were treated with miR-106b-5p mimetics, cellular KCNQ2 expression was reduced by 15%, and conversely, when miR-106b-5p inhibitor was treated, KCNQ2 expression was increased by 20%. And it was found.

As shown in Figure 5, TuD-miR-106-5p increased the expression of KCNQ2 by more than 85% compared to the TuD control group, and also pre-miR-106b-5p decreased the KCNQ2 protein expression by 50% compared to the Pre control group. And it was found.

Example 3. Correlation between epilepsy phenotype and miRNA expression

The effect of miRNA expression regulation on the regulation of neuronal activation was analyzed using the plasmid construct of miR-106b-5p.

Specifically, the plasmid construct of miR-106b-5p was transformed using Ca Phosphate 3 days after plating the neurons isolated from the hippocampal tissue on the coverslip. Two days after transformation, the cells were transferred to a whole cell patch clamp chamber. GFP-positive neurons were checked under a microscope and immediately measured. The tip of a recording pipette drawn from glass capillaries was injected into the soma of the neuron, and the action potential firing rate was measured while maintaining the neuron at -60 mv. The recording analysis was performed using Clampfit software, and the results are shown in FIGS. 6 and 7 .

The results of the pre-miR-106b-5p are shown in FIG. 6 , and the results of the TuD-miR-106b-5p are shown in FIG. 7 .

6 is a diagram showing changes in neuronal excitability in neurons according to an increase in the expression of miR-106b-5p.

7 is a diagram showing changes in neuronal excitability in neurons according to a decrease in the expression of miR-106b-5p.

As shown in FIG. 6 , when pre-miR-106b-5p was expressed in neurons, the number of action potentials, the holding potential, and the AP frequency were increased compared to the control group. This means that the expression of KCNQ2 in neurons was decreased due to the increase of pre-miR-106b-5p, and the basic excitability of the neurons itself was greatly increased.

On the other hand, as shown in FIG. 7 , when TuD-miR-106-5p was expressed in neurons, the number of action potentials, maintenance potentials, and AP frequency were significantly reduced compared to the control group. These results suggest that suppressing miR-106-5p in epilepsy patients can induce an increase in KCNQ2 expression in neurons, thereby regulating excessive neuronal excitability such as seizures.

As a result of the above, miR-106b-5p regulates KCNQ2 related to epilepsy, and the miR-106b-5p can be utilized as a biomarker for epilepsy, as well as substances that inhibit miR-106b-5p It can be seen that it can be usefully used as a therapeutic agent for epilepsy.

Example 4. Construction of Epilepsy Animal Model Mice

Based on the result that the phenotype of epilepsy appears according to the change in the expression of miR-106b-5p in Example 3. did

Specifically, by constructing AAV pre-miR-106b-5p and injecting it into the hippocampus, neurons were transduced to allow the long-term stable expression of pre-miR-106b-5p. Therefore, an epilepsy model in which KCNQ2 expression is reduced and neuronal excitability is easily induced was prepared.

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Claims (6)

A nucleic acid molecule inhibiting the activity of miR-106b-5p by complementary binding to all or part of the nucleotide sequence of miR-106b-5p as an active ingredient, wherein the miR-106b-5p is SEQ ID NO: 1 or SEQ ID NO: It has a nucleotide sequence of 3, and the target mRNA of miR-106b-5p is a 3'-untranslated region (UTR) of KCNQ2. A pharmaceutical composition for preventing or treating epilepsy. The method according to claim 1, wherein the nucleic acid molecule is RNA, DNA, antagomir, antisense molecule, siRNA, shRNA, 2'-O-modified oligonucleotide, phosphorothioate-backbone deoxyribonucleotide, phosphorothioate- A pharmaceutical composition that is a backbone ribonucleotide, a decoy oligonucleotide, a tough decoy oligonucleotide, a peptide nucleic acid (PNA) oligonucleotide, or a locked nucleic acid (LNA) oligonucleotide. The pharmaceutical composition according to claim 1, wherein the nucleic acid molecule increases the expression of KCNQ2. delete delete The pharmaceutical composition of claim 1, wherein the composition is delivered to the brain.

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