KR101890874B1 - A composition for repression of muscle-aging and regeneration of old muscle - Google Patents

Abstract

The present invention relates to a pharmaceutical composition for preventing or treating muscle aging-related diseases comprising miR-431 or mimic thereof as an active ingredient. The present invention also relates to miR-431 or an analogue thereof, and a cell therapy agent for treating muscle aging-related diseases, comprising a source cell as an active ingredient. The present invention can inhibit muscle aging and treat muscle aging-related diseases.

Description

TECHNICAL FIELD [0001] The present invention relates to a composition for inhibiting muscle senescence or for treating muscular aging-related diseases,

The present invention relates to a pharmaceutical composition for preventing or treating muscle aging-related diseases comprising miR-431 or an analogue thereof as an active ingredient. The present invention also relates to a cell therapy agent for treatment of muscle aging-related diseases comprising miR-431 or an analogue thereof, and a source cell or a source cell having enhanced differentiation ability as an active ingredient. Further, the present invention relates to a composition for regenerating aged muscle, a composition for inhibiting muscle senescence, and a composition for promoting the differentiation of aged muscle into extracellular source cells, comprising miR-431 or an analogue thereof as an active ingredient. Further, there is provided a method for measuring the muscle differentiation ability of aged root cells, comprising measuring a miR-431 expression level of a source cell, and a method for measuring the expression level of miR-431, And to a measurement kit.

Skeletal muscle is characterized by a decrease in muscle volume and function during aging of the muscle, which is also called age-related sarcopenia (Narici and Maffulli 2010). It is known that adult stem cells, also known as satellite cells, gradually decrease in number as they get older and that their differentiation potential also decreases (Brack et al., 2005;

Previous studies on muscle differentiation and regeneration have focused on the regulation of myogenic regulatory factors (MRFs) such as MyoD, Mef2, and Myogenin (Sartorelli and Caretti 2005; Wang and Rudnicki 2012). Recently, a number of studies have been published that show that microRNAs are involved not only in muscle differentiation but also in muscle disorders including skeletal muscle hypertrophy (Eisenberg et al. 2007; Thum et al. 2007; Dey et al. 2012; Liu et al. 2012). The present inventors have recently discovered a miRNA-mRNA regulatory network of skeletal muscles involved in aging, showing that miRNAs participate in the aging process of muscles (Kim et al. 2014b).

On the other hand, aging dwarfism, which occurs in senescent individuals due to the differentiation ability of the myoblasts due to aging, is a disease differentiated from neuromuscular diseases caused by damages of autonomic nerve cells. Specifically, examples of the neuromuscular diseases include muscle dystrophy, neuromuscular junction disease, and motor neuropathy, and there is a difference in aging-related myopenia, pathogenesis mechanism and treatment method.

Accordingly, the present inventors have made intensive researches to inhibit aging of muscles and to treat diseases related to muscle aging. As a result, miRNA expression is analyzed by comparing between the neonatal myocytes and aged endothelial cells, so that delayed muscle differentiation and regeneration of aged muscles And the present invention has been completed.

It is an object of the present invention to provide a pharmaceutical composition for preventing or treating muscle aging-related diseases comprising miR-431 or mimic thereof as an active ingredient.

Another object of the invention is miR-431 or an analogue thereof, and a source cell; Or miR-431 or an analogue thereof, as an active ingredient, for treating a muscle aging-related disease.

Another object of the present invention is to provide a composition for regenerating aged muscle comprising miR-431 or an analogue thereof as an active ingredient.

It is still another object of the present invention to provide a composition for inhibiting muscle senescence comprising miR-431 or an analogue thereof as an active ingredient.

Yet another object of the present invention is to provide a composition for improving the ability to multiply extracorporeal myoblast differentiation comprising miR-431 or an analogue thereof as an active ingredient.

Yet another object of the present invention is to provide a method for measuring the muscle differentiation ability of aged source cells, comprising measuring the expression level of miR-431 in a source cell.

Another object of the present invention is to provide a kit for measuring the muscle differentiation ability of aged source cells comprising an agent for measuring the expression level of miR-431.

In one aspect of the present invention, the present invention provides a pharmaceutical composition for preventing or treating muscle aging-related diseases comprising miR-431 or mimic thereof as an active ingredient.

The term " microRNA (miRNA) " of the present invention is a small untranslated RNA molecule consisting of about 22 nucleotides, which functions to finely control the expression of an individual gene through inhibition of mRNA degradation or translation I have. In the present invention, attention has been paid to the fact that miRNAs participate in the aging process of muscles, and efforts have been made to find miRNAs that can be used to inhibit muscle aging and treat muscle damage.

The term " miR-431 " of the present invention can be targeted to Smad4 as a miRNA that exists in a number of young myoblasts, and specifically targets Smad4 mRNA, particularly 3'UTR, but is not limited thereto. MiR-431 of the present invention may have the sequence (5'-UGUCUUGCAGGCCGUCAUGCA-3`) of SEQ ID NO: 1, but is not limited thereto.

In one embodiment of the present invention, the expression patterns of the miRNAs between the neonatal stem cell and the senescent source cell were compared using the next generation sequencing (NGS) The expression level of 47 miRNAs was up-regulated in the myoblast, whereas 71 miRNAs were down-regulated. In particular, it was confirmed that miR-431 has the strongest enhancing activity for differentiation (Experimental Example 1).

The term " miR-431 analog " of the present invention may be a single-stranded RNA that is similar to miR-431 in the body and is identical to the miR-431 sequence, but is not limited thereto . For the purposes of the present invention, miR-431 analogs can specifically enhance the ability of the aged source cells to differentiate into muscle.

According to a specific embodiment of the present invention, miR-431 may be SEQ ID NO: 1, but is not limited thereto.

In one embodiment of the present invention, it was confirmed that the analog of miR-431 improves the levels of MyHC and Myogenin mRNA of the senescent source cells (Experimental Example 2).

The term " muscle aging " of the present invention means that the density and function of muscles are gradually weakened with aging. The term " muscle aging-related diseases " Particularly, age-related sarcopenia, is also referred to as age-related sarcopenia. People who experience muscle aging generally experience falls or fractures easily.

According to a specific embodiment of the present invention, the muscle aging-related diseases may be age related sarcopenia, neuron diseases, metabolic muscle diseases, inflammatory myopathies, neuromuscular junction diseases, endocrine myopathies, It does not.

Such aging-related diseases including aging-related muscular dysgenesis are diseases distinguishing from muscular dystrophy and muscle atrophy.

More specifically, muscular dystrophy patients show muscle necrosis in muscular tissue examination, the size of muscle fibers is uneven and varied, and the muscle fibers are replaced with fat and fibrous tissue in the necrotic area. The muscular dystrophies include Becker's muscular dystrophy, , Congenital muscular dystrophy, Emery-Dreifuss muscular dystrophy, etc. (Alan EH Emery, Lancet. 2002 Feb 23; 359 (9307): 687-95). In particular, the muscular dystrophy is caused by a deficiency or mutation of a specific gene such as DMD or EMD, and is known to be caused by genetic factors.

In addition, muscular dystrophy refers to atrophy of muscles in the limbs, and is representative of amyotrophic lateral sclerosis and spinal proximal muscular atrophy, which is known to be caused by progressive denaturation of motor nerve fibers and cells in the spinal cord.

Specifically, the spinal muscular atrophy is a genetic disorder caused by degeneration of motor neurons in the spinal cord, and is known to be a neuromuscular disease that occurs in infants and children. In addition, amyotrophic lateral sclerosis is characterized by intractable and irreversible neurodegenerative changes caused by the death of the upper motor neurons and lower motor neurons in the cerebrum and spinal cord, and it is known that the nerve growth factor deficiency and nerve inflammation are the main causes, And the cause and symptoms of onset are different from those of aging.

Currently, there are several methods for treating myopenia due to aging, including urocortin II (urocortin II) and hormone replacement therapy, but some side effects have been reported. Therefore, fundamental treatment for muscle-related diseases There is a lack of aspect.

However, the present inventors have confirmed that miR-431 or its analog can particularly effectively enhance the differentiation ability of the senescent source cells. Thus, miR-431 or an analogue thereof for preventing or treating the above-described muscle aging- Was found to be effective.

In one embodiment of the present invention, it was confirmed that overexpression of miR-431 does not increase the ability of the myocyte to differentiate in the neonatal myocytes, and inhibition of miR-431 does not decrease the ability of myocytes to differentiate in the senescent myocytes 2). This suggests that the basal level of miR-431 is saturated in the neonatal myocytes, and that mR-431 is sufficiently reduced in the senescent source cells.

From the above example, it was confirmed that miR-431 of the present invention is particularly effective for the treatment of aged muscle regeneration and aged muscle related diseases.

The term " myoblast " of the present invention is a muscle cell in an undifferentiated state, and proliferates into a source cell when a satellite cell (mononuclear cell) is activated. When the differentiation of myoblasts progresses, they fuse with other cells to form myotube, which is thin and long, to make muscle fibers. The senescent myofibroblasts decrease the differentiation ability with aging, which inhibits the formation of muscle fibers as described above, resulting in symptoms of muscle weakness.

The term " source cell differentiation " of the present invention is a process in which monocyte myoblasts form myotubes through fusion. Myocyte precursor cells correspond to Pax7 ⁺ markers in self-renewal and Pax7 ⁺ / MyoD ⁺ in proliferation. Cells at the differentiation stage that form the root canal can be distinguished using the Pax7 ^- MyoD ⁺ MyoG ⁺ marker. The cells at the early stage of differentiation to form the canal increase the expression of myogenic transcription factors such as MyoD (D), and increase myosin G (MyoG) in the middle stage. The expression of Myosin Heavy Chain is increased in the late stage of differentiation.

Such differentiation of myoblast can be induced in skeletal muscle, myocardium and smooth muscle, and most preferably can be induced in skeletal muscle, but is not limited thereto.

The miR-431 or derivatives thereof of the present invention can increase root canal and muscle fiber by increasing the ability of differentiation of source cells, especially aged source cells, thereby increasing muscle mass reduced by aging.

According to another specific embodiment of the present invention, miR-431 or its analogue can target mRNA of Smad4, more specifically target 3'UTR (untranslated region) of Smad4 mRNA .

According to another specific embodiment of the present invention, miR-431 or an analogue thereof can inhibit the expression of Smad4.

In one embodiment of the present invention, it was confirmed that miR-431 inhibits Smad4 expression through the 3'UTR of Smad4, and it was confirmed that the muscle differentiation ability of the source cells was increased through the inhibition of Smad4 (Experimental Examples 3 to 6) Experimental Example 4).

In another embodiment of the present invention, it was confirmed that the expression level of Smad4 was increased in senescent root cells having low regenerative activity of muscle, and miR-431 or siSmad4, which inhibits Smad4, restored the muscle differentiation ability of root cells Experimental Examples 5 to 6).

In another embodiment of the present invention, when miR-431 inhibitor reduces muscle differentiation in human skeletal muscle cell (HSMM), when miR-431 inhibitor inhibits miR-431 expression in HSMM , And the HSMM differentiation was reduced (Experimental Example 7).

The term " prophylactic " of the present invention refers to any action that inhibits or delays the onset of a muscle-related disease associated with administration of the pharmaceutical composition of the present invention.

The term " treatment " of the present invention means any action that improves or alleviates symptoms of muscle aging-related diseases by the administration of the pharmaceutical composition of the present invention.

According to a specific embodiment of the present invention, miR-431 or an analogue thereof contained in the pharmaceutical composition of the present invention may be contained in the form of a recombinant plasmid or viral vector expressing miR-431 or an analogue thereof, But is not limited to.

The expression level of miR-431 in an individual is increased due to miR-431 or its analogue delivered and expressed by the recombinant plasmid, viral vector, or non-viral carrier, thereby enhancing the ability to differentiate aged source cells.

Invention miR-431 or analogues thereof used in the various embodiments provided herein may be included in complex with various nucleic acid carriers (viral or nonviral carriers) known in the art to enhance in vivo delivery efficiency. For example, it may be included as a recombinant plasmid or viral vector expressing miR-431. Plasmids usable for this purpose include pSilencer (Ambion), pSiEx (Novagen), siXpress (Takara Bio), pBLOCK-iT (Invitrogen), pcDNA3.1 (Invitrogen), pCEP4 (Invitrogen), SilenCircle Allele, and the like, but are not limited thereto. As the viral carrier, retroviral vectors, adenovirus vectors, adeno-associated viral vectors, vaccinia virus vectors, lentivirus vectors, herpes virus vectors, alphavirus vectors, EB virus vectors, papilloma virus vectors, But is not limited thereto. As a non-viral carrier, it is also possible to use Mirus TrasIT-TKO lipophilic reagent, lipofectin, lipofectamine, cellfectin, G-fectin, cationic phospholipid nanoparticles, cationic macromolecule, cationic micelle , Cationic emulsions or liposomes, ligand-DNA complexes, geneguns, but are not limited thereto. The form of the liposome is combined with an amphipathic agent, such as a micelle, an insoluble monolayer, a liquid crystal, or a lipid present as a lamellar layer present in an aqueous solution. Lipids for liposomal formulation include, but are not limited to, monoglyceride, diglyceride, sulfatide, lysolecithin, lecithin phospholipid, saponin, bile acid, lipofectin, and the like.

Further, in order to enhance the stability of miR-431 in the body, it can be formulated so as to utilize general ribonucleic acid intracellular delivery technology known in the art such as conjugation of a biocompatible polymer such as polyethylene glycol to increase intracellular absorption.

The method also includes suspending the cells in a microRNA-containing solution by electroporation and introducing them into cells by passing a pulse of a DC high voltage. In the living body, the solution containing the microRNA can be administered to the desired site, and the DC voltage can be pulsed through the electrode to be introduced into the cell.

The term " individual " of the present invention refers to all animals, including humans, that may have a muscle aging-related disorder, and may be, but not limited to, an animal other than a human. By administering a composition comprising miR-431 or an analogue thereof of the present invention to an individual, the above-mentioned muscle aging-related diseases can be effectively prevented and treated. The composition of the present invention can be administered in combination with a therapeutic agent for existing muscle-related diseases.

According to one particular embodiment of the present invention, the pharmaceutical composition of the present invention may further include a pharmaceutically acceptable carrier, but is not limited thereto.

The term " pharmaceutically acceptable carrier " as used herein means a carrier or diluent which does not disturb the biological activity and properties of the compound to be administered, without stimulating the organism.

The type of the carrier that can be used in the present invention is not particularly limited, and any carrier conventionally used in the art and pharmaceutically acceptable may be used. Non-limiting examples of the carrier include saline, sterilized water, Ringer's solution, buffered saline, albumin injection solution, dextrose solution, maltodextrin solution, glycerol, ethanol and the like. These may be used alone or in combination of two or more.

In addition, if necessary, other conventional additives such as an antioxidant, a buffer and / or a bacteriostatic agent can be added to the composition, and a diluent, a dispersant, a surfactant, a binder and / or a lubricant may be additionally added thereto to form an aqueous solution, a suspension, Capsules, granules, tablets or the like. The pharmaceutical composition of the present invention can be manufactured into various formulations depending on whether the intended administration mode is oral administration or parenteral administration.

Non-limiting examples of formulations for oral administration include troches, lozenges, tablets, aqueous suspensions, oily suspensions, prepared powders, granules, emulsions, hard capsules, soft capsules, syrups, .

Such as lactose, saccharose, sorbitol, mannitol, starch, amylopectin, cellulose, or gelatin, for formulation into oral dosage forms such as tablets, capsules and the like. Binder; Excipients such as dicalcium phosphate and the like; Disintegrating agents such as corn starch or sweet potato starch; Lubricating oils such as magnesium stearate, calcium stearate, sodium stearyl fumarate or polyethylene glycol wax, and the like. Furthermore, in the case of a capsule formulation, in addition to the above-mentioned substances, a liquid carrier such as a fatty oil may be further contained.

Formulations for parenteral administration include, for example, injectable forms such as subcutaneous, intravenous, or intramuscular injections; Suppository injection system; Or an aerosol formulation that allows for inhalation through the respiratory tract, but is not limited thereto. In order to formulate the injectable preparation, the composition of the present invention may be mixed with a stabilizer or a buffer in water to prepare a solution or suspension, which may be formulated for unit administration of an ampoule or a vial. When formulated for spraying with an aerosol or the like, a propellant or the like may be blended with the additive such that the water-dispersed concentrate or the wet powder is dispersed.

 The pharmaceutical composition of the present invention can be administered in a pharmaceutically effective amount. The term " pharmaceutically effective amount " of the present invention means the amount of the compound of the present invention that is effective in treating or preventing a disease at a reasonable benefit / risk ratio applicable to medical treatment or prevention And the effective dose level refers to the amount of the effective amount of the composition of the present invention, and the effective dose level is determined depending on the severity of the disease, the activity of the drug, the age, weight, health, sex, sensitivity of the patient to the drug, The duration of the treatment, the factors including the drugs used in combination or concurrently with the composition of the present invention used, and other factors well known in the medical field.

The pharmaceutical composition of the present invention may be administered as an individual therapeutic agent or in combination with another therapeutic agent, and may be administered sequentially or simultaneously with a conventional therapeutic agent. And can be administered singly or multiply. It is important to take into account all of the above factors and administer an amount that will achieve the maximum effect in the least amount without side effects.

According to another aspect of the present invention, the present invention provides miR-431 or an analogue thereof and a source cell; Or miR-431 or an analogue thereof, as an active ingredient. The present invention also provides a cell therapy agent for treating muscle aging-related diseases.

The terms "miR-431", "analog of miR-431", "source cell", and "muscle aging-related disease" of the present invention are as described above.

The term " cell therapeutic agent " of the present invention is a pharmaceutical (US FDA regulation) used for the purpose of treatment, diagnosis and prevention with cells and tissues prepared by separation, culture and special manipulation from human, In order to restore function, these cells are used for the purpose of the treatment, diagnosis and prevention of diseases through a series of actions such as proliferation selection of living, homologous, or xenogeneic cells in vitro or other ways of changing the biological characteristics of the cells Medicines.

The preferred dosage of the cell therapy agent of the present invention varies depending on the condition and the weight of the individual, the degree of disease, the type of drug, the administration route and the period of time, but can be appropriately selected by those skilled in the art. The administration may be carried out once a day or divided into several doses, and the dosage is not limited in any way to the scope of the present invention.

The cell treatment agent of the present invention includes miR-431 or miR-431 analogues and source cells or source cells that are enhanced in differentiation by miR-431, thereby promoting regeneration of aged muscles and treating aging-related diseases .

According to another aspect of the present invention, there is provided an aged muscle regeneration composition comprising miR-431 or an analogue thereof as an active ingredient.

The terms " miR-431 " and " miR-431 analogs " of the present invention are as described above.

The composition for regenerating the aged muscle comprising miR-431 of the present invention is characterized by being able to regenerate and recover effectively aged muscle tissue by maintaining the steminess of the aged source cells and increasing the differentiation ability.

According to another aspect of the present invention, there is provided a composition for inhibiting muscle senescence comprising miR-431 or an analogue thereof as an active ingredient.

The terms " miR-431 " and " miR-431 analogs " of the present invention are as described above.

The composition for inhibiting muscle senescence comprising miR-431 of the present invention is characterized by being able to effectively inhibit aging of muscles by maintaining stem cell stem cells.

According to another aspect of the present invention, there is provided a composition for improving the ability to multiply extracorporeal myoblast cells comprising miR-431 or an analogue thereof as an active ingredient.

The terms " miR-431 " and " miR-431 analogs " of the present invention are as described above.

The composition for improving the pluripotent stem cell differentiation ability comprising miR-431 of the present invention is characterized by being able to increase the ability to differentiate aged stem cells.

According to another aspect of the present invention, there is provided a method for detecting miR-431 expression in a source cell, And comparing the amount of miR-431 expression measured in the above step with the expression level of miR-431 expressed in control source cells, to a method for measuring the muscle differentiation ability of aged source cells.

The step of measuring the expression level may be, but not limited to, measuring mRNA expression level of miR-431.

The measurement of the mRNA expression level of miR-431 may be performed using an antisense oligonucleotide that specifically binds to the mRNA of the gene, a primer pair, a probe, or a combination thereof, which may be a reverse transcriptase polymerase, a competitive reverse transcriptase polymerase But is not limited to, using an assay selected from the group consisting of enzyme reaction, real-time reverse transcriptase polymerase reaction, RNase protection assay, Northern blotting and DNA microarray chip.

The control source cells may be source cells that have not been impaired in their ability to differentiate, and may be source cells or purchased source cells collected from young mammals for the purpose of the present invention. Especially, in the case of humans, the source cells of young donors younger than 20 years But is not limited thereto.

In the step of comparing the expression level of miR-431, it can be judged that when the expression level of miR-431 is lower than the expression level of miR-431 of the control source cells, the differentiation ability of the source cells is decreased.

The step of comparing the expression level of miR-431 may further include the step of measuring the expression level of Myosin heavy chain (MyHC) or Myogenin.

The amount of expression of MyHC or Myogenin may be determined by measuring mRNA expression level or protein expression level of a gene encoding MyHC or Myogenin.

The step of measuring the mRNA expression level is as described above.

The measurement of the protein expression level may be performed using an antibody, an aptamer or a combination thereof that specifically binds to MyHC or Myogenin protein, which may be Western blotting, ELISA, radioimmunoassay, radioimmunoprecipitation, But are not limited to, immunoassays, immunoassays, immunocytochemistry, immunoassay analysis, complement fixation, FACS, and protein chips.

According to another aspect of the present invention, there is provided a kit for measuring the muscle differentiation ability of an aged source cell comprising an agent for measuring the expression level of miR-431 in a source cell.

The agent for measuring the expression level of miR-431 includes an antisense oligonucleotide that specifically binds to the mRNA of the gene encoding miR-431, a primer pair, a probe, an antibody specifically binding to miR-431, And combinations thereof. However, the present invention is not limited thereto.

The kit may further comprise an agent for measuring the expression level of Myogenic heavy chain (MyHC) or Myogenin.

The formulation for measuring the expression level is as described above.

The kit for measuring muscle differentiation ability may further include an instruction manual describing an optimal reaction performing condition. The instructions include instructions on the surface of the package, including the brochure or leaflet in the form of a brochure, a label attached to the kit, and a kit. In addition, the brochure may include information that is disclosed or provided through an electronic medium, such as the Internet.

The present invention confirms that miR-431 enhances the differentiation ability of the senescing source cells, and the miR-431 or its analogue has an effect of preventing or treating muscle aging-related diseases, and has the effect of regenerating aged muscle and inhibiting muscle aging .

FIG. 1 is a diagram showing miRNA expression profiles of the neonatal myocytes and the senescent myocytes. FIG. (A) Hierachical clustering of 118 differentially regulated miRNAs with aging; 47 miRNAs were up-regulated and 71 miRNAs were down-regulated. Each column shows miRNA expression in parental cells isolated from each of three-month-old young mice (n = 3) and 27-month old mice (n = 3). Strengths represent different degrees. Red and green indicate high and low expression, respectively. (B) After infection with the indicated miRNA analogs, senescent root cells were induced to differentiate in differentiation medium. Relative expression of differentiation marker genes such as MyHC (white bar, n = 3 for each group) and myogenin (black bar, n = 3 for each group) was determined by qRT-PCR. The results were normalized to the amount of beta -actin mRNA. Data are presented as mean ± standard deviation. ** P < 0.01.
Figure 2 is a diagram showing muscle differentiation of miR-431-induced senescent root cells. (A) Myocytes (green) and DAPI (blue), immunoprophoresis showed that the senescent source cells infected with miR-431 inhibitor-infected neonatal cells (upper panel) or miR- ) Exhibit a reduced or increased number of differentiated root canal cells, respectively, as compared to the control group. The scale bar represents 200 mu m. (B) The fusion index was determined as the percentage of MyHC-positive cells with two or more nuclei for all MyHC-positive cells. Six different views were randomly selected to measure the fusion index. * P <0.05 (C) After infection with the control-analog or miR-431 analog, senescent source cells were induced to differentiate in differentiation medium and mRNA levels of differentiation marker genes such as myogenin or MyHC were analyzed. (D) Neonatal myocytes infected with the miR-431 inhibitor showed reduced mRNA levels of the indicated differentiation marker genes. The results were normalized by the mean of [beta] -actin mRNA. Data are presented as mean ± standard deviation. ** P < 0.01
Fig. 3 shows the differentiation of senescent root cells promoted by knockdown of Smad4. (A, supra). Immunoblot analysis was performed with antibodies to the lysed labeled proteins of four independently isolated neonatal and senescent source cells. β-Actin was used as a loading control (below). Quantification of Western bands was expressed as mean ± standard deviation. * P < 0.05. (B) After senescence source cells were infected with si control group, siSmad3 or siSmad4, the cells induced differentiation in differentiation medium. Smad4 knockdown promoted differentiation, which induced upregulation of differentiation marker genes such as myogenin and MyHC (below). On the other hand, there was no change in Smad3 knockdown (above). (C) Immunofluorescent staining of MyHC (green) and DAPI (blue) showed that the number of myotubes differentiated from Smad4 aged myocytes was increased compared to control cells. The scale bar is 200 um. (D) The fusion index was calculated as the percentage of MHC-positive cells with two or more nuclei for total MHC-positive cells. Six different views were randomly selected to measure the fusion index. * P < 0.05 (E) Up to 3 days (up to 3 days) Expression patterns of the expressed proteins in the neonatal and senescent myocytes cultured in the differentiation medium were indicated by Western blot analysis. beta -actin was used as a loading control. GM is a culture medium; DM is a differentiation medium. (F) shows the expression of relative miR-431 in the cell during differentiation. The data were normalized to the amount of beta -actin mRNA and expressed as relative standardized values for the neonatal source cells of the growth medium.
4 is a diagram showing miR-431 that directly binds to 3'UTR of Smad4 and regulates expression of Smad4. (A) In the luciferase activity assay, miR-431 inhibited the WT 3'UTR of Smad4, and when the miR-431 binding site in the Smad4 3'UTR was deleted, the inhibition was eliminated. WT means wild type, and MT means mutant. (B) The miR-431 binding site (positions 1067 to 1073) in mouse Smad4 3'UTR is conserved in human Smad4 3'UTR (positions 1121 to 1127). (C) miR-431 inhibitor-infected neonatal cells showed increased levels of Smad4 protein (left). On the other hand, senescent source cells infected with the miR-431 analog decreased Smad4 levels. (D) Neonatal muscle or agar (300 ug) of aging muscle tissue was spin-cultured with biotinylated (Bi) miR431-5p or Bi-cel-miR-67 (control) at 4 ° C. After 4 hours, the RNA was isolated from the pulldown material using streptavidin beads and the Smad4 mRNA enrichment was analyzed by qRT-PCR. Standardization was performed using GADPH mRNA for neoplastic or aged muscle tissue. Data are presented as mean ± standard deviation of three independent experiments. * P < 0.05. (E) were cultured with antisense oligo (ASO) or negative control RNA (Gm14635 lincRNA) complementary to Smad4 mRNA from lysates from the neonatal source cells. The pull-down separated RNA using streptavidin beads was analyzed using qRT-PCR for Smad4 mRNA (normalized to GADPH mRNA) (left) or miR431-5p (normalized to RNU6B) (right).
Figure 5 is a diagram showing rapidly increased Smad4 and low expression levels of miR-431 in the regenerating muscle tissue of old mice after muscle injury. (A, supra) expression patterns of Smad4 were analyzed for 14 days in CTX-damaged neonatal TA and aged TA muscle tissues using Western blot analysis. GADPH was used as a loading control (below). Quantitative RT-PCR analysis was used to analyze the miR-431 of neoplastic and aging TA muscle tissue damaged by CTX. Expression was measured three times and data were normalized to the amount of U6 snoRNA and expressed as a relative value to the normalized value of the new TA muscle tissue on the day of measurement. (B) On day 14 after CTX injury, the sections of the new TA muscle tissue and the aging TA muscle tissue section were stained with antibodies against Pax7 (green) and Smad4 (red), and the same positions of the two antigens (colocalization). The scale bar is 20 um. White dashed lines indicate muscle fibers. The percentage of Pax7 ⁺ source cells expressing the Smad4 protein was significantly increased in the aged TA muscle tissue damaged by CTX. *** P <0.001
Figure 6 shows enhanced regeneration ability of senescent muscle treated with siSmad4 or miR-431. (A) is a schematic diagram showing an experimental procedure. Cardiotoxin (CTX, 20 uM) was injected into the TA muscle tissue of the aged mouse at day 0. After 2 and 5 days, the injured muscle tissue was injected with the siSmad4 or miR-431 analog and the control siRNA or miRNA analog was injected into the contralateral muscle tissue. (B) is a representative IHC image (left panel) stained with regenerated fibers (n = 4) treated with siSmad4 in laminin (red) and DAPI (blue) on day 14. The cross-sectional area (CSA) was measured using ImageJ software. Six views were randomly selected for CSA measurements. * P < 0.05. The scale bar is 100 um. (C) is a representative IHC image (left panel) stained with regenerating fibers (n = 4) treated with miR-431 in laminin (red) and DAPI (blue) on day 14. The cross-sectional area (CSA) was measured using ImageJ software. Six views were randomly selected for CSA measurements. * P < 0.05. The scale bar is 100 um.
Figure 7 shows muscle differentiation of human skeletal muscle root cells (HSMM) reduced by inhibition of miR-431. (A) shows that the level of Smad4 protein was increased or decreased, respectively, as compared with the control, when the HSMM was infected with an inhibitor or analog of miR-431. The protein levels were analyzed by western blot analysis and beta -actin was used as a loading control. (B) After HSMM infection with inhibitor-control or inhibitor-miR-431, differentiation of the cells was induced in differentiation medium. Inhibitor-miR-431-infected HSMMs analyzed by immunofluorescence staining with MyHC (green) and DAPI (blue) decreased the number of differentiated canaliculus cells compared to control cells. The scale bar represents 200 mu m. (C) mRNA levels of differentiation marker genes such as myogenin and MyHC of inhibitor-miR-431 infected HSMM were decreased. Data are presented as means ± SD. ** P < 0.01 (D) is a schematic representation of miR-431-mediated regulation of myocyte aging. Reduction of miR-431 in senescent Pax7 ⁺ MyoD ⁺ source cells induces an increase in Smad4, a downstream effector of TGF-β or BMP signaling, thereby interfering with normal muscle differentiation.
8 is a diagram showing the separation of neonatal and senescent myocytes. (A) Neonatal primitive myocytes and senescent myocytes were isolated from the hindlimb muscles of mice at 3 and 27 months of age. The separated cells were subjected to immunofluorescence using antibodies against Pax7 (red) and MyoD (green), and stained with DAPI (blue). The scale bar is 200 um. (B) Representative images of enlarged dyed new and old age cells. The scale bar is 20 um.
Figure 9 is a diagram showing delayed differentiation of senescent source cells. (Green) and DAPI (blue) during early differentiation in the (A) DIC image and (B) differentiation medium (DM, 36 hours). The scale bar is 200 um. The fusion index is the percentage of MyHC-positive cells with two or more nuclei for total MHC-positive cells. Six different views were randomly selected to measure the fusion index. * P < 0.05 (C) After incubation in differentiation medium for 36 hours, protein levels of MyHC in four independently isolated neonatal and senescent myocytes were determined by immunoblot analysis (left). beta -actin was used as a loading control. The figure on the right is a quantitative graph of MyHC specific bands. * P < 0.05. (D) The mRNA levels of differentiation marker genes such as myogenin and MyHC were decreased in senescent source cells. Data are presented as means ± SD. * P < 0.05, *** P < 0.001
Figure 10 shows that the differentiation potential is not altered in the miR-431 inhibitor-treated senescent source cells infected with miR-431 analogs. Neonatal myocytes infected with control-analog or miR-431 analogs were induced to differentiate in differentiation medium and mRNA levels of differentiation marker genes such as myogenin and MyHC were analyzed. Aging source cells infected with the miR-431 inhibitor showed similar mRNA levels of differentiation marker genes. The results were normalized to the mean of? -Actin mRNA. M is an analog; I represents an inhibitor. Data are presented as means ± SD.
Fig. 11 shows that miR-431 is presumed to be a miRNA regulating Smad4. (A) Among the down-regulated miRNAs that were negatively correlated with the increased Smad4 protein, four miRNAs containing miR-431 (red) were estimated to be regulatory miRNAs of the 3'UTR of Smad4 mRNA. (B) Only miR-431 inhibited the WT Smad4 3'UTR in the luciferase assay. ** P < 0.01
Figure 12 shows that miR-431 regulates Smad4 expression in C2C12 cells. (A) C2C12 cells infected with the miR-431 analog decreased the level of Smad4 protein compared to the control (left), whereas cells infected with the miR-431 inhibitor increased the level of Smad4 compared to the control. (B) C2C12 cells were infected with biotinylated (Bi) -miR431-5p or Bi-cel-miR-67 (small control RNA without a known mammalian target) and after 24 hours streptavidin beads RNA was isolated by pull-down and Smad4 mRNA content was analyzed using RT-qPCR. Standardization was performed using GADPH mRNA. The graph shows the results of three independent experiments as mean ± standard deviation (SEM) (C, left). C2C12 cell lysates were incubated with antisense oligomers (ASO) or negative control RNA (Gm14635 lincRNA) complementary to Smad4 mRNA. The RNA was isolated from streptavidin pulldown and analyzed by RT-qPCR to detect Smad4 mRNA (normalized to GADPH mRNA) or miR431-5p (normalized to RNU6B) (right). The graph shows the results of three independent experiments as mean ± standard deviation (SEM).
Fig. 13 is a view showing CTX-damaged neonatal TA muscle tissue and aged TA muscle tissue. (A) Representative image of CTX injury 7th day or 14th day of TA muscle tissue of newly regenerated TA muscle and aging regeneration TA muscle tissue. (B) Expression pattern of Smad4 in CTX-injured neonatal TA muscle tissue and aging muscle tissue was determined using Western blot analysis for 14 days. GADPH was used as a loading control.

Hereinafter, the present invention will be described in more detail with reference to the following examples. However, the following examples are intended to illustrate the present invention, but the scope of the present invention is not limited thereto

Example 1. Preparation of mouse

Young (3 months old) C57BL / 6 mice and old (27 months old) C57BL / 6 mice were purchased from the Laboratory Animal Resource Center (Korea Research Institute of Bioscience and Biotechnology, KRIBB). All animal experiments were carried out according to protocols approved by the Korean Society for Biotechnology Research (KRIBB) Animal Care and Use Committee.

Example 2. Isolation and culture of primordial source cells

Primary myoblasts were isolated from the hind limb muscles of wild-type and Prx3 KO mice as described in previous studies (Rando and Blau 1994). Briefly, the muscle was thinned with scissors and incubated in a dissociation buffer at 37 ° C for 20 minutes. The dissociation buffer contains distearase II (2.4 U / ml, Roche), collagenase D (1%, Roche) and 2.5 uM calcium chloride. The slurry was pulverized using a serum pipette and passed through a 70-uM nylon mesh (BD Bioscience) to remove debris. Cells were harvested by centrifugation at 1,000 rpm for 3 min and resuspended in growth medium containing Ham's F-10 (HyClone) supplemented with 20% FBS, antibiotics and 5 ng / ml bFGF. To remove fibroblasts, the cells were plated on uncoated plates for 1 hour, and the floating cells were transferred to collagen-coated culture dishes. To maintain primordial source cells, the growth medium was replaced every two days. Differentiation of the myoblast cells was induced by culturing the cells in differentiation medium containing DMEM (HyClone) supplemented with 5% antibiotic and 5% horse serum for 5 days.

Human skeletal muscle origin cells (Lonza) from 17-year-old donors were treated with basal medium supplemented with antibiotics, human epidermal growth factor (hEGF), dexamethasone, L-glutamine and 10% fetal bovine serum medium) 2. Differentiation was initiated after 24-48 hours of seeding by alternating with differentiation medium containing DMEM / F12 (Gibco), antibiotics and 2% horse serum.

C2C12 cells were purchased from ATCC and cultured in growth medium containing DMEM (Gibco) with antibiotics and 10% fetal bovine serum. Differentiation was initiated after 24-48 hours of seeding by alternating with differentiation medium containing DMEM / F12 (Gibco), antibiotics and 5% horse serum.

Example 3. miRNA sequencing and data analysis

Small RNA-enriched total RNA was extracted from myocytes (n = 12) using the mirVana miRNA isolation kit (Ambion, Austin, Tex.) According to the manufacturer's protocol. miRNA libraries were prepared following the Illumina library construction protocol (Illumina Inc, San Diego, CA, USA). Each library was indexed using an Illumina adapter and the small RNA library was sized on a 6% TBE urea polyacrylamide gel. From this a 140-160 base pair (bp) size fragment was cut from the gel. The purified miRNA library was quantified on an Agilent DNA 1000 chip. All indexed samples were collected and sequenced in a lane of Illumina GAIIx (50 basepairs read). Prior to mapping the unprocessed small RNA sequencing leads to the reference genome, the quality of the leads was assessed using FastQC (http://www.bioinformatics.babraham.ac.uk/projects/fastqc) The in-house PERL script was used to remove the adapter sequence attached to the 3 'end. Small RNA leads trimmed with adapters are loaded on the reference genome mm10, downloaded from the UCSC genome (http://genome.ucsc.edu) using bwa software (http://bio-bwa.sourceforge.net) with no options Respectively. For the known miRNA (mirbase ver 20), the FPKM of each precursor and mature miRNA from the mapped leads was calculated using a custom python script. Mirbase was used to filter out already known miRNAs, and Rattus norvegicus was used as an associated species. The data obtained as a result of the above analysis was stored in the Gene Expression Omnibus (GEO) under the number GSE66267.

Example 4. Quantitative RT-PCR and mature miRNA expression assay

Quantitative RT-PCR was performed to determine the expression level of miRNA. RNA preparation and cDNA synthesis were performed according to standard protocols. Quantitative RT-PCR analysis was performed using a 20-μl reaction volume of StepOnePlus ™ (Applied Biosystems) containing cDNA, primers, SYBR master mix (Master Mix, Applied Biosystems). Data were normalized to the abundance of β-actin mRNA in each reaction. The sequences of the primers are shown in SEQ ID NOs: 2 to 15 and summarized in Table 1.

For the expression analysis of mature miRNA, TaqMan MicroRNA assays were performed according to the protocol recommended by the manufacturer (Applied Biosystems). Briefly, for each reverse transcriptase (RT) reaction, a total of 10 ng of RNA, 50 nM stem-loop RT primer, 0.25 mM each dNTP, 3.33 units / ul MultiScribe RT enzyme separated using a miRVana miRNA isolation kit , 0.25 units / ul RNase inhibitor and 1 RT buffer (all purchased from Applied Biosystems). The above-mentioned qRT-PCR reaction was carried out by incubating in 96-well plate of Applied Biosystem at 95 ° C for 10 minutes and then repeating 40 times at 95 ° C for 15 seconds and 60 ° C for 1 minute. The threshold value period (Ct) is defined as the number of partial cycles during which fluorescence passes through a fixed threshold value. U6 snRNA was used as an endogenous control for normalization.

Example 5. Infection of cells and luciferase assay

30 nM siRNA, miRNA analogs, and miRNA-inhibitors were infected with primary source cells, C2C12, or human musculoskeletal source cells using RNAiMax (Invitrogen) according to the manufacturer's recommended protocol. miRNA-analogs and miRNA-inhibitors were either mir Vana (Invitrogen) or AccuTarget (TM ) Human miRNA analogs (Bioneer) and siRNAs were purchased from Dhamacon (Thermo Scientific). The analogs and inhibitors are listed in Tables 2 to 4, and the siRNAs are listed in Table 5.

The 3'UTR fragment (position 0 to 1,265 bp) of Smad4 containing the miR-431 binding site was cloned into the pcDNA3-luc vector in which a sequence encoding luc was located between HindIII and BamHI in a multiple restriction enzyme cloning site Respectively. The resulting plasmid construct contains a luc-3'UTR driven by a strong CMV promoter, which allows the ability to analyze the function of miRNAs that regulate mRNA translation. In addition, a Smad4 3'UTR variant (mutant) with deletion of the miR-431 binding site (position 1067 to 1073 bp) was cloned into the same pcDNA 3-luc vector for luciferase assay.

Example 6. A pulldown assay using biotinylated miR 431-5p or antisense oligo as a bait

C2C12 cells were infected with biotinylated (Bi) miR431-5p or control Bi-cel-miR-67 (IDT, 60 nM each) in 100-mm plates. After 24 hours of infection, the cells were washed with PBS and resuspended in 1 ml lysis buffer [20 mM Tris (pH 7.5), 100 mM potassium chloride, 5 mM magnesium chloride, 0.3% NP-40, 100 U of RNase OUT (Invitrogen) (Roche). As already known (Lal et al. 2011), the cells were cultured on ice for 10 minutes. The cytoplasmic lysate was removed by centrifugation at 12,000 g for 10 min. Dynabead (100 μl) was added and incubated for 4 hours at 4 ° C with rotation. The beads were washed 4 times with 1 ml of ice-cold lysis buffer, and the RNA was isolated using TRIzol. The enrichment of Smad4 mRNA was measured by qRT-PCR and normalized to Gapdh mRNA. Before pulling down by the above method, 300 μg of the lysate in the neonatal muscle sample and the aging muscle sample was incubated with 4 μl of biotinylated (Bi) -miR431-5p or Bi-cel-miR -67 (control, ctrl).

C2C12 cells (four 100-mm plates with ~70% confluence) were solubilized with PEB containing protease inhibitor (Roche) and RNase inhibitor (Thermo Fisher), using complementary antisense oligos to Smad4 as bait. The lysates were incubated in 1X TENT buffer (10 mM Tris-HCl [pH 8.0], 1 mM EDTA [pH 8.0], 250 mM sodium chloride, 0.5% [v / v] 100 ng of biotin-labeled oligomer complementary to Smad4 mRNA or lncRNA Gm14635 in Triton X-100, protease and RNase inhibitor for 1 hour at room temperature. Dynabead (50 μl) was added and further incubated for 30 min at room temperature. The beads were washed three times with cold PBS buffer, RNA was isolated using TRIzol, and the enrichment of Smad4 mRNA was analyzed by qRT-PCR standardized to GAPDH mRNA. The same RNA as above was used to analyze the increased abundance of miR431-5p in Smad4 mRNA enriched samples using the QuantiMir kit (SBI). For neonatal and senescent muscles, 600 ug of lysate was used for pulldown using the method described above.

Example 7. Immunoblot analysis

Muscle tissue and isolated myoblasts were homogenized in PRO-PREP lysis buffer (Intron Biotech) containing protease and phosphatase inhibitors. The lysate was centrifuged at 15,000 g for 20 minutes at 4 ° C. SDS-PAGE followed by immunoblot analysis was performed with the resulting supernatant. Antibodies against MyHC (Santa Cruz Biotechnology), Myogenin (Santa Cruz Biotechnology), Santa Cruz Biotechnology, Smad2 / 3 (Cell Signaling Technology), Smad4 (Santa Cruz Biotechnology), GADPH (Santa Cruz Biotechnology) Included in the blotting.

Example 8. Regeneration of CTX-induced muscle

As previously described (Conboy and Rando 2012), CTX (Sigma) was injected into the tibialis anterior muscle (TA) at 3 and 27 months of age to induce muscle regeneration. Briefly, TA muscle was injected with 50 uL of 20 uM CTX and a mixture of miRNA-analog or siRNA and RNAiMAX reagent was prepared to over-express miR-431 or siSmad4 in TA muscle (Dey et al. 2014). 3 ul of 1 mM miRNA-analog or siRNA and 37 ul of Nuclease-depleted water were mixed with 40 ul RNAiMAX reagent (Invitrogen) and incubated for 30 minutes. Finally, 50 ul of the mixture was injected into the TA muscle on the 3rd and 5th days after CTX injection.

Example 9. Immunohistochemical analysis and immunofluorescence.

Skeletal muscle tissue was fixed in 4% paraformaldehyde and section samples frozen or paraffin-embedded were prepared. Frozen sections (10 [mu] M) were stained with DAPI and antibody as per standard protocol. Antibodies for immunohistochemical analysis include Laminin (Sigma-Aldrich) Pax7 (Developmental Studies Hybridoma Bank) and Smad4 (Abcam) antibodies. The paraffin section samples were stained according to standard protocols with H & E. Cross-sectional (CSA) measurements were performed with NIH ImageJ software (http://rsb.info.nih.gov/ij/). For immunofluorescence, primordial myocytes were grown on Laminin (Invitrogen) -coated cover slips with growth or differentiation medium. The cells were fixed in 4% paraformaldehyde, washed with PBS buffer, and permeabilized with 0.3% Triton X-100 in PBS buffer. Cells were blocked with 3% bovine serum albumin in PBS buffer and one more blotted with primary antibody diluted in blocking solution. The cells were washed with PBS buffer and incubated with secondary antibody. After washing with PBS buffer, cover slips are mounted on glass slides using VectoLab Mounting Medium. Antibodies for immunofluorescence include MyHC (Santa Cruz Biotechnology), MyoD (Santa Cruz Biotechnology), and Pax7 (Developmental Studies Hybridoma Bank). The fusion index was calculated as the ratio of the number of MyHC-positive cells to the total number of MyHC-positive cells.

Example 10. Statistical analysis

The quantification data are expressed as mean ± SD unless otherwise noted. The mean difference was assessed by the student's unpaired t test. A p value <0.05 was considered statistically significant.

The results of the experiments of the above examples were analyzed as in the following experimental examples.

Experimental Example 1. Comparative analysis of miRNA expression profiles between the neonatal and senescent cells using next generation sequencing (NGS)

MiRNA expression profiles were analyzed in neonatal and senescent myocytes using next-generation sequencing (NGS) to characterize the role of miRNAs in the myogenic capability of senescent myoblasts.

Primary myoblasts were isolated from the hindlimb muscles of 3-month-old (hereinafter referred to as 'newborn') and 27-month old (hereinafter referred to as 'aging') mice. Two proteins, Pax7 and MyoD, were used as specific markers of primordial source cells (Wang and Rudnicki, 2012). In immunostaining analysis, about 99% of isolated myocytes expressed both Pax7 and MyoD proteins (FIG. 8).

The differentiation potentials were compared in neonatal and senescent myocytes. As a result, the senescent myoblasts showed a decreased differentiation potential, consistent with previous reports (Schultz and Lipton 1982; Fulle et al. 2005; Wagers and Conboy 2005). Compared with the neonatal myocytes, the senescent myocytes decreased the number of myocyte heavy chain (MyHC) -specific differentiated canalicular cells (Fig. 9A, 9B). Protein levels of MyHC in senescent source cells also decreased. The mRNA levels of differentiation marker genes such as MyHC and Myogenin were also significantly down-regulated (Figure 9D).

Of the 118 mature miRNAs (Fig. IA) that showed significant differences (> 2-fold) between the neonatal and senescent myocytes, 47 miRNAs were significantly upregulated (P <0.05) ) And 71 miRNAs were down-regulated (Table 7).

We have reported that 57% of down-regulated miRNAs in aging muscle tissue are located in the Dlk-Dio3 region on chromosome 12 (Kim et al. 2014a). Interestingly, 63 of the 71 down-regulated miRNAs (89%) were also located in the Dlk-Dio3 region of senescent source cells.

These results suggest that miRNAs located in the genomic region may play an important role in the aging of muscle tissue and myocytes. Thus, the present inventors screened 13 miRNAs located in the genomic region and infected the senescent source cells to analyze the mRNA levels of the two differentiation markers (MyHC and Myogenin). miR-431 showed the most potent activity in enhancing the differentiation ability of senescent source cells (Fig. 1B).

Experimental Example 2: Increased root differentiation of miR-431 in the senescent source cells

In addition to the increase of the differentiation marker genes, as shown by the morphology and number of MyHC-positive differentiated root canal cells, exogenous miR-431 increased the differentiation of senescent source cells. In contrast, inhibition of miR-431 decreased the ability of the neoplastic stem cells to differentiate (Figs. 2A and B). The mRNA levels of MyHC and Myogenin increased in the senescent root cells when infected with the miR-431 mimic (Fig. 2C), whereas the mRNA levels of both MyHC and Myogenin decreased when the miR- (Fig. 2D).

These results suggest that miR-431, one of the miRNAs that decrease in senescent myocytes, plays an important role in the regulation of differentiation potentials upon aging.

On the other hand, the over-expression of miR-431 in the neonatal myocytes did not increase the differentiation ability, and the miR-431 inhibitor did not decrease the differentiation ability in the senescent source cells (FIG. 10).

These results suggest that the basal levels of miR-431 in neonatal myoblasts are saturated in the normal differentiation process (program). Conversely, it suggests that miR-431 is reduced enough to induce delayed differentiation in senescent myocytes.

Experimental Example 3: Differentiation of Smad4 in aging root cells by knockdown

In order to understand the mechanism of miR-431-induced damage during muscle aging, the target of miR-431 was searched using TargetScan (www.targetscan.org) and miRanda (www.microRNA.org). As a result, Smad4 was one of the genes presumed to be the target gene of miR-431.

Several previous studies have reported that TGF-β signaling inhibits muscle differentiation, and thus, older mice exhibit delayed muscle regeneration (Carlson et al. 2008; Carlson et al., 2009). Activated TGF-β receptors in the TGF-β signal pathway first phosphorylate Smad2 or Smad3. Subsequently, phosphorylated Smad2 / 3 forms a heterotrimeric complex with Smad4, a common Smad protein, and migrates to the nucleus to turn the target gene (Schmierer and Hill 2007).

In accordance with these reports, we examined the expression of the three Smad proteins (Smad2, 3, and 4) in neonatal and senescent myocytes. Protein levels of Smad3 and Smad4, downstream factors of the TGF-beta signaling pathway, were up-regulated in senescent myocytes, whereas protein levels of Smad2 did not change (Figure 3A).

In order to confirm whether upregulation of Smad3 and Smad4 is associated with reduced differentiation potential in senescent source cells, we used siRNA to knock down Smad3 or Smad4 in senescent source cells and then infect control siRNA We compared muscle differentiation with aging source cells.

Interestingly, Smad4 knockdown increased MyHC and Myogenin mRNA levels and reversed delayed muscle differentiation of senescent source cells to a significant level. Whereas Smad3 knockdown was not (Fig. 3B). The number of MyHC-positive differentiated root canal cells of senescent source cells was significantly increased in siSmad4 infected source cells (Fig. 3C, D), as compared to control siRNA infected ones.

Expression levels of miR-431 and Smad4, which are presumed to be regulated miRNAs of Smad4, during the differentiation of neoplastic and senescent source cells were analyzed. During differentiation, protein levels of Smad4 in senescent source cells were higher than that of neonatal source cells, while Smad4 protein was induced to induce muscle differentiation and progressively decreased until 3 days (Fig. 3E). On the other hand, miR-431 was gradually up-regulated in both neonatal and senescent myocytes during differentiation (Fig. 3F), although miR-431 expression levels remained low in senescent myocytes. These results suggested that expression levels of Smad4 protein and miR-431 were negatively correlated with each other. Thus, the present inventors confirmed whether miR-431 actually regulates Smad4 expression.

Experimental Example 4. Regulation of Smad4 Expression via 3'UTR of miR-431

In the 3'UTR of Smad4 mRNA, we found four down-regulated miRNAs, including miR-431, that are negatively correlated with increased protein levels of Smad4 during aging. Among them, only miR-431 inhibited the luciferase activity of Smad4 3'UTR (Fig. 11). The mutated Smad4 3'UTR was not inhibited by miR-431, indicating that the inhibition of Smad4 by miR-431 is specific (Fig. 4A).

Importantly, the Smad4 3'UTR site to which miR-431 binds is conserved between humans and mice (Fig. 4B). As expected, Smad4 protein was down-regulated in overexpression of miR-431 in senescent myocytes, whereas it was upregulated in miR-431 inhibitor-infected neonatal myocytes (Fig. 4C). Similar results were obtained in C2C12 cells treated with miR-431 analog or inhibitor (Fig. 12A).

The direct interaction of miR-431 with endogenous Smad4 mRNA was confirmed by pull-down experiments using biotinylated (Bi) miR-431 or Smad4 antisense oligo as bait. C2C12 cells were first infected with Bi-miR-431 or Bi-cel-miR-67 (control) and RNA was isolated by streptavidin-coupled beads. As a result of confirming using qRT-PCR, it was confirmed that Smad4 mRNA was enriched in RNA isolated from Bi-miR-431-infected cells (Fig. 12B).

Smad4 mRNA was abundant in the pull-down experiments of Bi-miR-431 after incubation of lysates of neonatal muscle and aging muscle tissue (Fig. 4D). In addition, biotinylated antisense oligo was used to confirm the abundance of miR-431 in an endogenous Smad4 mRNA pull-down experiment.

As a result, miR-431 was considerably abundant in lysates of Bi-Smad4 ASO cultured root cells and C2C12 cells (Fig. 4E; Fig. 12C). Taken together, these results suggest that miR-431 acts directly on the 3'UTR and inhibits Smad4 expression.

Experimental Example 5: Sudden increase of Smad4 protein in aged muscle tissue regeneration

Expression levels of Smad4 and miR-431 were examined in skeletal muscle tissue regeneration (Charge and Rudnicki 2004; Liu et al. 2012) induced by cadiotoxin (CTX) injected into the tibialis anterior (TA) muscle.

First, CTX was injected into the TA muscle of young and old mice to induce muscle injury and regeneration thereof. Thereafter, Smad4 protein and miR-431 expression were compared for 14 days after CTX injection.

As a result, as in previous studies (Conboy and Rando 2012), aging TA muscles showed impaired regenerative ability after CTX injection, whereas neonatal TA muscle had no problems in regeneration (Fig. 13A). Smad4 protein levels were rapidly elevated in aged TA muscle, which was sustained for 14 days (Fig. 5A), although the level of Smad4 protein was upregulated in both senescent TA and neonatal TA muscles after the first day of CTX injection. In contrast, expression of miR-431 remained low in senescent TA muscle than in neonatal TA muscle (Fig. 5A; Fig. 13B).

Next, at 14 days after CTX injection, the tissues were analyzed by immunohistochemical staining to identify regions where Smad4 was up-regulated. Interestingly, Smad4 is strongly stained in Pax7 positive myocytes (FIG. 5B).

Collectively, such in vitro data suggest that up-regulated Smad4 may be one of the causes of damaged regeneration in aging muscle.

Experimental Example 6. Regeneration of TA muscle by miR-431

In order to confirm the potential of miR-431 as a therapeutic substance for restoring senescent muscle regeneration ability, miR-431 or siSmad4 was tested in vivo to improve the ability to regenerate damaged muscle in old mice.

In order to over-express miR-431 or siSmad4 in senescent TA muscle damaged by CTX, the RNAiMAX mixture was injected two times, two days and five days after CTX injury as described in previous studies (Dey et al. 6A). Two weeks later, the cross-sections of the control muscle tissue and the cross-sections of the regenerating fibers were compared.

As a result, it was predicted that newly generated muscle fibers with a core were significantly larger in Smad4 knockdown TA muscle tissue than in control muscle tissue (Fig. 6B). Furthermore, miR-431 enhanced muscle regeneration in old mice, resulting in significantly larger regenerated fibers than control mice (Fig. 6C).

Taken together, miR-431 can restore regeneration of delayed or damaged muscles in old mice.

Experimental Example 7. Increase in muscle differentiation of human skeletal muscle cell (HSMM) by miR-431

Since the 3'UTR seed sequence of Smad4 is conserved between human and mouse (Fig. 4B), miR-431 was expected to regulate the level of human Smad4 protein. To confirm this, levels of Smad4 protein in human skeletal muscle source cells (HSMM) infected with miR-431 analog or inhibitor were analyzed.

As a result, the hSmad4 protein level was down-regulated in the HSMM over-expressing miR-431, whereas the inhibitor of miR-431 increased the Smad4 protein level (Fig. 7A). This was consistent with results from mouse myoblast cells.

In order to confirm whether miR-431 can increase HSMM differentiation, HSMM was infected with miR-431 inhibitor and the differentiation index was analyzed.

As a result, as in the case of mouse stem cells, the miR-431 inhibitor decreased HSMM differentiation (FIGS. 7B and 7C).

From the above description, it will be understood by those skilled in the art that the present invention may be embodied in other specific forms without departing from the spirit or essential characteristics thereof. In this regard, it should be understood that the above-described embodiments are to be considered in all respects as illustrative and not restrictive. The scope of the present invention should be construed as being included in the scope of the present invention without departing from the scope of the present invention as defined by the appended claims.

<110> KOREA RESEARCH INSTITUTE OF BIOSCIENCE AND BIOTECHNOLOGY <120> A COMPOSITION FOR REPRESION OF MUSCLE-AGING AND REGENERATION OF OLD MUSCLE <130> KPA150327KR-P1 <150> KR10-2015-0063399 <151> 2015-05-06 <160> 15 <170> Kopatentin 2.0 <210> 1 <211> 21 <212> RNA <213> miR-431 in C57BL / 6 mouse myoblast <400> 1 ugucuugcag gccgucaugc a 21 <210> 2 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Forward primer for Myogenin <400> 2 ctacaggcct tgctcagctc 20 <210> 3 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Reverse primer for Myogenin <400> 3 acgatggacg taagggagtg 20 <210> 4 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> Forward primer for MyHC <400> 4 tccaaaccgt ctctgcactg tt 22 <210> 5 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> Reverse primer for MyHC <400> 5 agcgtacaaa gtgtgggtgt gt 22 <210> 6 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> Forward primer for b-actin <400> 6 ugucuugcag gccgucaugc a 21 <210> 7 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> Reverse primer for b-actin <400> 7 ccagttggta acaatgccat g 21 <210> 8 <211> 19 <212> DNA <213> Artificial Sequence <220> <223> Forward primer for Smad3 <400> 8 cacgcagaac gtgaacacc 19 <210> 9 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> Reverse primer for Smad3 <400> 9 ggcagtagat aacgtgaggg a 21 <210> 10 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> Forward primer for Smad4 <400> 10 acaccaacaa gtaacgatgc c 21 <210> 11 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> Reverse primer for Smad4 <400> 11 gcaaaggttt cactttcccc a 21 <210> 12 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> Forward primer for Human MyHC <400> 12 caagagttct caggatggga ag 22 <210> 13 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Reverse primer for Human MyHC <400> 13 gccatgtctt cgatcctgtc 20 <210> 14 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> Forward primer for Human Myogenin <400> 14 ggggaaaact acctgcctgt c 21 <210> 15 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Reverse primer for Human Myogenin <400> 15 aggcgctcga tgtactggat 20

Claims (16)

A pharmaceutical composition for the prevention or treatment of aging-related sarcopenia caused by an increase in Smad4 in an aging subject comprising miR-431 or mimic thereof as an active ingredient.
delete The method according to claim 1,
Wherein the sequence of miR-431 is SEQ ID NO: 1.
The method according to claim 1,
Wherein said miR-431 or analog thereof is targeted to Smad4 mRNA.
5. The method of claim 4,
Wherein said miR-431 or analog thereof targets the 3'UTR (untranslated region) of said Smad4 mRNA.
The method according to claim 1,
Wherein said miR-431 or analog thereof inhibits Smad4.
The method according to claim 1,
Wherein said composition comprises a recombinant plasmid or viral vector expressing said miR-431 or an analogue thereof, or further comprises a non-viral carrier.
The method according to claim 1,
Wherein the pharmaceutical composition further comprises a pharmaceutically acceptable carrier.
delete delete delete delete delete delete delete delete

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