KR20150131559A - MicroRNA-34 for the diagnosis and treatment of muscle aging - Google Patents

Abstract

The present invention relates to miRNA-34 as a diagnostic marker for muscle aging, and more specifically, to a composition for diagnosing muscle aging comprising an agent for measuring the expression level of miRNA-34 or fragments thereof, a kit for diagnosing muscle aging comprising the composition, and a method for providing information for diagnosing muscle aging including a step for measuring the expression level of the miRNA-34 or fragments thereof. The muscle aging can cause muscular atrophy and deterioration of muscle functions associated with the same, and diseases such as obesity, diabetes mellitus, and the like due to the accompanying energy hypometabolism. Various miRNAs including the miRNA-34, provided in the present invention, can be used as a marker for diagnosing the progress of muscle aging, whether the muscle aging has been detected or not, and the degree of the muscle aging, thereby being able to be widely used as a marker having excellent effects on developing a kit for diagnosing the muscle aging, and searching for a muscle aging inhibitory substance.

Description

MicroRNA-34 for the diagnosis and treatment of muscle aging.

The present invention relates to microRNA-34 as a diagnostic marker for muscle aging. More specifically, the present invention relates to a composition for diagnosing muscle aging comprising an agent for measuring the expression level of miRNA-34 or a fragment thereof, a muscle aging And a method for providing information for diagnosing muscle aging comprising the step of measuring the expression level of a diagnostic kit and miRNA-34 or fragment thereof.

Aging is largely divided into physiological aging and pathological aging. Physiological aging is an age-related aging that accompanies, for example, changes in hair, skin, eyes, bones, brain, exercise capacity and energy metabolism.

On the other hand, the decrease in strength and endurance caused by aging lowers daily life ability. In addition, degradation of energy metabolism causes unbalance of energy consumption and energy consumption, and may cause lifestyle diseases such as obesity and diabetes.

In general, muscular atrophy, in which muscle mass or muscle strength is reduced, includes disuse muscle atrophy and Sarcopenia. When muscle atrophy occurs, muscular atrophy accompanies the muscular atrophy . ≪ / RTI > In particular, elderly people are susceptible to muscle atrophy or muscle weakness, and are liable to muscle damage or fracture. When placed under restricting conditions such as stable state or casting fixation for treatment and care, The myocardial function is rapidly declining.

Aging is driven by genetic or environmental factors, but it is considered possible to slow down by improving environmental factors, such as eating habits and exercise. As a means to prevent exercise capacity accompanied by aging and to prevent muscle atrophy and decrease in myocardial function, it is particularly desirable to exercise exercise or rehabilitation in the case of the elderly.

In addition, in recent years, not only exercise and physical therapies but also researches for components capable of inhibiting aging and more specifically inhibiting muscle aging such as muscle atrophy and accompanying deterioration of muscle function have been conducted.

In addition to this, it is possible to diagnose the degree and the degree of muscle aging which can accompany not only muscle atrophy and accompanying muscle weakness but also metabolic degeneration resulting from obesity and diabetes, It is necessary to develop a diagnostic marker having an excellent effect in the diagnosis of cancer.

Recently, micro RNA (miRNA) has been recognized as a major factor controlling various life phenomena including aging. miRNA is a short non-coding RNA of about 22 nucleotides in length and is a genome found in various animals from plants. It is known that miRNAs regulate over 30% protein production gene expression by interfering with transcription of complementary messenger RNA (mRNA) or by degrading mRNA in post-transcriptional stages. Many studies have shown that these miRNAs are involved in various biological processes. Recently, miRNA expression changes have been reported to play an important role in various aging processes such as cell, tissue, and life span of an individual.

Although it is not yet known how miRNAs play a role in aging diseases, studies on the expression of miRNAs in the aging process are actively under way. For example, US Pat. No. 4,030,091, which describes aging-related genes and miRNAs to reduce signs of skin aging (e.g., wrinkles), aged liver and brain of mice (Smith-Vikos T. et al., J Cell < ( R) >) describing changes and roles of genes and miRNA expression levels associated with aging expressed in organs such as brain Sci . , ≪ / RTI > 125 (1): 7-17, 2012) and changes in expression levels of miRNA and miRNA clusters associated with aging expressed in aged hearts of mice (Zhang X. et al., PLoS One . , 7 (4): e34688, 2012).

It is expected that the miRNA associated with aging can be usefully used as a substance search for the prevention and treatment of diseases caused by aging of various organs such as skin aging, heart and brain, but miRNA Has not been disclosed.

Under these circumstances, the present inventors have made intensive researches to develop a method for diagnosing muscle aging, and as a result, it is possible to diagnose muscle aging by detecting that the expression levels of various miRNAs can be changed in aged skeletal muscle And completed the present invention.

It is an object of the present invention to provide a composition for diagnosing muscle aging comprising an agent that measures the level of expression of miRNA-34 or a fragment thereof.

Another object of the present invention is to provide a kit for diagnosing muscle aging comprising the composition.

Yet another object of the present invention is to provide a method for providing information for diagnosis of muscle aging comprising the step of measuring the expression level of miRNA-34 or fragment thereof.

In one aspect to achieve the above object, the present invention provides a composition for diagnosing muscle aging comprising an agent for measuring the expression level of miRNA-34 or a fragment thereof.

The term "miRNA-34" used in the present invention is composed of miRNA-34a, miRNA-34b and miRNA-34c. Biologically, the miRNA-34 family pathologically indicates hypertrophy and destruction of myocardial cells, It is a miRNA known to enhance cardiac function by weakening the process of cardiac remodeling, which is characterized by destruction of collagen between myocardial cells.

In the present invention, the miRNA-34 may include miRNA-34a or miRNA-34c, preferably miRNA-34a is a nucleotide sequence of SEQ ID NO: 13 and miRNA-34c is a poly Lt; / RTI > nucleotides.

As used herein, the term "fragment" of miRNA-34 may be deleted when compared to a reference sequence of a miRNA, or may comprise a segment of the same sequence as the position corresponding to the reference sequence, The term "reference sequence" is a sequence designated to be used as a basis for sequence comparison. The miRNA-34a fragment of the present invention may preferably be a polynucleotide having the sequence of SEQ ID NO: 14 or SEQ ID NO: 15 and the miRNA-34c fragment of SEQ ID NO: 17 or SEQ ID NO: 18.

microRNA order SEQ ID NO: 1
(miRNA-136) GAGGACUCCAUUUGUUUUGAUGAUGGAUUCUUAAGCUCCAUCAUCGUCUCAAAUGAGUCUUC SEQ ID NO: 2
(miRNA-136-3p) AUCAUCGUCUCAAAUGAGUCUUU SEQ ID NO: 3
(miRNA-136-5p) ACUCCAUUUGUUUUGAUGAUGG SEQ ID NO: 4
(miRNA-337) CAGUGUAGUGAGAAGUUGGGGGGUGGGAACGGCGUCAUGCAGGAGUUGAUUGCACAGCCAUUCAGCUCCUAUAUGAUGCCUUUCUUCACCCCCUUCA SEQ ID NO: 5
(miRNA-337-3p) UUCAGCUCCUAUAUGAUGCCU SEQ ID NO: 6
(miRNA-337-5p) GAACGGCGUCAUGCAGGAGUU
SEQ ID NO: 7
(miRNA-127) CCAGCCUGCUGAAGCUCAGAGGGCUCUGAUUCAGAAAGAUCAUCGGAUCCGUCUGAGCUUGGCUGGUCGG SEQ ID NO: 8
(miRNA-127-3p) UCGGAUCCGUCUGAGCUUGGCU SEQ ID NO: 9
(miRNA-127-5p) CUGAAGCUCAGAGGGCUCUGAU SEQ ID NO: 10
(miRNA-411) UGGUACUUGGAGAGAUAGUAGACCGUAUAGCGUACGCUUUAUCUGUGACGUAUGUAACACGGUCCACUAACCCUCAGUAUCA SEQ ID NO: 11
(miRNA-411-3p) UAUGUAACACGGUCCACUAACC SEQ ID NO: 12
(miRNA-411-5p) UAGUAGACCGUAUAGCGUACG SEQ ID NO: 13
(miRNA-34a) CCAGCUGUGAGUAAUUCUUUGGCAGUGUCUUAGCUGGUUGUUGUGAGUAUUAGCUAAGGAAGCAAUCAGCAAGUAUACUGCCCUAGAAGUGCUGCACAUUGU SEQ ID NO: 14
(miRNA-34a-3p) AAUCAGCAAGUAUACUGCCCU SEQ ID NO: 15
(miRNA-34a-5p) UGGCAGUGUCUUAGCUGGUUGU SEQ ID NO: 16
(miRNA-34c) AGUCUAGUUACUAGGCAGUGUAGUUAGCUGAUUGCUAAUAGUACCAAUCACUAACCACACAGCCAGGUAAAAAGACU SEQ ID NO: 17
(miRNA-34c-3p) AAUCACUAACCACACAGCCAGG SEQ ID NO: 18
(miRNA-34c-5p) AGGCAGUGUAGUUAGCUGAUUGC SEQ ID NO: 19
(miRNA-143) CCUGAGGUGCAGUGCUGCAUCUCUGGUCAGUUGGGAGUCUGAGAUGAAGCACUGUAGCUCAGG SEQ ID NO: 20
(miRNA-143-3p) UGAGAUGAAGACACUGUAGCUC SEQ ID NO: 21
(miRNA-143-5p) GGUGCAGUGCUGCAUCUCUGG SEQ ID NO: 22
(miRNA-144) GGCUGGGAUAUCAUCAUAUACUGUAAGUUUGUGAUGAGACACUACAGUAUAGAUGAUGUACUAGUC SEQ ID NO: 23
(miRNA-144-3p) UACAGUAUAGAUGAUGUACU SEQ ID NO: 24
(miRNA-144-5p) GGAUAUCAUCAUAUACUGUAAGU SEQ ID NO: 25
(miRNA-146a) AGCUCUGAGAACUGAAUUCCAUGGGUUAUAUCAAUGUCAGACCUGUGAAAUUCAGUUCUUCAGCU SEQ ID NO: 26
(miRNA-146a-3p) CCUGUGAAAUUCAGUUCUUCAG SEQ ID NO: 27
(miRNA-146a-5p) UGAGAACUGAAUUCCAUGGGUU SEQ ID NO: 28
(miRNA-148a) AGCCAGUUUGGUCUUUUGAGACAAAGUUCUGAGACACUCCGACUCUGAGUAUGAUAGAAGUCAGUGCACUACAGAACUUUGUCUCUAGAGGCUGUGGUC SEQ ID NO: 29
(miRNA-148a-3p) UCAGUGCACUACAGAACUUUGU SEQ ID NO: 30
(miRNA-148a-5p) AAAGUUCUGAGACACUCCGACU SEQ ID NO: 31
(miRNA-185) AGGGAUUGGAGAGAAAGGCAGUUCCUGAUGGUCCCCUCCCAGGGGCUGGCUUUCCUCUGGUCCUU SEQ ID NO: 32
(miRNA-185-3p) AGGGGCUGGCUUUCCUCUGGU SEQ ID NO: 33
(miRNA-185-5p) UGGAGAGAAAGGCAGUUCCUGA SEQ ID NO: 34
(miRNA-190a) CUGUGUGAUAUGUUUGAUAUAUUAGGUUGUUAUUUAAUCCAACUAUAUAUCAAGCAUAUUCCUACAG SEQ ID NO: 35
(miRNA-190a-3p) ACUAUAUAUCAAGCAUAUUCCU SEQ ID NO: 36
(miRNA-190a-5p) UGAUAUGUUUGAUAUAUUAGGU SEQ ID NO: 37
(miRNA-193a) GAGAGCUGGGUCUUUGCGGGCAAGAUGAGAGUGUCAGUUCAACUGGCCUACAAAGUCCCAGUCUCUCUC SEQ ID NO: 38
(miRNA-193a-3p) AACUGGCCUACAAAGUCCCAGU SEQ ID NO: 39
(miRNA-193a-5p) UGGGUCUUUGCGGGCAAGAUGA SEQ ID NO: 40
(miRNA-196b) AACUGGUCGGUGAUUUAGGUAGUUUCCUGUUGUUGGGAUCCACCUUUCUCUCGACAGCACGACACUGCCUUCAUUACUUCAGUUG SEQ ID NO: 41
(miRNA-196b-3p) UCGACAGCACGACACUGCCUUC SEQ ID NO: 42
(miRNA-196b-5p) UAGGUAGUUUCCUGUUGUUGGG SEQ ID NO: 43
(miRNA-215) AGCUCUCAGCAUCAACGGUGUACAGGAGAAUGACCUAUGAUUUGACAGACCGUGCAGCUGUGUAUGUCUGUCAUUCUGUAGGCCAAUAUUCUGUAUGUCACUGCUACUUAAA SEQ ID NO: 44
(miRNA-215-3p) UCUGUCAUUCUGUAGGCCAAU SEQ ID NO: 45
(miRNA-215-5p) AUGACCUAUGAUUUGACAGAC SEQ ID NO: 46
(miRNA-223) UCUGGCCAUCUGCAGUGUCACGCUCCGUGUAUUUGACAAGCUGAGUUGGACACUCUGUGUGGUAGAGUGUCAGUUUGUCAAAUACCCCAAGUGUGGCUCAUGCCUAUCAG SEQ ID NO: 47
(miRNA-223-3p) UGUCAGUUUGUCAAAUACCCCA SEQ ID NO: 48
(miRNA-223-5p) CGUGUAUUUGACAAGCUGAGUUG SEQ ID NO: 49
(miRNA-369) GGUACUUGAAGGGAGAUCGACCGUGUUAUAUUCGCUUGGCUGACUUCGAAUAAUACAUGGUUGAUCUUUUCUCAGUAUC SEQ ID NO: 50
(miRNA-369-3p) AAUAAUACAUGGUUGAUCUUU SEQ ID NO: 51
(miRNA-369-5p) AGAUCGACCGUGUUAUAUUCGC SEQ ID NO: 52
(miRNA-483) GAGGGGGAAGACGGGAGAAGAGAAGGGAGUGGUUUUUGGGUGCCUCACUCCUCCCCUCCCGUCUUGUUCUCUC SEQ ID NO: 53
(miRNA-483-3p) UCACUCCUCCCCUCCCGUCUU SEQ ID NO: 54
(miRNA-483-5p) AAGACGGGAGAAGAGAAGGGAG SEQ ID NO: 55
(miRNA-615) UCGGGAGGGGCGGGAGGGGGGGUCCCCGGUGCUCGGAUCUCGAGGGUGCUUAUUGUUCGGUCCGAGCCUGGGUCUCCCUCUUCCCCCCAUCCC SEQ ID NO: 56
(miRNA-615-3p) UCCGAGCCUGGGUCUCCCUCUU SEQ ID NO: 57
(miRNA-615-5p) GGGGGUCCCCGGUGCUCGGAUC SEQ ID NO: 58
(miRNA-155) CUGUUAAUGCUAAUUGUGAUAGGGGUUUUGGCCUCUGACUGACUCCUACCUGUUAGCAUUAACAG SEQ ID NO: 59
(miRNA-155-3p) CUCCUACCUGUUAGCAUUAAC SEQ ID NO: 60
(miRNA-155-5p) UUAAUGCUAAUUGUGAUAGGGGU SEQ ID NO: 61
(miRNA-203) GCCUGGUCCAGUGGUUCUUGACAGUUCAACAGUUCUGUAGCACAAUUGUGAAAUGUUUAGGACCACUAGACCCGGC SEQ ID NO: 62
(miRNA-203-3p) GUGAAAUGUUUAGGACCACUAG SEQ ID NO: 63
(miRNA-203-5p) AGUGGUUCUUGACAGUUCAACA SEQ ID NO: 64
(miRNA-434) UCGACUCUGGGUUUGAACCAAAGCUCGACUCAUGGUUUGAACCAUUACUUAAUUCGUGGUUUGAACCAUCACUCGACUCCUGGUUCGAACCAUC SEQ ID NO: 65
(miRNA-434-3p) UUUGAACCAUCACUCGACUCCU SEQ ID NO: 66
(miRNA-434-5p) GCUCGACUCAUGGUUUGAACCA SEQ ID NO: 67
(miRNA-92b) GGUGGGCGGGAGGGACGGGACGUGGUGCAGUGUUGUUCUUUCCCCUGCCAAUAUUGCACUCGUCCCGGCCUCCGGCCCCCUCG SEQ ID NO: 68
(miRNA-92b-3p) UAUUGCACUCGUCCCGGCCUCC SEQ ID NO: 69
(miRNA-92b-5p) AGGGACGGGACGUGGUGCAGUGUU

As used herein, the term "muscle aging" refers to a decrease in muscular strength caused by aging, such as a decrease in muscular strength (muscle strength, muscle endurance, muscle strength, etc.) or muscle atrophy, This means that the muscle mass is decreased by the decrease or reduction of muscle cells.

As used herein, the term "diagnosis" means identifying the presence or characteristic of a pathological condition. In the present invention, the diagnosis can be interpreted as confirming whether or not the progression of muscle aging has occurred.

In the present invention, the composition for diagnosing muscle aging includes miRNA-136, miRNA-337, miRNA-127, miRNA-411, miRNA-143, miRNA-144, miRNA-146, miRNA- One or more members selected from the group consisting of miRNA-193, miRNA-215, miRNA-223, miRNA-369, miRNA-483, miRNA-615, miRNA- lt; RTI ID = 0.0 > miRNA < / RTI > or fragment thereof.

In the present invention, miRNA-146, miRNA-148, miRNA-190, miRNA-193, miRNA-193, miRNA-196a and miRNA- 92 may contain miRNA-92b.

But are not limited to, miRNA-136, miRNA-337, miRNA-127, miRNA-411, miRNA-143, miRNA-144, miRNA-146a, miRNA- 148a, miRNA- sequence of miRNA-196b, miRNA-215, miRNA-223, miRNA-369, miRNA-483, miRNA-615, miRNA-155, miRNA-203, miRNA- May be composed of the nucleotide sequence shown in Table 1.

In the present invention, the miRNAs may be the nucleotide sequences shown in SEQ ID NOS: 1 to 69, and the nucleotide sequences shown in SEQ ID NOS: 1 to 69 may be used as diagnostic markers to diagnose progression or development of muscle aging. The sequence may be modified to some degree in diagnosing progression or onset of muscle aging. Those skilled in the art will appreciate that nucleotides that retain more than 80% homology, preferably more than 90% homology, more preferably more than 95% homology, and most preferably 98% homology, As long as it is possible to compare the difference in the expression level between the normal individuals and the aging individuals as the muscle aging diagnostic markers, it will be easily understood that they are equivalent to the above base sequences of the present invention.

As used herein, the term "marker or diagnosis marker" refers to a substance capable of differentiating individuals having muscle aging cells or aging diseases from normal cells or normal individuals, Such as polypeptides, proteins or nucleic acids (such as mRNA), lipids, glycolipids, glycoproteins or sugars (monosaccharides, disaccharides, oligosaccharides, etc.) which show an increase or decrease in the progressed or developed cells or individuals . For the purpose of the present invention, the marker for diagnosing muscle aging according to the present invention is a miRNA or a fragment of the miRNA which shows a difference in expression level specifically in muscle aging cells as compared with normal cells or tissues.

The term "agent for measuring the level of miRNA-34 or a fragment thereof " used in the present invention means a preparation used in a method for confirming the expression of miRNA-34 or a fragment thereof contained in a sample, RT-PCR, Competitive RT-PCR, Real-time RT-PCR, RNase protection assay, Northern blotting, A primer or a probe capable of specifically binding to a target gene used in the method of the present invention, but the present invention is not particularly limited thereto.

The term "primer" as used herein refers to a nucleic acid sequence having a short free 3 'hydroxyl group and capable of forming a base pair with a complementary template, Refers to a short nucleic acid sequence that functions as a starting point. The primers can initiate DNA synthesis in the presence of reagents and four different nucleoside triphosphates for polymerization reactions (i. E., DNA polymerase or reverse transcriptase) at appropriate buffer solutions and temperatures.

As used herein, the term "probe" refers to a nucleic acid fragment such as RNA or DNA corresponding to a few nucleotides or hundreds of nucleotides, which can specifically bind to a gene or mRNA. An oligonucleotide, A probe, a single stranded DNA probe, a double stranded DNA probe, an RNA probe, or the like, and can be labeled for easier detection.

The primers or probes of the present invention can be chemically synthesized using the phosphoramidite solid support method, or other well-known methods. Such nucleic acid sequences may also be modified using many means known in the art. Non-limiting examples of such modifications include, but are not limited to, methylation, capping, substitution of one or more natural nucleotides with one or more homologues, and modifications between nucleotides, such as uncharged linkers (e.g., methylphosphonate, phosphotriester, (E.g., phosphoramidate, carbamate, etc.) or charged linkages (e.g., phosphorothioate, phosphorodithioate, etc.).

Specifically, one embodiment of the present invention derives an miRNA that can be used as a marker for diagnosing muscle aging. The miRNA can be obtained by using a next generation sequence (NGS) method, which is a new method for analyzing the nucleotide sequence of a DNA in a large amount at the same time using a Solexa or SOLID sequencer. And the amount of expression of miRNA expressed in the skeletal muscle of the aged mouse was estimated to derive the aged mouse skeletal muscle specific miRNA.

As shown in Table 2 and Table 3, miRNAs expressed in skeletal muscle of young mice and aged mice were able to derive miRNAs whose expression levels were down-regulated or up-regulated specifically in skeletal muscle of aging mice .

The miRNAs up-regulated in the skeletal muscle of the aged mouse include miRNA-34 as well as miRNA-146a, miRNA-185, miRNA-196b, miRNA-215, miRNA-223, miRNA-369, miRNA- MiRNA-143, miRNA-144, miRNA-148a, miRNA-131, miRNA-203 and miRNA-92 were detected in the downregulated miRNA, miRNA-190a, miRNA-193a and miRNA-434, and the like (Fig. 3).

In addition, real-time quantitative PCR was performed to reexamine the differences in the expression levels of miRNAs expressed in skeletal muscle of young and aged mice as described above. As a result, miRNAs in skeletal muscle of aging mice MiRNA-146, miRNA-147, miRNA-143, and miRNA-143, as well as miRNA-146a, miRNA- -148a and the like were down-regulated (Fig. 4).

These results indicate that the expression levels of the various miRNAs are decreased or increased as the muscle aging progresses. When the agent for measuring the expression levels of various miRNAs including miRNA-34 of the present invention is used, It can be diagnosed effectively.

According to another aspect of the present invention, there is provided a kit for diagnosing muscle aging comprising the composition for diagnosing muscle aging.

MiRNA-144, miRNA-146, miRNA-144, miRNA-144, miRNA-147, miRNA-143, miRNA-144, miRNA-183, miRNA-193, miRNA-196, miRNA-215, miRNA-223, miRNA-369, miRNA-483, miRNA-615, miRNA- 434 and miRNA-92 or fragments corresponding to the miRNAs. The expression of the various miRNAs or their corresponding fragments, including but not limited to miRNA-34, One or more other component compositions, solutions or devices suitable for the assay method may be included, as well as primers or probes for measuring the level.

As a specific example, miRNA-34 or a fragment thereof of the present invention and miRNA-136, miRNA-337, miRNA-127, miRNA-411, miRNA-143, miRNA-144, miRNA- miRNA-193, miRNA-196, miRNA-215, miRNA-223, miRNA-369, miRNA-483, miRNA-615, miRNA-155, miRNA-203, miRNA- A diagnostic kit for determining the level of expression of a fragment corresponding to miRNAs may be a kit containing the necessary elements to perform RT-PCR. The RT-PCR kit can be used in combination with a test tube or other appropriate container, a reaction buffer (pH and magnesium concentration varies), deoxynucleotides (dNTPs), Taq polymerase and reverse transcriptase , DNase, RNAse inhibitor, DEPC-water, sterile water, and the like. In addition, it may contain a primer pair specific to a gene used as a quantitative control.

As another example, the kit of the present invention may contain necessary elements necessary for performing gene chip analysis. The kit for gene chip analysis may include a substrate on which a cDNA corresponding to a gene or a fragment thereof is attached as a probe, and a reagent, a preparation, and an enzyme for producing a fluorescent-labeled probe. In addition, the substrate may contain a cDNA corresponding to a quantitative control gene or a fragment thereof.

In yet another aspect, the present invention provides a method for providing information for the diagnosis of muscle aging using the diagnostic composition or kit, comprising: (a) contacting miRNA-34 or fragments thereof, And miRNA-146, miRNA-185, miRNA-196, miRNA-215, miRNA-223, miRNA-369, miRNA-483, miRNA-615, miRNA-155, miRNA-203 and miRNA- Measuring the expression level of a fragment or the like that is to be detected; And (b) if the level of expression of the various miRNAs or fragments thereof, including the measured miRNA-34, is significantly increased compared to the level measured from a normal control sample, it is determined that the aging of the muscle has progressed or developed .

MiRNA-143, miRNA-144, miRNA-148, miRNA-190, miRNA-193, and miRNA-193 in a biological sample isolated from an individual, 434 or fragments corresponding to the miRNAs; And (b) if the level of expression of the various miRNAs or fragments thereof, including the measured miRNA-411, is significantly reduced compared to the level measured from a normal control sample, it is determined that the aging of the muscle has progressed or developed .

The miRNA-34 or its fragment and the miRNA-136, miRNA-337, miRNA-127, miRNA-411, miRNA-143, miRNA-144, miRNA-146, miRNA-148, miRNA- , miRNA-193, miRNA-196, miRNA-215, miRNA-223, miRNA-369, miRNA-483, miRNA-615, miRNA-155, miRNA-203, miRNA-434 and miRNA- The method for measuring the expression level of the fragment is the same as described above.

In another aspect of the present invention, there is provided a method for preventing or treating progression or onset of muscle aging, comprising administering a candidate substance which is expected to prevent or treat the progression of muscle aging and measuring the expression level of miRNA-34 or fragment thereof A method for screening an agent for preventing or treating muscle aging which comprises

Specifically, the method for screening a preparation for preventing or treating muscle aging according to the present invention comprises the steps of: (a) measuring the expression level of miRNA-34 or fragment thereof from a sample of an experimental animal in which muscle aging has occurred; (b) administering a candidate agent expected to be able to treat muscle aging in said experimental animal; (c) measuring the expression level of miRNA-34 or fragment thereof from a sample of an experimental animal to which said candidate agent is administered; And (d) comparing the level of miRNA-34 or fragment thereof measured in the control with that measured in the experimental group, compared to that measured in the experimental group, the candidate agent is selected from the group of muscle aging Or prophylactic agent for preventing progression or onset, or a therapeutic agent for treating muscle aging.

The present invention provides a method for measuring the expression level of miRNA-34 or fragment thereof of the present invention in a cell, more preferably a muscle cell, in the absence of a candidate substance capable of preventing or treating muscle aging, Or the fragment thereof of the present invention is measured in the presence of the candidate substance and the expression level of the miRNA-34 or fragment thereof of the present invention when the candidate substance is present is measured in the absence of the candidate substance Of the total body weight can be predicted as a preventive agent for muscle aging.

In addition to miRNA-34, which is an up-regulated miRNA in the skeletal muscle of aging mice, miRNA-146, miRNA-185, miRNA-196, miRNA-215, miRNA-223, miRNA-369, miRNA- miRNA-155, miRNA-203 and miRNA-92 or fragments corresponding to the miRNAs can be predicted by agents for preventing or treating muscle aging.

In addition, miRNA-411, miRNA-136, miRNA-337, miRNA-127, miRNA-143, miRNA-144, miRNA-148, miRNA-190, miRNA-193 and miRNA- -434 or fragments corresponding to the miRNAs can be predicted by agents for preventing or treating muscle aging.

In another aspect of the present invention, there is provided a composition for inhibiting muscle senescence comprising an inhibitor targeting miRNA-34 or a fragment thereof as an active ingredient.

The composition for inhibiting muscle senescence is selected from the group consisting of miRNA-136, miRNA-337, miRNA-127, miRNA-411, miRNA-143, miRNA-144, miRNA-148, miRNA- A miRNA-146, miRNA-185, miRNA-196, miRNA-215, miRNA-223, miRNA-369, miRNA-483, miRNA- 615, miRNA-155, miRNA-203 and miRNA-92, or fragments corresponding to the miRNAs, and the like.

Specifically, miRNA-411, miRNA-136, miRNA-337, miRNA-127, miRNA-143, miRNA-144, miRNA-148, miRNA-190 and miRNA-193, which are downregulated in the skeletal muscle of the aged mouse, enhancers targeting miRNA-434 or fragments corresponding to the miRNAs or the like can inhibit muscle aging by increasing the expression level of the miRNA and the corresponding fragments.

In addition, miRNA-146, miRNA-185, miRNA-196, miRNA-215, miRNA-223, miRNA-369, miRNA-483, miRNA- Inhibitors targeting miRNA-155, miRNA-203, and miRNA-92 or fragments corresponding to such miRNAs, etc., can inhibit muscle aging by decreasing the expression levels of the miRNA and corresponding fragments.

Promoters targeting the miRNAs or fragments thereof may include, but are not limited to, compounds or mimic forms specific for the miRNAs or fragments thereof.

In addition, inhibitors targeting such miRNAs or fragments thereof may include, but are not limited to, siRNAs, aptamers, antisense oligonucleotides, ribozymes, or compound forms specific for the miRNAs or fragments thereof .

As used herein, the term "mimic" is a polynucleotide that mimics miRNA action and can be therapeutically targeted by the analog.

The term "siRNA" used in the present invention is a nucleic acid molecule capable of mediating RNA interference or gene silencing, and can inhibit the expression of a target gene. Therefore, an effective gene knockdown method or gene therapy method .

As used herein, the term "aptamer " refers to a single-stranded oligonucleotide which is about 20 to 60 nucleotides in size and has a binding activity to a given target molecule. Depending on the sequence, it has various three-dimensional structures and can have a high affinity with a specific substance such as an antigen-antibody reaction. The aptamer can inhibit the activity of a given target molecule by binding to a predetermined target molecule. The aptamers of the present invention may be RNA, DNA, modified nucleic acids, or mixtures thereof, and may also be in linear or cyclic form.

As used herein, the term "antisense " refers to a molecule designed to interfere with gene expression and recognize or bind to a particular desired target polynucleotide sequence. Antisense molecules typically include oligonucleotides or oligonucleotide analogs capable of specifically binding to a target sequence present on the RNA molecule.

As used herein, the term "oligonucleotide" is a molecule consisting of DNA, RNA or DNA / RNA hybrids, and the term "oligonucleotide analogue" is a molecule comprising an oligonucleotide-like structure.

The term "ribozyme " as used in the present invention means an RNA molecule having an enzyme activity, and means a nucleic acid molecule capable of catalyzing a chemical reaction intramolecularly or intermolecularly. The ribozymes that can be used in the present invention include all types of ribozymes capable of catalyzing nuclease and nucleic acid polymerase type reactions based on ribozymes found in natural systems and are engineered to catalyze specific reactions in vivo Lt; / RTI > are also available. A ribozyme can cleave an RNA or DNA substrate and can cleave the RNA substrate for the purposes of the present invention. Ribozymes typically cleave the nucleic acid substrate through recognition and binding and subsequent cleavage of the target substrate. This recognition is based on base pair interaction, and thus can have a characteristic that target specific cleavage is possible.

Various miRNAs, including miRNA-34 provided by the present invention, can be used not only for the reduction of muscular atrophy and accompanying muscular dysfunction due to aging, but also for the reduction of energy metabolism resulting from aging and muscle aging which can accompany diseases such as obesity and diabetes. It can be widely used as a marker having excellent effect in development of a muscle aging diagnostic kit and searching for substances inhibiting muscle aging because it can be used as a marker for diagnosing the progress or invention and the degree of aging.

Brief Description of the Drawings Fig. 1 is a diagram showing the difference in the skeletal muscle skeletal muscle between a young mouse and an aged mouse.
FIG. 2 is a diagram showing an analysis outline of a small RNA transcriptome expressed in the skeletal muscle of the young mouse and the skeletal muscle of the aged mouse derived by the next generation sequencing (NGS) method.
Figure 3 compares differences in the expression levels of the final miRNAs found in the skeletal muscle of young and aged mice by high throughput small-RNA sequencing.
FIG. 4 is a graph comparing differences in expression amounts of the final miRNAs found in the skeletal muscle of young and aged mice by real-time quantitative PCR (PCR).

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

Example  1: Preparation of muscle tissue samples and measurement of muscle mass

To study aging in skeletal muscle, muscle mass was compared between young mice (6 months old) and aged mice (24 months old).

Specifically, 6 young male C57BL / 6 mice (6 months) and 6 aged male C57BL / 6 mice (24 months old) were purchased from the Laboratory Animal Resource Center (Korea Research Institute of Bioscience and Biotechnology). Before sacrificing for four types of muscle dessection, the soleus muscle (SOL), the extensor digitorum longus (EDL), the tibialis anterior (TA), and the gastrocnemius (gastroc.) , And the mice were weighed. Each muscle mass was recorded and gastrocnemius (gastroc.) Was immediately collected for the next step of microRNA (miRNA) profiling experiments.

As shown in FIG. 1, when comparing muscle weights between young mice (6 months old) and aged mice (24 months old), it was found that compared to young mice, the tibialis anterior (TA) and gastrocnemius ) Showed significant muscle mass loss, and in particular, the muscle mass of the skeletal muscle of the skeletal muscle gradually decreased with age until the age of 32 months.

These results indicate that muscle loss is one of the main characteristics of muscle aging.

Example  2: Micro RNA ( miRNA ) Sequence analysis ( sequencing ) And data analysis

In order to elucidate the molecular mechanism of muscle aging, small RNA sequencing is used to analyze and compare genome-wide miRNAs expressed in skeletal muscle of young mice and in skeletal muscle of aged mice Respectively.

Specifically, small RNA-enriched total RNAs from gastrocnemius (gastroc.) (N = 12) using the mir vAna miRNA isolation kit (Ambion, Austin, Tex.) According to the manufacturer's protocol (total RNA), and the miRNA library was prepared according to the Illumina library preparation protocol (Illumina Inc., San Diego, CA, USA). Each library was indexed with an Illumina adapter (a 6-base barcode). The small RNA library was size fractionated on a 6% TBE urea polyacrylamide gel and a 140-160 base pair fraction was cut from the gel And purified. The purified miRNA library was quantified using an Agilent DNA 1000 chip. All indexed samples were pooled and sequenced in one lane of Illumina GAIIx (36 bp reads).

Raw small RNAs that have not been processed Before decoding, the reference genome is decoded and the quality of the sequence is decoded by FastQC (http://www.bioinformatics.babraham.com) .ac.uk / projects / fastqc), and the adapter sequence attached at the 3 'end was removed with an in-house PERL script. After trimming the adapter sequence, the read size distribution was analyzed with the R (http://www.r-project.org.) Script. A small adapter-trimmed ribonucleic acid fragment (adapter) trimming the adapter sequence is available in the UCSC Genome Browser (http: // bio-bwa. Sourceforge.net.) Software with the default option BWA // genome. ucsc.edu) (Li & Durbin 2009). In order to classify the mapped sequence fragments into RNA classes, a microRNA (miRNA), a transfer RNA (tRNA), a ribosomal RNA (rRNA) and other non-coated RNA ) In the annotation file from the UCSC Genome Browser and fRNAdb (http://www.ncrna.org/frnadb.) To serialize the genomic position information (ID) of small ribonucleic acid and repeats ). ≪ / RTI > The mapping position and annotation information are compared and sequenced into RNA types consistent with all mapped sequence fragments and the relative frequency of expression of each RNA type is assessed based on the number of sequence fragments specified Respectively. To determine the major miRNA expression site in total small RNA sequencer data, the top 10 miRNAs expressed from a pooled sequence fragment set of six young mouse samples were selected and pooled into a pool of young mice The relative proportion of the 10 largest miRNAs to the smallest portion of the sequence fragments in the set of sequenced fragments of aged mice was assessed. For known miRNAs (miRBase ver. 20), fragments per kilobase of transcript per million fragments mapped (FPKM) to each precursor and final miRNA (mature microRNA) from sequence fragments mapped using Python scripts ) Were calculated. For new miRNA searches, mir Deep2 software with default parameters was used and MiRBase was used to exclude known miRNAs. The rat ( Rattus norvegicus ) was used as a relative.

As shown in FIG. 2, most of the sequences showed 96.9% of the final miRNA (mature miRNA) having a length of 20 to 23 nt with a peak at 22 nt (nucleotide) (Fig. 2A) 3.1% of small RNAs are composed of small nuclear RNA (snRNA), small cytoplasmic RNA (scRNA), signal recognition particle RNA (srpRNA), ribosomal RNA (rRNA) , Transfer RNA (tRNA), and piwi-interacting RNA (piRNA) interacting with the target protein (Fig. 2B). The piRNA found in germ cells was found to be up-regulated in the skeletal muscle of aging mice (Fig. 2C). In order to identify the abundance of the final miRNA in the skeletal muscle of young and aged mice, the top 10 miRNAs expressed in the top 10 miRNAs of the skeletal muscle were 75% of the total miRNAs (Fig. 2D). Of the top 10 final miRNAs analyzed, the remaining miRNAs, except miR-10b which occupied most of them, were found to be miRNAs already reported to have a role in muscle.

Example  3: ribonucleic acid ( RNA ) Sequence analysis ( sequencing ) And data analysis

To explore miRNAs associated with skeletal muscle senescence, we found a final miRNA with at least five sequence fragments from the skeletal muscle of young and aged mice by high throughput mini-RNA sequencing. Of these, 20 miRNAs were up-regulated in the aged muscle and 19 miRNAs were down-regulated (> 1.5-fold difference, t- test, p <0.05). Among the selected miRNAs, miRNAs such as miRNA-206, miRNA-434 and miRNA-34, which were previously reported to be associated with muscle aging, were also included.

More specifically, total RNA was extracted from gastrocnemius (gastrocn.) (N = 5) of young and aged rats using the RNeasy Fibrous Tissue Mini Kit (Qiagen) according to the manufacturer's protocol. RNA quality and integrity were determined by agarose gel electrophoresis and ethidium bromide (EtBr) staining followed by visual inspection under ultraviolet light. Respectively. The sequencing library was prepared using the TruSeq RNA Sample Preparation kit v2 (Illumina, CA, USA) according to the manufacturer's protocol. Briefly, messenger RNA (mRNA) was purified from total RNA using poly-T oligo-attached magnetic beads and fragmented into cDNA. After ligation of the adapter, the fragment was amplified by polymerase chain reaction (PCR) and sequencing was performed using the Genome Analyzer IIx (Illumina) End sequence fragment (2 x 76 bp).

Mouse ( Mus The reference genome sequence from the mouse was obtained from the University of California, Santa Cruz Genome Browser Gateway (assembly ID: mm10). The reference standard genetic index was designed using the Bowtie2-build component of Bowtie2 (ver. 2.0) and SAMtools (ver. 0.1.18). Tophat2 (ver. 2.0.8) was applied to a tissue sample for mapping sequence fragments for reference standard genomes. Gene expression was calculated by using the standard sequence gene (Ref 10q gene, mm10) model downloaded from the UCSC Browser gateway and the FPKM (fragments per kilobase of transcript per million fragments mapped) for each gene. And converted to log ₂ for further analysis.

As shown in Fig. 3, the expression level of miRNA-34 of the present invention among the final miRNAs having at least 5 sequence fragments found in the skeletal muscle of young and aged mice was significantly lower than that of young mice It was confirmed that the expression level in muscle was upregulated.

In addition, as shown in the following Table 2 and Table 3, 20 miRNAs up-regulated in aged muscle and 19 down-regulated miRNA-34, including miRNA-34 of the present invention miRNA could be identified (> 1.5-fold difference, t- test, p <0.05)

From these results, it was confirmed that the miRNAs including the miRNA-34 of the present invention can be diagnosed by detecting up-regulation or down-regulation of the miRNAs according to muscle aging.

Example  3-1: Final miRNA ( mature microRNA ) Expression analysis verification

Real-time quantitative PCR was performed in order to re-verify differences in expression levels of the final miRNAs detected from the skeletal muscle of young and aged mice by high throughput small-RNA sequencing.

More specifically, the TaqMan MicroRNA assay for miRNA expression analysis was performed according to the manufacturer's suggested protocol (Applied Biosystems). Briefly, each reverse transcriptase (RT) reaction contained 50 nM stem-loop RT primer, 0.25 mM dNTP, 3.33 units / ul MultiScribe RT enzyme, 0.25 units / ul RNase inhibitor and 1 × 10 ng of total RNA was isolated using the mir Vana miRNA isolation kit containing RT buffer (Applied Biosystems). The reaction was incubated at 16 ° C for 30 minutes, 42 ° C for 30 minutes, 85 ° C for 5 minutes, and the reaction was stopped at 4 ° C. Each quantitative PCR (qPCR) reaction consisted of 1.33 ul of reverse transcription product, 1 μl of 20 × primer from TaqMan MicroRNA assay, probe mix, and 0.5 μl of H-Taq DNA polymerase and 10 μl of buffer mixture mM Tris-HCL (pH 8.4) , were made, including _{100 mM KCl, 6 mM MgCl 2} , 0.5 mM dNTP, and 10% DMSO). The reactions were incubated on a 96-well plate at 95 DEG C for 10 minutes using a Bio-Rad CFX96 System, and PCR was performed for 40 cycles each at 95 DEG C for 15 seconds and 60 DEG C for 1 minute. At this time, in the PCR reaction step, TaqMan® universal reverse primer was used as the reverse primer and the forward primer as shown in Table 4 below. The threshold cycle (Ct) is defined as the fractional cycle number at which the fluorescence emission has a fixed threshold. U6 snRNA (small nuclear RNA) was provided as an endogenous control for normalization.

microRNA Primer sequence SEQ ID NO: miRNA-136-5p primer ACUCCAUUUGUUUUGAUGAUGG 70 miRNA-337-3p primer UUCAGCUCCUAUAUGAUGCCU 71 miRNA-127-3p primer UCGGAUCCGUCUGAGCUUGGCU 72 miRNA-34a-5p primer UGGCAGUGUCUUAGCUGGUUGU 73 miRNA-146a-5p primer UGAGAACUGAAUUCCAUGGGUU 74 miRNA-148a-3p primer UCAGUGCACUACAGAACUUUGU 75 miRNA-411-5p primer UAGUAGACCGUAUAGCGUACG 76 miRNA-92b-3p primer UAUUGCACUCGUCCCGGCCUCC 77 miRNA-155-5p primer UUAAUGCUAAUUGUGAUAGGGGU 78 miRNA-203-3p primer GUGAAAUGUUUAGGACCACUAG 79 miRNA-434-3p primer UUUGAACCAUCACUCGACUCCU 80 miRNA-434-5p primer GCUCGACUCAUGGUUUGAACCA 81

As shown in Fig. 4, miRNA-34 of the present invention, which was confirmed to be up-regulated in the senescent muscle by sequencing, was compared with the amount of expression in muscle of young mice by real-time quantitative PCR, It was reaffirmed that the expression level was upregulated in aging muscle.

In the case of miRNA-92, miRNA-146, miRNA-155 and miRNA-203 which were confirmed to be up-regulated in the senescent muscle by sequencing, (Fig. 4A). As a result, it was confirmed that the expression level was upregulated in aging muscle (Fig. 4A).

In addition, in the case of miRNA-337, miRNA-148, miRNA-136, miRNA-127, miRNA-434 and miRNA-411 whose expression levels in aging muscle were confirmed to be down-regulated by sequencing, PCR showed that the amount of expression in the muscle of the young mouse was compared with that of the muscle in the young mouse (Fig. 4B).

Thus, through the real-time quantitative PCR results, it can be clearly seen that the miRNAs including the miRNA-34 of the present invention can be used as muscle aging markers.

<110> Korea Research Institute of Bioscience and Biotechnology <120> MicroRNA-34 for the diagnosis and treatment of muscle aging <130> KPA140127-KR <160> 81 <170> Kopatentin 2.0 <210> 1 <211> 62 <212> RNA <213> Artificial Sequence <220> <223> miRNA-136 <400> 1 gaggacucca uuuguuuuga ugauggauuc uuaagcucca ucaucgucuc aaaugagucu 60 uc 62 <210> 2 <211> 22 <212> RNA <213> Artificial Sequence <220> <223> miRNA-136-3p <400> 2 aucaucgucu caaaugaguc uu 22 <210> 3 <211> 22 <212> RNA <213> Artificial Sequence <220> <223> miRNA-136-5p <400> 3 acuccauuug uuuugaugau gg 22 <210> 4 <211> 97 <212> RNA <213> Artificial Sequence <220> <223> miRNA-337 <400> 4 caguguagug agaaguuggg gggugggaac ggcgucaugc aggaguugau ugcacagcca 60 uucagcuccu auaugaugcc uuucuucacc cccuuca 97 <210> 5 <211> 21 <212> RNA <213> Artificial Sequence <220> <223> miRNA-337-3p <400> 5 uucagcuccu auaugaugcc u 21 <210> 6 <211> 21 <212> RNA <213> Artificial Sequence <220> <223> miRNA-337-5p <400> 6 gaacggcguc augcaggagu u 21 <210> 7 <211> 70 <212> RNA <213> Artificial Sequence <220> <223> miRNA-127 <400> 7 ccagccugcu gaagcucaga gggcucugau ucagaaagau caucggaucc gucugagcuu 60 ggcuggucgg 70 <210> 8 <211> 22 <212> RNA <213> Artificial Sequence <220> <223> miRNA-127-3p <400> 8 ucggauccgu cugagcuugg cu 22 <210> 9 <211> 22 <212> RNA <213> Artificial Sequence <220> <223> miRNA-127-5p <400> 9 cugaagcuca gagggcucug au 22 <210> 10 <211> 82 <212> RNA <213> Artificial Sequence <220> <223> miRNA-411 <400> 10 ugguacuugg agagauagua gaccguauag cguacgcuuu aucugugacg uauguaacac 60 gguccacuaa cccucaguau ca 82 <210> 11 <211> 22 <212> RNA <213> Artificial Sequence <220> <223> miRNA-411-3p <400> 11 uauguaacac gguccacuaa cc 22 <210> 12 <211> 21 <212> RNA <213> Artificial Sequence <220> <223> miRNA-411-5p <400> 12 uaguagaccg uauagcguac g 21 <210> 13 <211> 102 <212> RNA <213> Artificial Sequence <220> <223> miRNA-34a <400> 13 ccagcuguga guaauucuuu ggcagugucu uagcugguug uugugaguau uagcuaagga 60 agcaaucagc aaguauacug cccuagaagu gcugcacauu gu 102 <210> 14 <211> 21 <212> RNA <213> Artificial Sequence <220> <223> miRNA-34a-3p <400> 14 aaucagcaag uauacugccc u 21 <210> 15 <211> 22 <212> RNA <213> Artificial Sequence <220> <223> miRNA-34a-5p <400> 15 uggcaguguc uuagcugguu gu 22 <210> 16 <211> 77 <212> RNA <213> Artificial Sequence <220> <223> miRNA-34c <400> 16 agucuaguua cuaggcagug uaguuagcug auugcuaaua guaccaauca cuaaccacac 60 agccagguaa aaagacu 77 <210> 17 <211> 22 <212> RNA <213> Artificial Sequence <220> <223> miRNA-34c-3p <400> 17 aaucacuaac cacacagcca gg 22 <210> 18 <211> 23 <212> RNA <213> Artificial Sequence <220> <223> miRNA-34c-5p <400> 18 aggcagugua guuagcugau ugc 23 <210> 19 <211> 63 <212> RNA <213> Artificial Sequence <220> <223> miRNA-143 <400> 19 ccugaggugc agugcugcau cucuggucag uugggagucu gagaugaagc acuguagcuc 60 agg 63 <210> 20 <211> 21 <212> RNA <213> Artificial Sequence <220> <223> miRNA-143-3p <400> 20 ugagaugaag cacuguagcu c 21 <210> 21 <211> 21 <212> RNA <213> Artificial Sequence <220> <223> miRNA-143-5p <400> 21 ggugcagugc ugcaucucug g 21 <210> 22 <211> 66 <212> RNA <213> Artificial Sequence <220> <223> miRNA-144 <400> 22 ggcugggaua ucaucauaua cuguaaguuu gugaugagac acuacaguau agaugaugua 60 cuaguc 66 <210> 23 <211> 20 <212> RNA <213> Artificial Sequence <220> <223> miRNA-144-3p <400> 23 uacaguauag augauguacu 20 <210> 24 <211> 23 <212> RNA <213> Artificial Sequence <220> <223> miRNA-144-5p <400> 24 ggauaucauc auauacugua agu 23 <210> 25 <211> 65 <212> RNA <213> Artificial Sequence <220> <223> miRNA-146a <400> 25 agcucugaga acugaauucc auggguuaua ucaaugucag accugugaaa uucaguucu 60 cagcu 65 <210> 26 <211> 22 <212> RNA <213> Artificial Sequence <220> <223> miRNA-146a-3p <400> 26 ccugugaaau ucagucuuc ag 22 <210> 27 <211> 22 <212> RNA <213> Artificial Sequence <220> <223> miRNA-146a-5p <400> 27 ugagaacuga auuccauggg uu 22 <210> 28 <211> 99 <212> RNA <213> Artificial Sequence <220> <223> miRNA-148a <400> 28 agccaguuug gucuuuugag acaaaguucu gagacacucc gacucugagu augauagaag 60 ucagugcacu acagaacuuu gucucuagag gcugugguc 99 <210> 29 <211> 22 <212> RNA <213> Artificial Sequence <220> <223> miRNA-148a-3p <400> 29 ucagugcacu acagaacuuu gu 22 <210> 30 <211> 22 <212> RNA <213> Artificial Sequence <220> <223> miRNA-148a-5p <400> 30 aaaguucuga gacacuccga cu 22 <210> 31 <211> 65 <212> RNA <213> Artificial Sequence <220> <223> miRNA-185 <400> 31 agggauugga gagaaaggca guuccugaug guccccuccc aggggcuggc uuuccucugg 60 uccuu 65 <210> 32 <211> 21 <212> RNA <213> Artificial Sequence <220> <223> miRNA-185-3p <400> 32 aggggcuggc uuuccucugg u 21 <210> 33 <211> 22 <212> RNA <213> Artificial Sequence <220> <223> miRNA-185-5p <400> 33 uggagagaaa ggcaguuccu ga 22 <210> 34 <211> 67 <212> RNA <213> Artificial Sequence <220> <223> miRNA-190a <400> 34 cugugugaua uguuugauau auuagguugu uauuuaaucc aacuauauau caagcauauu 60 ccuace 67 <210> 35 <211> 22 <212> RNA <213> Artificial Sequence <220> <223> miRNA-190a-3p <400> 35 acuauauauc aagcauauuc cu 22 <210> 36 <211> 22 <212> RNA <213> Artificial Sequence <220> <223> miRNA-190a-5p <400> 36 ugauauguuu gauauauuag gu 22 <210> 37 <211> 66 <212> RNA <213> Artificial Sequence <220> <223> miRNA-193a <400> 37 gagagcuggg ucuuugcggg caagaugaga gugucaguuc aacuggccua caaaguccca 60 guccuc 66 <210> 38 <211> 22 <212> RNA <213> Artificial Sequence <220> <223> miRNA-193a-3p <400> 38 aacuggccua caaaguccca gu 22 <210> 39 <211> 22 <212> RNA <213> Artificial Sequence <220> <223> miRNA-193a-5p <400> 39 ugggucuuug cgggcaagau ga 22 <210> 40 <211> 85 <212> RNA <213> Artificial Sequence <220> <223> miRNA-196b <400> 40 aacuggucgg ugauuuaggu aguuuccugu uguugggauc caccuuucuc ucgacagcac 60 gacacugccu ucauuacuuc aguug 85 <210> 41 <211> 22 <212> RNA <213> Artificial Sequence <220> <223> miRNA-196b-3p <400> 41 ucgacagcac gacacugccu uc 22 <210> 42 <211> 22 <212> RNA <213> Artificial Sequence <220> <223> miRNA-196b-5p <400> 42 uagguaguuu cguguuguug gg 22 <210> 43 <211> 112 <212> RNA <213> Artificial Sequence <220> <223> miRNA-215 <400> 43 agcucucagc aucaacggug uacaggagaa ugaccuauga uuugacagac cgugcagcug 60 uguaugucug ucauucugua ggccaauauu cuguauguca cugcuacuua aa 112 <210> 44 <211> 21 <212> RNA <213> Artificial Sequence <220> <223> miRNA-215-3p <400> 44 ucugucauuc uguaggccaa u 21 <210> 45 <211> 21 <212> RNA <213> Artificial Sequence <220> <223> miRNA-215-5p <400> 45 augaccuaug auuugacaga c 21 <210> 46 <211> 110 <212> RNA <213> Artificial Sequence <220> <223> miRNA-223 <400> 46 ucuggccauc ugcaguguca cgcuccgugu auuugacaag cugaguugga cacucugugu 60 gguagagugu caguuuguca aauaccccaa guguggcuca ugccuaucag 110 <210> 47 <211> 22 <212> RNA <213> Artificial Sequence <220> <223> miRNA-223-3p <400> 47 ugucaguuug ucaaauaccc ca 22 <210> 48 <211> 22 <212> RNA <213> Artificial Sequence <220> <223> miRNA-223-5p <400> 48 ugucaguuug ucaaauaccc ca 22 <210> 49 <211> 79 <212> RNA <213> Artificial Sequence <220> <223> miRNA-369 <400> 49 gguacuugaa gggagaucga ccguguuaua uucgcuuggc ugacuucgaa uaauacaugg 60 uugaucuuuu cucaguauc 79 <210> 50 <211> 21 <212> RNA <213> Artificial Sequence <220> <223> miRNA-369-3p <400> 50 aauaauacau gguugaucui u 21 <210> 51 <211> 22 <212> RNA <213> Artificial Sequence <220> <223> miRNA-369-5p <400> 51 agaucgaccg uguuauauuc gc 22 <210> 52 <211> 73 <212> RNA <213> Artificial Sequence <220> <223> miRNA-483 <400> 52 gagggggaag acgggagaag agaagggagu gguuuuuggg ugccucacuc cuccccuccc 60 gucuuguucu cuc 73 <210> 53 <211> 21 <212> RNA <213> Artificial Sequence <220> <223> miRNA-483-3p <400> 53 ucacuccucc ccucccgucu u 21 <210> 54 <211> 22 <212> RNA <213> Artificial Sequence <220> <223> miRNA-483-5p <400> 54 aagacgggag aagagaaggg ag 22 <210> 55 <211> 92 <212> RNA <213> Artificial Sequence <220> <223> miRNA-615 <400> 55 ucgggagggg cgggaggggg guccccggug cucggaucuc gagggugcuu auuguucggu 60 ccgagccugg gcccccc uccccccauc cc 92 <210> 56 <211> 22 <212> RNA <213> Artificial Sequence <220> <223> miRNA-615-3p <400> 56 uccgagccug ggucucccuc uu 22 <210> 57 <211> 22 <212> RNA <213> Artificial Sequence <220> <223> miRNA-615-5p <400> 57 gggggucccc ggugcucgga uc 22 <210> 58 <211> 65 <212> RNA <213> Artificial Sequence <220> <223> miRNA-155 <400> 58 cuguuaaugc uaauugugau agggguuuug gccucugacu gacuccuacc uguuagcauu 60 aac 65 <210> 59 <211> 21 <212> RNA <213> Artificial Sequence <220> <223> miRNA-155-3p <400> 59 cuccuaccug uuagcauuaa c 21 <210> 60 <211> 23 <212> RNA <213> Artificial Sequence <220> <223> miRNA-155-5p <400> 60 uuaaugcuaa uugugauagg ggu 23 <210> 61 <211> 76 <212> RNA <213> Artificial Sequence <220> <223> miRNA-203 <400> 61 gccuggucca gugguucuug acaguucaac aguucuguag cacaauugug aaauguuuag 60 gaccacuaga cccggc 76 <210> 62 <211> 22 <212> RNA <213> Artificial Sequence <220> <223> miRNA-203-3p <400> 62 gugaaauguu uaggaccacu ag 22 <210> 63 <211> 22 <212> RNA <213> Artificial Sequence <220> <223> miRNA-203-5p <400> 63 agugguucuu gacaguucaa ca 22 <210> 64 <211> 94 <212> RNA <213> Artificial Sequence <220> <223> miRNA-434 <400> 64 ucgacucugg guuugaacca aagcucgacu caugguuuga accauuacuu aauucguggu 60 uugaaccauc acucgacucc ugguucgaac cauc 94 <210> 65 <211> 22 <212> RNA <213> Artificial Sequence <220> <223> miRNA-434-3p <400> 65 uuugaaccau cacucgacuc cu 22 <210> 66 <211> 22 <212> RNA <213> Artificial Sequence <220> <223> miRNA-434-5p <400> 66 gcucgacuca ugguuugaac ca 22 <210> 67 <211> 83 <212> RNA <213> Artificial Sequence <220> <223> miRNA-92b <400> 67 ggugggcggg agggacggga cguggugcag uguuguucuu uccccugcca auauugcacu 60 cgucccggcc uccggccccc ucg 83 <210> 68 <211> 22 <212> RNA <213> Artificial Sequence <220> <223> miRNA-92b-3p <400> 68 uauugcacuc gucccggccu cc 22 <210> 69 <211> 24 <212> RNA <213> Artificial Sequence <220> <223> miRNA-92b-5p <400> 69 agggacggga cguggugcag uguu 24 <210> 70 <211> 22 <212> RNA <213> Artificial Sequence <220> <223> miRNA-136-5p primer <400> 70 acuccauuug uuuugaugau gg 22 <210> 71 <211> 21 <212> RNA <213> Artificial Sequence <220> <223> miRNA-337-3p primer <400> 71 uucagcuccu auaugaugcc u 21 <210> 72 <211> 22 <212> RNA <213> Artificial Sequence <220> <223> miRNA-127-3p primer <400> 72 ucggauccgu cugagcuugg cu 22 <210> 73 <211> 22 <212> RNA <213> Artificial Sequence <220> <223> miRNA-34a-5p primer <400> 73 uggcaguguc uuagcugguu gu 22 <210> 74 <211> 22 <212> RNA <213> Artificial Sequence <220> <223> miRNA-146a-5p primer <400> 74 ugagaacuga auuccauggg uu 22 <210> 75 <211> 22 <212> RNA <213> Artificial Sequence <220> <223> miRNA-148a-3p primer <400> 75 ucagugcacu acagaacuuu gu 22 <210> 76 <211> 21 <212> RNA <213> Artificial Sequence <220> <223> miRNA-411-5p primer <400> 76 uaguagaccg uauagcguac g 21 <210> 77 <211> 22 <212> RNA <213> Artificial Sequence <220> <223> miRNA-92b-3p primer <400> 77 uauugcacuc gucccggccu cc 22 <210> 78 <211> 23 <212> RNA <213> Artificial Sequence <220> <223> miRNA-155-5p primer <400> 78 uuaaugcuaa uugugauagg ggu 23 <210> 79 <211> 22 <212> RNA <213> Artificial Sequence <220> <223> miRNA-203-3p primer <400> 79 gugaaauguu uaggaccacu ag 22 <210> 80 <211> 22 <212> RNA <213> Artificial Sequence <220> <223> miRNA-434-3p primer <400> 80 uuugaaccau cacucgacuc cu 22 <210> 81 <211> 22 <212> RNA <213> Artificial Sequence <220> <223> miRNA-434-5p primer <400> 81 gcucgacuca ugguuugaac ca 22

Claims (17)

A composition for diagnosing muscle aging, comprising an agent that measures the level of expression of miRNA-34 or a fragment thereof.
The composition of claim 1, wherein said miRNA-34 is miRNA-34a or miRNA-34c.
3. The composition of claim 2, wherein the miRNA-34a comprises the nucleotide sequence of SEQ ID NO:
3. The composition of claim 2, wherein the miRNA-34c comprises the nucleotide sequence of SEQ ID NO: 16.
3. The composition of claim 2, wherein the fragment of miRNA-34a comprises the nucleotide sequence of SEQ ID NO: 14 or 15.
3. The composition of claim 2, wherein the fragment of miRNA-34c consists of the nucleotide sequence of SEQ ID NO: 17 or 18.
The composition of claim 1, wherein the agent that measures the level of expression of miRNA-34 or fragment thereof comprises a primer or a probe that specifically binds to the miRNA-34 or fragment thereof.
8. The composition of claim 7, wherein the primer specifically binding to the fragment of miRNA-34 comprises the nucleotide sequence of SEQ ID NO: 73.
The method according to claim 1, wherein the miRNA-136, miRNA-337, miRNA-127, miRNA-411, miRNA-143, miRNA-144, miRNA- one or more miRNAs selected from the group consisting of miRNA-196, miRNA-215, miRNA-223, miRNA-369, miRNA-483, miRNA-615, miRNA-155, miRNA- Wherein the composition further comprises an agent that measures the level of expression of the fragment.
9. A kit for diagnosing muscle aging, comprising the composition of any one of claims 1 to 9.
11. The kit for diagnosing muscle aging according to claim 10, wherein the kit is an RT-PCR kit or a gene chip kit.
(a) determining the level of expression of miRNA-34 or fragment thereof in a biological sample separated from the individual; And
(b) determining that muscle aging has progressed if the expression level of the measured miRNA-34 or fragment thereof is increased above the expression level of the corresponding miRNA-34 or fragment thereof in a normal control sample. A method for providing information for a user.
13. The method of claim 12, wherein said miRNA-34 is miRNA-34a or miRNA-34c.
The method according to claim 12, wherein the step of measuring the expression level of the miRNA-34 comprises the steps of RT-PCR, competitive RT-PCR, real-time RT- time quantitative RT-PCR, RNase protection method, Northern blotting or gene chip.
14. The method of claim 12, wherein the step (a) comprises the steps of: (a) culturing the miRNA-136, miRNA-337, miRNA-127, miRNA-411, miRNA- 190, miRNA-193, miRNA-196, miRNA-215, miRNA-223, miRNA-369, miRNA-483, miRNA-615, miRNA-155, miRNA-203, miRNA- RTI ID = 0.0 &gt; miRNA &lt; / RTI &gt; or fragment thereof.
18. The method of claim 15, wherein the miRNA-136, miRNA-337, miRNA-127, miRNA-411, miRNA-143, miRNA- 144, miRNA- 148, miRNA- 190, miRNA- 434 &lt; / RTI &gt; is less than the expression level of the corresponding miRNA or fragment thereof in a normal control sample.
18. The method of claim 15, wherein, in step (b), miRNA-146, miRNA-185, miRNA-196, miRNA-215, miRNA-223, miRNA-369, miRNA- 203 and miRNA-92 is determined to be higher than the expression level of the corresponding miRNA or fragment thereof in a normal control sample, determining that the aging of the muscle has progressed Way.

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