KR20160139849A - Pharmaceutical composition comprising miR-7, miR-18a or miR-18b for prevention or treatment of metabolic disease - Google Patents

Abstract

The present invention relates to a use of miRNA as a pharmaceutical composition for preventing or treating metabolic diseases. In the present invention, a candidate substance of metabolic diseases using miRNA is developed, and it is confirmed that the substance is effectively down-regulated in the RNA level and protein level through specific binding to the target area of LXR. Accordingly, miRNA defined in the present invention, i.e. miR-7, miR-18a or miR-18b can be used for treatment of metabolic diseases such as atheriosclerosis, a fatty liver, diabetes, hyperlipidemia, hypertension, angina, myocardial infarction, etc.

Description

The present invention relates to a pharmaceutical composition for preventing or treating a metabolic disease comprising miR-7, miR-18a or miR-18b as an active ingredient.

The present invention relates to a pharmaceutical composition for the prevention or treatment of metabolic diseases comprising one or more miRs selected from the group consisting of miR-7, miR-18a and miR-18b as an active ingredient.

Metabolic diseases, including obesity and type 2 diabetes, are a disease caused by the inability of metabolism in each organ of our body, which is one of the biggest health threats in the world today. Atherosclerosis is caused by the accumulation of cholesterol in endothelium, which mainly covers the innermost part of the blood vessel, and the proliferation of endothelial cells, as a result of the old water pipe being rusted and foreign substances being deposited, atheroma 'is formed. Atheroma is thinned like porridge, and the surrounding area is surrounded by a hard fibrous membrane, a hardening half. When the hardening half becomes unstable, thrombus (blood cake) is formed in the blood vessel. In addition, if hemorrhage occurs inside the atheroma, the diameter of the inside of the blood vessel becomes sharply narrowed or the blood vessel becomes completely blocked, resulting in an obstacle to peripheral blood circulation. Arteriosclerosis is a kind of aging phenomenon in which degenerative changes occur in the middle layer of blood vessels, fibrosis progresses, and blood vessel elasticity decreases. This leads to systolic hypertension and cardiac hypertrophy, which results in thickening of the heart muscle. Recently, atherosclerosis and atherosclerosis are mixed with atherosclerosis. The treatment of atherosclerosis can be classified into two broad categories: treatment to prevent disease progression, enlargement of narrowed blood vessels when symptoms have already occurred due to the obstruction of blood supply to organs, There is a treatment that attaches blood vessels and makes a detour. An angiogram is performed to improve blood circulation (blood circulation) of the narrowed blood vessels. If there is a narrowed area, the blood vessel forming balloon is inflated through a catheter to inflate and narrow the narrowed part due to arteriosclerosis. Or a mesh-like stent to support the vessel wall of the coronary artery to prevent it from narrowing again, which is called angioplasty. In addition, statin is being developed as a therapeutic agent, and LDLdmf lowering and HDL uptake are the main methods, but many patients suffer from a decrease in coronary arteries. Therefore, there is a need to develop a new therapeutic agent that can be effectively treated without side effects.

Meanwhile, microRNAs (miRNAs), which are untranslated RNAs, have been known as powerful modulators of cell phenotype, function, and fate through transcriptional and epigenetic regulation. The microRNA gene is transcribed by RNA polymerase II to become large primary transcripts (pri-microRNA), and the pri-microRNA is a precursor microRNA (pre-microRNA) of about 70 nt in length by a protein complex containing Drosha, an RNase III enzyme . The pre-microRNA migrates to the cytoplasm and becomes a mature microRNA of about 22 nt by the second RNase III enzyme, DICER. Mature microRNA enters the ribonuclear particle and becomes an RNA-induced silencing complex, RISC, where the microRNA binds to the target site of the mRNA and inhibits translation or gene silencing by degrading the target mRNA. MicroRNAs are known to be involved in cell cycle regulation, cell death, development and physiological stages. It is also involved in stem cell differentiation, hematopoiesis, hypoxia, myocardial and skeletal muscle development, neurogenesis, insulin secretion, cholesterol metabolism, aging, immune response and virus proliferation. During the embryogenesis of embryogenesis, microRNAs are expressed either in a tissue-specific manner or at a specific stage, which implies that the microRNA also plays a role in differentiation and tissue specificity. MicroRNA binds to the target site in 3 'UTR of target mRNA to inhibit protein synthesis or induce gene silencing by inducing mRNA degradation. Because most target sites in mRNA are only partially complementary to the microRNA sequences, one microRNA can regulate several different mRNAs. In addition, one mRNA may have multiple binding sites for several microRNAs, thus having complex gene regulation relationships. In addition, many microRNAs are part of a very similar microRNA family. As described above, miRNAs are involved in various gene expression and also involved in various metabolic regulation processes including adipocyte differentiation, metabolic integration and appetite regulation. As a result, miRNA is highly likely to be used as a therapeutic candidate for metabolic diseases, and it is a promising area for development as a therapeutic agent.

Korean Patent Application No. 2012-0047868

The present inventors have developed a candidate substance that can be used as a preventive and therapeutic agent for metabolic diseases such as diabetes and obesity by using miRNA, and confirmed that miRNA having a specific sequence has such a pharmacological effect while searching for various candidate substances, It is finished.

Accordingly, the present invention aims at providing a pharmaceutical composition for preventing and treating metabolic diseases such as obesity and type 2 diabetes, which contains miRNA as an active ingredient.

For the purpose of this invention, the present invention provides a pharmaceutical composition for the prevention or treatment of metabolic diseases comprising as an active ingredient at least one miR selected from the group consisting of miR-7, miR-18a and miR-18b.

In one embodiment of the present invention, the miR-7 may be represented by SEQ ID NO: 1, miR-18a may be represented by SEQ ID NO: 2, and miR-18b may be represented by SEQ ID NO:

In one embodiment of the present invention, the miR-7, miR-18a and miR-18b may have an activity to specifically inhibit Liver X receptor beta.

In one embodiment of the present invention, the metabolic diseases may be selected from the group consisting of atherosclerosis, fatty liver, diabetes, hyperlipidemia, hypertension, angina pectoris and myocardial infarction.

In addition, the present invention includes miR (microRNA) as an active ingredient which binds at nucleotide positions 178 to 185 in the 3 'UTR sequence of Liver X receptor beta (SEQ ID NO: 4) .

In one embodiment of the present invention, the miR may be miR-7 represented by SEQ ID NO: 1.

The present invention also relates to a method for the metabolism of miR (microRNA), which binds at the 274th to 280th nucleotide positions in the 3'UTR sequence of Liver X receptor beta (SEQ ID NO: 5) A pharmaceutical composition for preventing or treating a disease is provided.

In one embodiment of the present invention, the miR may be miR-18a represented by SEQ ID NO: 2 or miR-18b represented by SEQ ID NO: 3.

The present invention also relates to a method of treating a liver X receptor beta (beta) in a cell, comprising the step of treating in vitro a miR of any one selected from the group consisting of miR-7, miR-18a and miR- lt; RTI ID = 0.0 > recptor < / RTI > beta).

In one embodiment of the present invention, miR-7 is represented by SEQ ID NO: 1, miR-18a is represented by SEQ ID NO: 2, and miR-18b is represented by SEQ ID NO: 3.

The inventors have developed candidate substances for metabolic diseases using miRNA according to the present invention and found that they effectively down-regulate RNA and protein levels through specific binding to the target site of LXRβ. Therefore, the candidate substance of the present invention may be useful for the treatment of metabolic diseases such as atherosclerosis, fatty liver, diabetes, hyperlipidemia, hypertension, angina pectoris and myocardial infarction.

Figure 1 shows the results of prediction of miRNA sites capable of regulating expression in miRWALK and other programmed mRNA 3'UTR regions.
Figure 2 shows the results of finding conserved sites for miRNAs that can regulate LXR [beta] expression. (a) shows the LXR beta 3 'UTR 178 to 185; (b) shows that 274-280 of LXR [beta] 3 ' UTR
Figure 3 is a schematic diagram of experiments performed to validate candidate miRNAs.
FIG. 4 is a schematic diagram of real-time PCR analysis for analyzing the expression of LXR? In Example 2. FIG.
FIG. 5 is a graph showing the results of real-time PCR of Example 2. FIG. (a) is the result of individual analysis of miRNAs, and (b) is the result of comparison with the control (Control). (* p < 0.05, ** p < 0.01, ns: not significant)
6 is a western blot analysis result for analyzing the expression of LXR? In Example 3. Fig. Expression of LXR [beta] was shown relative to the expression level of mRNA encoding GAPDH (glyceraldehyde phosphate dehydrogenase).
Fig. 7 is a graph showing the results of Example 3. Fig. (a) is the result of individual analysis of miRNAs, and (b) is the result of comparison with the control (Control). (* p < 0.05, ** p &lt; 0.01, ns: not significant)
Fig. 8 is a schematic diagram showing construction of LXR beta 3 'UTR inserted into a vector.
FIG. 9 shows the results of Luciferase reporter assay of Example 4. FIG.
FIG. 10 is a schematic diagram of inserting the LXR beta gene next to the furefly gene to perform Example 4. FIG.
FIG. 11 (a) is a schematic diagram of the Luciferase reporter assay of Example 4, and FIG. 11 (b) shows the MOA (mechanism of action) of pmirGLO Vector.
12 is a graph showing the results of the fourth embodiment. (a) is pmirGLO / h_LXR? 3'UTR FWD and (b) is pmirGLO / h_LXR? 3'UTR REV.

The terms used in the present invention are defined as follows.

"Nucleic acid" is meant to include any DNA or RNA, such as chromosomes, mitochondria, viruses and / or bacterial nucleic acids present in a tissue sample. Includes one or both strands of a double-stranded nucleic acid molecule and includes any fragment or portion of the intact nucleic acid molecule.

"Gene" means any nucleic acid sequence or portion thereof that has a functional role at the time of protein coding or transcription, or in the control of other gene expression. The gene may consist of only a portion of the nucleic acid encoding or expressing any nucleic acid or protein that encodes the functional protein. The nucleic acid sequence may comprise an exon, an intron, an initiation or termination region, a promoter sequence, another regulatory sequence, or a gene abnormality within a particular sequence adjacent to the gene.

The term "gene expression" generally refers to a cellular process in which a biologically active polypeptide is produced from a DNA sequence and exhibits biological activity in the cell. In this sense, gene expression includes post-transcriptional and post-transcriptional processes that not only involve transcription and translation processes, but can also affect the biological activity of the gene or gene product. Such procedures include, but are not limited to, RNA synthesis, processing and transport as well as polyp peptide synthesis, transport and post-translational modification of the polypeptide. In the case of a gene that does not encode a protein product, such as an miRNA gene, the term "gene expression" refers to a process in which a precursor miRNA is produced from a gene. Generally, this process is referred to as transcription, although the transcription product of the miRNA gene is not translated to produce a protein, unlike the transcription induced by RNA polymerase II on the protein coding gene. Nevertheless, the generation of mature miRNAs from miRNA genes is encompassed by the term "gene expression" as that term is used herein

"miR" or "microRNA" refers to 21 to 23 noncoding RNAs that regulate gene expression after transcription by promoting degradation of target RNA or inhibiting their translation. The maturation sequences of the miRNAs used herein can be obtained from the miRNA database (http://www.mirbase.org). Generally, microRNAs are transcribed into precursors of about 70-80 nt (nucleotide) long with a hairpin structure called pre-miRNA, and then matured by cleavage by the RNAse III enzyme Dicer. MicroRNAs form a ribonucleotide complex called miRNP, which cleaves the target gene by complementary binding to the target site, or inhibits translation. Over 30% of human miRNAs are present in clusters, which are transcribed into a single precursor and then cleaved to form the final mature miRNA.

The term "target gene" refers to a gene that is targeted for modulation using methods and compositions of the subject matter disclosed herein. Thus, the target gene comprises a nucleic acid sequence whose expression level is down-regulated by the miRNA at the mRNA or polypeptide level. Similarly, the term "target RNA" or "target mRNA" refers to a transcript of a target gene that will bind to miRNAs and direct the modulation of expression of the target gene

The term "transcription" refers to a cellular process involving the interaction of an RNA polymerase with a gene that induces expression as RNA of structural information present in the coding sequence of the gene.

The term "down-regulation" refers to the expression of a specific gene into mRNA or the expression level of a protein by an intracellular transcription or gene translation in an activated cell, .

"Introduction" means introducing foreign DNA into a cell by transfection or transduction. The transfection can be carried out in the presence or absence of a sugar such as calcium phosphate-DNA coprecipitation, DEAE-dextran-mediated transfection, polybrene-mediated transfection, electroporation, microinjection, liposomal fusion, lipofectamine and protoplast fusion Can be carried out by various methods known in the art. Transfection also refers to the transfer of genes into cells using virus or viral vector particles as a means of infection.

An "effective amount" is an appropriate amount that affects a beneficial or desired clinical or biochemical outcome. An effective amount may be administered one or more times. For purposes of the present invention, an effective amount of an inhibitor compound is an amount sufficient to temporarily alleviate, ameliorate, stabilize, reverse, slow down, or delay the progression of a disease state. If the recipient animal is capable of enduring the administration of the composition, or the administration of the composition to the animal is suitable, the composition will be "pharmaceutically or physiologically acceptable &quot;. If the dose administered is physiologically significant, it can be said that the formulation is administered in a "therapeutically effective amount &quot;. The formulation is physiologically relevant if the presence of the formulation results in a physiologically detectable change in the recipient.

"Treatment" means an approach to obtaining beneficial or desired clinical results. For purposes of the present invention, beneficial or desired clinical results include, but are not limited to, alleviation of symptoms, reduction in the extent of disease, stabilization (i.e., not worsening) of the disease state, (Either partially or totally), detectable or undetected, whether or not an improvement or temporary relief or reduction Also, "treatment" may mean increasing the survival rate compared to the expected survival rate when not receiving treatment. Treatment refers to both therapeutic treatment and prophylactic or preventative measures. Such treatments include treatments required for disorders that have already occurred as well as disorders to be prevented.

"Prevention" means any act that inhibits or delays the onset of a related disease. It will be apparent to those skilled in the art that the compositions herein may prevent early onset symptoms, or related disorders when administered prior to appearance.

"About" means that the reference quantity, level, value, number, frequency, percentage, dimension, size, quantity, weight or length is 30, 25, 20, 25, 10, 9, 8, 7, , Level, value, number, frequency, percent, dimension, size, quantity, weight, or length that varies from one to three, two, or one percent.

Throughout this specification, the words " comprising "and" comprising &quot;, unless the context requires otherwise, include the stated step or element, or group of steps or elements, but not to any other step or element, And that they are not excluded.

Hereinafter, the present invention will be described in detail.

The present invention provides a pharmaceutical composition for preventing or treating metabolic diseases comprising at least one miR selected from the group consisting of miR-7, miR-18a and miR-18b as an active ingredient. Preferably, miR-7 is represented by SEQ ID NO: 1, miR-18a is represented by SEQ ID NO: 2, and miR-18b is represented by SEQ ID NO: 3. In particular, miR-7, miR-18a and miR-18b have the activity of specifically inhibiting Liver X receptor beta.

The miRNA of the present invention targets LXR beta (Liver X receptor beta) to treat atherosclerosis and metabolic diseases such as diabetes. LXRβ is a transcription factor, a member of the nuclear receptor family. These transcriptional charges promote lipid metabolism and differentiation of adipocytes in adipocytes, and are thought to be a major target for various metabolic diseases including diabetes, which plays a key role in regulating insulin through PPARγ and lowering blood cholesterol. Accordingly, various substances that effectively inhibit the expression of LXR [beta] have been studied. In the present invention, miRNAs have been found to effectively inhibit expression at the RNA and protein level.

MicroRNAs (hereinafter referred to as miRNAs or miRs) that can be used in the present invention include animals including humans, for example, monkeys, pigs, horses, cows, sheeps, Mice, dogs, cats, mice, rabbits and the like, preferably selected from the group consisting of human-derived miR-7, miR-18a and miR-18b MiR-18a and miR-18b are SEQ ID NOS: 1, 2 and 3, respectively, and more preferably miR-7, miR-18a and miR- The miRNA nucleic acid molecules of the present invention may have a length of 18 to 100 nt (nucleotide), preferably 19 to 25 nt in length as mature miR-7, miR-18a and miR-18b, more preferably 21, Length. The miR-7, miR-18a and miR-18b nucleic acid molecules of the present invention may also be provided as precursor miR-7, miR-18a and miR-18b nucleic acid molecules having a length of 50-100 nt, preferably 65-95 nt have.

For example, human miR-7 (Mirbase Accession No. MIMAT000252) plays a very important role in the expression of mRNA, and mature miRNA is fully conserved from annelids to humans, It is presumed that it plays a role in. While human miR-18a (Mirbase Accession No. MIMAT0000072) and miR-18b (Mirbase Accession No. MIMAT0001412) exhibit mutations activated in non-Hodgkin's lymphoma in humans, these clusters are associated with multiple cancer ) Overexpression was observed.

In one embodiment according to the present disclosure, the substance capable of inhibiting the expression of LXR [beta] may comprise all or part of the mature sequence and / or of the mature sequence of any one or more of the miRs selected from the group consisting of miR-7, miR-18a, miR- And is an miRNA molecule capable of binding complementarily to a seed sequence of a target gene and inhibiting its activity.

In one embodiment according to the present application, any one or more miR molecules selected from the group consisting of miR-7, miR-18a and miR-18b of the present application contain a sequence complementary to all or a part of the seed sequence of LXR ?. Seed sequences are conserved sequences in a variety of species that are critical to the recognition of the target molecule. Since miRNA binds to the target through the seed sequence, it can effectively suppress the translation of the target mRNA when the interaction of the seed sequence with the target is inhibited. As a result of the seed sequence analysis, a site conserved in the species in Fig. 2 was confirmed. In the 3'UTR sequence of Liver X receptor beta (SEQ ID NO: 4) at positions 178 to 185 and at position 274 (SEQ ID NO: 5) in the 3'UTR sequence of Liver X receptor beta The nucleotide sequence of ~280 was confirmed, and miRNA candidate that specifically binds to this site was derived through various bioinformatic tolls.

Thus, in one embodiment according to the present invention, the miR-7 binds at nucleotide positions 178 to 185 in the 3 'UTR sequence of Liver X receptor beta (SEQ ID NO: 4) The miR-18a or miR-18b binds at the 274th to 280th nucleotide positions in the 3'UTR sequence of Liver X receptor beta (SEQ ID NO: 5).

Therefore, the present invention can be used for the prevention or treatment of various metabolic diseases by inhibiting the expression of LXR [beta]. Metabolic disease refers to diseases caused by imbalance such as carbohydrates, lipids, proteins, vitamins, minerals and moisture, and includes hypertension, diabetes, impaired bowel function, gastrointestinal disorders, gout, hyperlipidemia, stroke, alcoholic liver disease and the like. Preferably, the present invention can be selected from the group consisting of atherosclerosis, fatty liver, diabetes, hyperlipidemia, hypertension, angina pectoris and myocardial infarction.

The miR-7, miR-18a, and miR-18b used in the present invention may also include functional groups of the nucleic acid molecules constituting the miR-7, miR-18a and miR-18b nucleic acid molecules, 18a and miR-18b nucleic acid molecules that have been modified by deletion, substitution or insertion of miR-7, miR-18a and miR-18b nucleic acid molecules, to be. In the present invention, the mutant is also referred to as mimics.

In addition, any one or more of the miRs selected from the group consisting of miR-7, miR-18a and miR-18b of the present invention may exist in a single-stranded or double-stranded form. The mature miRNA molecules are predominantly single stranded, but the precursor miRNA molecules are at least partially self-complementary (e.g., stem- and loop-structure), which can form mainly double-stranded portions. In addition, the nucleic acid molecule of the present invention can be configured in the form of RNA, DNA, 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 addition, the composition of the present invention may include not only the miRNA nucleic acid molecule itself, but also other substances capable of increasing the expression rate of the miRNA of the present invention in a cell, for example, a compound, a natural product, a novel protein and the like.

Meanwhile, any one or more miR molecules selected from the group consisting of miR-7, miR-18a and miR-18b in the composition of the present invention may be provided as a vector for intracellular expression.

Any one or more miR molecules selected from the group consisting of miR-7, miR-18a and miR-18b of the present invention can be used as a variety of miRNAs such as a complex of DNA and DEAE-dextran, a complex of DNA and nuclear protein, The miR-7, miR-18a and miR-18b nucleic acid molecules may be in a form that is contained in a carrier that enables efficient introduction into the cell. The carrier is preferably 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, Preferably a lentiviral vector, but is not limited thereto. Lentiviruses are a type of retrovirus that is not only a mitotic cell but also a mitotic cell due to the nucleophilicity of a nucleopore or a pre-integrated complex (a virus "shell") that allows active incorporation into a complete nuclear membrane There is a feature that can be made.

In addition, the vector comprising any one or more of the miR molecules selected from the group consisting of miR-7, miR-18a and miR-18b preferably further comprises a selection marker. The term "selection marker" in the present invention is intended to facilitate screening of transformed cells by introduction of miR-7, miR-18a and miR-18b nucleic acid molecules. The selection marker which can be used in the vector of the present invention is not particularly limited as long as it is a gene capable of easily detecting or measuring the introduction of a vector, but it is typically a gene having resistance to drug resistance, nutritional requirement, cytotoxic agent, Such as GFP (green fluorescent protein), puromycin, neomycin (Neo), hygromycin (Hyg), histidinol Histidinol dehydrogenase gene hisD) and guanine phosphosribosyltransferase (Gpt). Preferably, GFP (green fluorescent protein) and puromycin marker can be used.

Meanwhile, the composition comprising the nucleic acid molecule of any one or more of the miRs selected from the group consisting of miR-7, miR-18a and miR-18b of the present invention may further comprise a pharmaceutically acceptable carrier, &Lt; / RTI &gt; As used herein, the term "pharmaceutically acceptable carrier" refers to a carrier or diluent that 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, sterile 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. In addition, diluents, dispersants, surfactants, binders, and lubricants may be additionally added to formulate into injectable solutions, pills, capsules, granules or tablets such as aqueous solutions, suspensions, emulsions and the like.

The composition of the present invention can be applied to any formulations containing nucleic acid molecules of any one or more of miRs selected from the group consisting of miR-7, miR-18a and miR-18b as an active ingredient and can be formulated into oral or parenteral formulations Can be manufactured. The pharmaceutical formulations of the present invention may be administered orally, rectally, nasal, topical (including under the ball and tongue), subcutaneous, vaginal or parenteral (intramuscular, subcutaneous And intravenous), or forms suitable for administration by inhalation or insufflation.

The composition according to the present application is administered in a pharmaceutically effective amount. Effective dose levels will depend on factors well known in the art and other medical disciplines including the type of disease, severity of the patient, activity of the drug, sensitivity to the drug, time of administration, route of administration and rate of release, duration of treatment, Can be determined. The composition of the present invention can be administered as an individual therapeutic agent or in combination with other therapeutic agents, and can be administered sequentially or simultaneously with conventional therapeutic agents, and can be administered singly or in multiple doses. It is important to take into account all of the above factors and to administer the amount in which the maximum effect can be obtained in a minimal amount without side effects, which can be easily determined by those skilled in the art.

The dosage ranges vary widely depending on the patient's body weight, age, sex, health condition, diet, time of administration, administration method, excretion rate, and severity of disease, and a proper dose may be, for example, And / or the degree of specific activity of the polynucleotide used. May be calculated on the basis of the EC50 generally measured as effective in the in vivo animal model and in vitro, for example from 0.01 [mu] g to 1 g per kg of body weight and may be divided into daily, weekly, monthly, May be administered once or several times per unit period, or may be continuously administered for a long period using an infusion pump. The number of repeated administrations is determined in consideration of the duration of the drug in the body, the drug concentration in the body, and the like. The composition may be administered for recurrence, even after treatment according to the course of the disease treatment.

The composition of the present invention may further comprise one or more active ingredients which exhibit the same or similar functions in relation to the treatment of the disease with one or more miRs selected from the group consisting of miR-7, miR-18a and miR- And / or a compound that maintains / increases the absorbency. Also optionally, it may further comprise a chemotherapeutic agent, an anti-inflammatory agent, an antiviral agent and / or an immunomodulator.

In addition, the compositions of the present invention may be formulated using methods known in the art so as to provide rapid, sustained or delayed release of the active ingredient after administration to the mammal. The formulations may be in the form of powders, granules, tablets, emulsions, syrups, aerosols, soft or hard gelatine capsules, sterile injectable solutions, sterile powders.

In addition, the present invention provides a method for inhibiting the expression of a liver X receptor beta (beta) in a cell, which comprises treating in vitro a miR of any one selected from the group consisting of miR-7, miR-18a and miR- beta &lt; / RTI &gt; In particular, miR-7 may be represented by SEQ ID NO: 1, miR-18a may be represented by SEQ ID NO: 2, and miR-18b may be represented by SEQ ID NO: 3.

The present invention also provides a food composition capable of improving or preventing the symptoms of metabolic diseases containing one or more miRs selected from the group consisting of miR-7, miR-18a and miR-18b as an active ingredient , The composition for food according to the present invention can be easily utilized as a food which is effective for improving or preventing symptoms of metabolic diseases, for example, as a raw material, additives, food additives, functional foods or beverages of foods.

Herein, the term &quot; food &quot; means a natural product or a processed product containing one or more nutrients, preferably a state of being able to be directly eaten through a certain processing step, , Food additives, functional foods and beverages.

Foods to which the composition for food according to the present invention can be added include, for example, various foods, beverages, gums, tea, vitamin complex, and functional food. In addition, in the present invention, the food may include special nutritive foods (e.g., crude oil, spirits, baby food, etc.), meat products, fish meat products, tofu, mackerel, noodles (Such as soy sauce, soybean paste, kochujang, mixed potatoes), sauces, confectionery (eg, snacks), candies, chocolate, gums, ice cream, milk products (eg, fermented milk, cheese, But are not limited to, pickled foods (various kinds of kimchi, pickles, etc.), beverages (e.g., fruit drinks, vegetable beverages, beverages, fermented beverages and the like) and natural seasonings (e.g. The food, beverage or food additive may be prepared by a conventional production method.

In addition, the functional food refers to a food group imparted with added value to function and express the function of the food by using physical, biochemical, biotechnological techniques, etc., or to control the biological defense rhythm of the food composition, Means a food which has been designed and processed so as to sufficiently express the body's control function with respect to the living body. Specifically, it may be a health functional food. The functional food may include a food-acceptable food-aid additive, and may further comprise suitable carriers, excipients and diluents conventionally used in the production of functional foods.

In addition, in the present invention, the beverage is a collective term for drinking thirst or for enjoying a taste, and includes a functional beverage. The beverage includes the composition for improving or preventing the symptom of metabolic diseases as an essential ingredient at the indicated ratio, and there is no particular limitation on the other ingredients. The beverage may contain various flavors or natural carbohydrates as an additional ingredient can do.

Further, in addition to the above-described foods, the food containing the food composition for improving or preventing the symptoms of metabolic diseases according to the present invention may contain various nutrients, vitamins, minerals (electrolytes), flavors such as synthetic flavors and natural flavors, And carbonating agents used in fillers (cheese, chocolate, etc.), pectic acid and its salts, alginic acid and its salts, organic acids, protective colloid thickeners, pH adjusting agents, stabilizers, preservatives, glycerin, And these components can be used independently or in combination.

In the food containing the food composition of the present invention, the amount of the composition according to the present invention may be 0.001% by weight to 90% by weight, preferably 0.1% by weight to 40% by weight, And may be contained in a ratio of 0.001 g to 2 g, preferably 0.01 g to 0.1 g, based on 100 ml in the case of beverage. However, in the case of long-term intake for health and hygiene purposes or for health control purposes May be less than the above range, and since the active ingredient has no problem in terms of safety, it can be used in an amount of more than the above range, so it is not limited to the above range.

Hereinafter, the present invention will be described in more detail with reference to Examples. It will be apparent to those skilled in the art that these embodiments are merely illustrative of the present invention and that the scope of the present invention is not limited to these embodiments.

< Example  1>

Excavation of candidate miRNAs

We investigated miRNA cadidates targeting human LXRβ using various miRNA prediction programs. Thus, miRWALK 2.0 program (http://www.umm.uni-heidelberg.de/apps/zmf/mirwalk) was used to identify several miRNA candidates targeting LXR 3'UTR. Using Targetscan program (http://www.targetscan.org) to identify interspecies conserved sequences of miRNA target sites Derived miR-7, miR-18a, miR-18b with conserved sites among several miRNA candidates Respectively. FIG. 1 shows the result of deriving the miRNA candidate using miRWALK and other programs, and FIG. 2 shows the result of finding conserved sites in the species. (a) shows the nucleotide sequence positions 178 to 185 in the 3 'UTR sequence of liver X receptor beta (SEQ ID NO: 4), (b) shows the nucleotide sequence of the liver The nucleotide sequence of the 274th to 280th nucleotides in the 3 'UTR sequence of the X receptor beta (Liver X recptor beta) is shown.

< Example  2>

Real - Time qPCR  Perform

To evaluate the post-transcriptional regulation of miRNA at the RNA level, Real-Time qPCR was performed in the experiment of Fig. Total RNA was extracted using TRIZOL (Invitrogen) and single strand cDNA was synthesized with Omni-script cDNA synthesis kit (Qiagen). HeLa cell lines delivered miR-7, miR-18a and miR-18b, the major miRNA cadidates, and mimic miRNA, miR-590, miR-613, which is expected to have a relatively small effect (Fig. Use LightCycler 480 DNA SYBRGreen I master mix (Roche) for quantitative PCR. Relative expression was calculated by the comparative ΔΔCt method. As a result, it was confirmed that all miRNAs except miR-613 inhibited the RNA of LXRβ as shown in FIG. As expected, miR-7, miR18a and miR-18b showed a relatively strong inhibitory effect (Figure 5 (b)) (* p <0.05, ** P <0.001 compared with the control group)

< Example  3>

Perform Western blotting

To evaluate the post-transcriptional regulation of miRNA at the protein level, Western blotting was performed in the experiment of FIG. Proteins were extracted using RIPA buffer (ELPIS Bio) and separated by 12% SDS-PAGE. For Western blotting, anti-LXRβ rabbit polyclonal (abcam) and anti-GAPDH (Santa Cruz) antibodies were used. Densitometry analysis was performed to compare protein expression. miRNAs were delivered to the miRNA cadidates miR-7, mir-18a, mir-18b, miR-590 and miR-613. As a result, as shown in FIG. 6 and FIG. 57, it was confirmed that five miRNA candidates down-regulate LXRβ protein expression at the protein level. (* p <0.05, ** P <0.001 compared with the control group)

< Example  4>

uciferase reporter assay  Perform

Human LXRβ 3'UTR amplified by PCR for plasmid constructions was inserted into the pmiRGLO vector. Figures 8 and 10 show a schematic diagram of the vector. Recombinant plasmids inserted in the forward and reverse directions were used. FIG. 11 (a) shows a schematic diagram of the dual-luciferase assay, and FIG. 11 (b) shows the MOA (mechanism of action) of the pmirGLO vector. For the miRNA validation assay, miR-7, miR-18a, and miR-18b were transfected into HeLa cells and tested 48 h later. MiR-18a, miR-18b and 20ng of pmirGLO / h_LXRb 3'UTR (Forward / Reverse form) were prepared for measurement of 3'UTR activity. The LXRβ 3'UTR forward and reverse forms were cloned into the pmiRGLO vector and co-transfected with the miRNA candidates in the HeLa cell line for 48 h. Luciferase assay was performed using Dual-Luciferase Assay kit (Promega). 3'UTR activity was calculated as Firefly luciferase activity and normalized to Renilla luciferase activity. The results are shown in FIG. 9 and FIG. FIG. 10 (a), which is a graph, shows that miR7, miR18a and miR18b are significantly reduced in expression in the form of forward insertion, and the expression is inhibited. In FIG. 10 (b) Because there is no activity change, miRNA candidates can be controlled by specific binding to LXRβ 3'UTR.

The present invention has been described with reference to the preferred embodiments. It will be understood by those skilled in the art that various changes in form and details may be made therein without departing from the spirit and scope of the invention as defined by the appended claims. Therefore, the disclosed embodiments should be considered in an illustrative rather than a restrictive sense. The scope of the present invention is defined by the appended claims rather than by the foregoing description, and all differences within the scope of equivalents thereof should be construed as being included in the present invention.

<110> Industry Academic Cooperation Foundation of Catholic University <120> Pharmaceutical composition comprising miR-7, miR-18a or miR-18b          for prevention or treatment of metabolic disease <130> pN1504-086 <160> 5 <170> Kopatentin 2.0 <210> 1 <211> 23 <212> RNA <213> miR-7 sequence <400> 1 uggaagacua gugauuuugu ugu 23 <210> 2 <211> 23 <212> RNA <213> miR-18a sequence <400> 2 uaaggugcau cuagugcaga uag 23 <210> 3 <211> 23 <212> RNA <213> miR-18b sequence <400> 3 uaaggugcau cuagugcagu uag 23 <210> 4 <211> 8 <212> RNA <213> seed position 8mer (178-185) of LXR beta 3-UTR <400> 4 gucuucca 8 <210> 5 <211> 7 <212> RNA <213> seed position 7mer (274-280) of LXR beta 3-UTR <400> 5 gcaccuu 7

Claims (10)

A pharmaceutical composition for preventing or treating metabolic diseases, which comprises as an active ingredient at least one miR selected from the group consisting of miR-7, miR-18a and miR-18b. The method according to claim 1,
Wherein said miR-7 is represented by SEQ ID NO: 1, miR-18a is represented by SEQ ID NO: 2, and miR-18b is represented by SEQ ID NO: 3. The method according to claim 1,
Wherein said miR-7, miR-18a and miR-18b have an activity of specifically inhibiting Liver X receptor beta. The method according to claim 1,
Wherein the metabolic disease is selected from the group consisting of atherosclerosis, fatty liver, diabetes, hyperlipidemia, hypertension, angina pectoris, and myocardial infarction. Prevention or treatment of a metabolic disease comprising miR (microRNA) as an active ingredient which binds at a nucleotide sequence position 178 to 185 within the 3'UTR sequence of liver X receptor beta shown in SEQ ID NO: 4 A pharmaceutical composition. 6. The method of claim 5,
Wherein the miR is miR-7 represented by SEQ ID NO: 1. Prevention or treatment of a metabolic disease comprising miR (microRNA) as an active ingredient which binds at a nucleotide sequence from 274th to 280th nucleotides in a 3'UTR sequence of liver X receptor beta shown in SEQ ID NO: 5 A pharmaceutical composition. 8. The method of claim 7,
Wherein the miR is miR-18a represented by SEQ ID NO: 2 or miR-18b represented by SEQ ID NO: 3, or a pharmaceutical composition for preventing or treating metabolic diseases. Expression of liver X receptor beta (beta) in a cell, comprising in vitro treatment of one miR selected from the group consisting of miR-7, miR-18a and miR-18b in a cell Or inhibiting the activity. 10. The method of claim 9,
Wherein said miR-7 is represented by SEQ ID NO: 1, miR-18a is represented by SEQ ID NO: 2, and miR-18b is represented by SEQ ID NO: 3.

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