KR101520545B1 - Composition for diagnosing muscle inflammation and uses thereof - Google Patents

Abstract

The present invention provides information for the diagnosis of muscle inflammation comprising a composition for diagnosing muscle inflammation comprising an agent for measuring the level of miR-494, a kit for diagnosing muscle inflammation comprising said composition, and a step of measuring the level of miR-494 And screening a substance capable of lowering the level of miR-494. The present invention also relates to a method for screening a drug for preventing or treating muscle inflammation. MiR-494 provided in the present invention can be used as a diagnostic marker of muscle inflammation that causes insulin resistance accompanying the onset of muscle inflammation, and thus it can be widely used for effective diagnosis and treatment of muscle inflammation.

Description

Composition for diagnosing muscle inflammation and uses thereof

More particularly, the present invention relates to a composition for diagnosing muscle inflammation comprising an agent for measuring the level of miR-494, a kit for diagnosing muscle inflammation comprising said composition, a kit for diagnosing muscle inflammation comprising miR-494 A method for screening a substance that can lower the level of miR-494, and a method for screening an agent for preventing or treating muscle inflammation, comprising the steps of: .

Weight bearing or resistance training exercises result in a widespread response in the active muscles. By muscle expansion and strain, anabolic signaling pathways activated by growth factors that are locally synthesized and circulating in the bloodstream can stimulate muscle growth, but excessive muscle activity causes muscle cell destruction Inflammatory disease can be caused thereby. Inflammation is a local reaction to tissue damage or destruction. Rapid casting is characterized by pain, fever, flushing, swelling and loss of function. In particular, the inflammatory response in muscle tissue counteracts muscle protein synthesis, and most people involved in intense exercise, including resistance training, experience mild or excessive physical discomfort.

Polymyositis, one of the inflammatory diseases of these muscles, is a disease in which the symptoms of limb weakness near the body are accompanied by an increase in muscle enzymes, signs of EMG abnormality, abnormality in muscle histology, etc. . Until now, a method to cure multiple myositis has not been developed because even though inflammation symptoms and immune symptoms are treated, there is a temporary effect, but it recurs again with the passage of time.

As a result, various studies have been conducted to identify the cause of recurrence of multiple myositis, and it has been reported that insulin resistance accompanying myositis is a major cause of recurrence. Insulin resistance refers to a symptom in which insulin functions to lower blood glucose levels are lowered so that glucose accumulates in the cells without consuming glucose. In particular, when inflammation occurs in muscle cells, insulin resistance appears, and the insulin resistance accumulates Glucose causes symptoms of worsening of muscle cell inflammation.

In order to more effectively treat muscle inflammation accompanied by such insulin resistance, it is expected that it will be effective to diagnose the onset of muscle inflammation at the early stage of muscle inflammation and prevent excessive accumulation of glucose. However, There has not been developed an early diagnosis method.

Under these circumstances, the present inventors have tried to develop a method for early diagnosis of muscle inflammation, which is one of the main causes of muscle damage, as a result of which miR-494 causes insulin resistance accompanying muscle inflammation, By detecting the level of miR-494, it was confirmed that muscle inflammation can be diagnosed early, and the present invention has been completed.

It is an object of the present invention to provide a composition for diagnosing muscle inflammation comprising an agent for measuring the level of miR-494.

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

Yet another object of the present invention is to provide a method for providing information for diagnosis of muscle inflammation comprising measuring the level of miR-494.

It is still another object of the present invention to provide a screening method for a prophylactic or therapeutic agent for muscle inflammation comprising the step of screening a substance capable of lowering the level of miR-494.

The present inventors have focused on insulin resistance while carrying out various studies to develop a method for more effectively detecting muscle inflammation, which is one of the main causes of muscle damage. Insulin resistance refers to a symptom in which insulin functions to lower blood glucose levels are lowered so that glucose accumulates in the cells without consuming glucose. In particular, when inflammation occurs in muscle cells, insulin resistance appears, and the insulin resistance accumulates Glucose causes symptoms of worsening of muscle cell inflammation. Thus, the causative agent that induces insulin resistance, which causes deterioration of muscle inflammation, can be a target for effectively detecting muscle inflammation, and thus, the causative agent was sought.

Accordingly, the inventors of the present invention found that treatment of TNF-a, which is involved in muscle inflammation, in differentiated root canal causes symptoms similar to those of chronic inflammation of the root canal, and that insulin resistance occurs in the root canal induced by chronic inflammation. It was confirmed that insulin resistance occurs when chronic inflammation is induced by treatment with TNF-α. In order to discover miRNAs involved in inflammation in the root canal induced by chronic inflammation by the treatment of TNF- ?, total RNAs obtained in inflamed root canals were treated with miRNAs whose expression amount increased as the onset of inflammation progressed Were selected. As a result, it was confirmed that expression levels of miR-494 and miR-10b increased as the onset of inflammation progressed. In particular, miR-494 has been shown to be elevated in the early stages of inflammation and is likely to be substantially involved in inflammation induction since it has been confirmed that such levels are maintained constantly.

To confirm this, miR-494 expression-induced myoblasts were treated with insulin and the level of phosphorylation of proteins involved in the Akt signaling pathway involved in signal transduction of insulin was measured. As a result, in the case of insulin treatment normally, the phosphorylation level of the protein contained in the Akt signaling pathway should increase. However, in the miR-494 expression-induced myoblasts, the phosphorylation level of the protein contained in the Akt signaling pathway , Suggesting that miR-494 attenuates insulin action by downregulating Akt activity. However, it was confirmed that miR-494 had no significant effect on TNF-a induced inflammatory action other than insulin signal transduction.

Thus, miR-494 can be used as a diagnostic marker for muscle inflammation accompanied by insulin resistance, since it specifically plays a role in inducing insulin resistance occurring in muscle inflammation.

As one embodiment for achieving the above object, the present invention provides a composition for diagnosing muscle inflammation comprising an agent for measuring the level of miR-494.

The term "miR-494 " of the present invention means a polynucleotide having a nucleotide sequence shown below in SEQ ID NO: 1, which is formed by cleaving pre-miR-494 (SEQ ID NO: 2) in a cell. Biologically, it can be used to control TRAIL-induced apoptosis in non-small cell lung cancer, to regulate the endometrial receptivity of the endometrium, to regulate nucleolin expression, to inhibit the proliferation of A549 lung cancer cells It is known to induce aging.

miR-494: 5'-UGAAACAUACACGGGAAACCUC-3 '(SEQ ID NO: 1)

pre-miR-494: 5'-GAUACUCGAAGGAGAGGUUGUCCUUGUUUUCUUCUCUUUAUUUAUGAUGAAACAUACACGGGAAACCUCUUUUUUAGUAUC-3 '(SEQ ID NO: 2)

The term "diagnosing" of the present invention means identifying the presence or characteristic of a pathological condition. In the present invention, the diagnosis can be interpreted as a measurement of the level of miR-494 from muscle cells or muscle tissue obtained through biopsy to confirm the onset of muscle inflammation.

The term "agent for measuring the level of miR-494" of the present invention means a preparation used for determining the expression of miR-494 contained in a sample, preferably RT-PCR, competitive RT-PCR Which is used in methods such as competitive RT-PCR, real-time RT-PCR, RNase protection assay (RPA), Northern blotting and DNA chip analysis, But it is not particularly limited to these.

The term "primer" of the present invention refers to a nucleic acid sequence having a short free 3 'hydroxyl group, capable of forming base pairs with a complementary template and having a starting point for template strand copying ≪ / RTI > 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.

The term "probe" of the present invention means a nucleic acid fragment such as RNA or DNA corresponding to a short period of a few nucleotides or several hundreds of nucleotides capable of specifically binding to a gene or an mRNA. The oligonucleotide probe, For example, in the form of a single stranded DNA probe, a double stranded DNA probe, an RNA probe, or the like, and may be labeled for easier detection.

According to one embodiment of the present invention, when 2 ng / ml of TNF-a is treated for 2 hours and 4 days in total RNA obtained from a root canal induced by chronic inflammation by the treatment of TNF-a, the onset of inflammation is progressed The expression levels of miR-494 and miR-10b increased with the onset of inflammation, and miR-494 showed a high level at the onset of inflammation. (Fig. 2). In addition, insulin treatment of miR-494-induced myoblasts and measurement of the phosphorylation level of the protein involved in the Akt signaling pathway involved in insulin signal transduction revealed that the miR-494 expression- It was confirmed that the phosphorylation level of the protein contained in the Akt signal transduction pathway was decreased in the parent cells.

As described above, since miR-494 increased the expression level as the onset of inflammation progressed and the insulin action was slowed down by downregulating Akt activity, the agent for measuring the level of miR-494 of the present invention can be used to prevent muscle inflammation It can be diagnosed effectively.

In another aspect of the present invention, there is provided a kit for diagnosing muscle inflammation comprising the composition for diagnosing muscle inflammation.

The kit of the present invention measures the level of miR-494 from a sample of a patient suspected of developing muscle inflammation, preferably muscle cells or muscle tissue obtained by performing biopsy on the patient, to determine whether or not insulin resistance has occurred May be used to diagnose muscle inflammation, including, but not limited to, primers or probes for measuring the level of miR-494, as well as one or more other component compositions, solutions or devices suitable for the assay It is possible.

As a specific example, a kit for measuring the level of miR-494 of the present invention may be a kit containing necessary elements necessary for performing 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 the necessary elements necessary for performing DNA chip analysis. The kit for DNA 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, an enzyme, and the like 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 another aspect, the present invention provides a method for providing information for diagnosing muscle inflammation using the diagnostic composition or kit, the method comprising the steps of: (a) detecting miR-1 from a biological sample of a patient suspected of having muscle inflammation; Measuring the level of 494; And (b) comparing the level of the measured miR-494 to the level measured from a sample of a normal control, and determining that the muscle inflammation has occurred if the level is significantly increased. At this time, muscle cells or muscle tissue obtained through biopsy may be used as the sample, and the method of measuring the level of miR-494 is the same as described above.

In another aspect of the present invention, the present invention provides a method for preventing muscle inflammation, comprising administering a candidate substance expected to prevent or treat the onset of muscle inflammation and measuring the level of miR-494 Or a method for screening a therapeutic agent.

Specifically, the method for screening an agent for preventing or treating muscle inflammation according to the present invention comprises the steps of: (a) measuring the level of miR-494 from a sample of an experimental animal in which muscle inflammation is a control; (b) administering a candidate agent expected to be able to treat muscle inflammation in said experimental animal; (c) measuring the level of miR-494 from a sample of an experimental animal to which the candidate agent is administered; And (d) comparing the level of miR-494 measured in the control with that measured in the experimental group, when the level of the miR-494 measured in the experimental group is lower than that measured in the control, the candidate substance is prevented from developing the muscle inflammation , Or a therapeutic agent for treating muscle inflammation.

The level of miR-494 of the present invention in a cell is measured in the absence of a candidate substance capable of preventing or treating muscle inflammation and the level of the miR-494 of the present invention is measured in the presence of the candidate substance, After comparison, a substance that reduces the level of the miR-494 of the present invention in the absence of the candidate substance in the absence of the candidate substance can be predicted as a prophylactic agent for muscle inflammation.

MiR-494 provided in the present invention can be used as a diagnostic marker of muscle inflammation that causes insulin resistance accompanying the onset of muscle inflammation, and thus it can be widely used for effective diagnosis and treatment of muscle inflammation.

Figure 1 shows the induction of inflammation and insulin resistance by treatment with TNF-a in C ₂ C ₁₂ root canal. (AC) was treated with ₂ ng / ml or 20 ng / ml TNF-α in C ₂ C ₁₂ cells treated with serum-free DMEM medium for 5 days after the differentiation and immunoblot analysis using cell lysate (D) quantitatively indicates NF-κB phosphorylation level, (E) pretreats 2 or 20 ng / ml TNF-α in C ₂ C ₁₂ root canal for 2 or 4 hours, Insulin is treated and insulin-induced p-Akt levels are measured by immunoblot analysis. (F) quantitatively indicates Akt phosphorylation level.
Figure 2 shows the expression levels of each microRNA selected in the C ₂ C ₁₂ root canal treated with TNF-α.
Figure 3 shows the effect of miR-494 on the insulin signaling pathway. The mimic of miR-494 was overexpressed in the cells, the cells were blocked for 4 hours and then treated with 0, 10 and 100 nM of insulin for 20 minutes. The levels of p-Akt (Ser473 and Thr308), p-AS160, p-p70S6K and p-GSK-3alpha were measured in C ₂ C ₁₂ myoblasts (A) and CHOIR / IRS-1 cells (D); GSK-3α / β, AS160 and p70S6K in C ₂ C ₁₂ myoblasts (B) and CHOIR / IRS-1 cells (E) (C) shows the results of measuring the level of p-IRS1 (Tyr608) in insulin-treated C ₂ C ₁₂ myoblasts. GAPDH and actin were used as a control.
Figure 4 shows the effect of miR-494 on predicted targets involved in inflammatory signaling and insulin signaling. (A) transfected a miR-494 mimetic polynucleotide into C ₂ C ₁₂ myoblasts and after 48 hours, the cells were stopped feeding, treated with TNF-α, and subjected to cell lysate , phosphor-NF-虜 B and I 虜 B, respectively. (B) quantitatively indicates the level of phosphorylation of NF-kB and IkB.

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  One: Root canal  For chronic inflammation symptoms TNF Effect of -α

To test the effect of inflammation on insulin resistance of muscle cells, we differentiated C ₂ C ₁₂ cells into root canals.

Specifically, mouse C ₂ C ₁₂ myoblasts expressing human insulin receptor were cultured at 37 ° C and 5 ° C using growth medium (DMEM containing 10% FBS, 1% penicillin / streptomycin and HAM's F-12 medium) It was cultured until saturation in% CO ₂ conditions. Then, differentiation medium (DMEM containing 2% horse serum) was cultured for 10 days instead of growth medium and changed into root canals by alternating every two days.

Then, the level of MyoD and myogenin, which are differentiation markers of muscle cells, was measured in the root canal. Specifically, the differentiated root canal was treated with lysis buffer (20 mM Tris pH 7.5, 5 mM EDTA, 10 mM Na ₄ P ₂ O ₇ , 100 mM NaF, 2 mM Na ₃ VO ₄ , 1% NP-40, 1 mM PMSF, 10 [mu] g / ml aprotinin and 10 [mu] g / ml leupeptin) and 20 [mu] g thereof was analyzed by SDS-PAGE and immunoblotting was performed using an antibody against myogenin and myoD. The resulting membrane was visualized by applying it to a ChemiDoc imaging system (Bio-Rad) and quantitated using Image Lab software (Bio-Rad) (Fig. 1a). As a result, as shown in FIG. 1A, since myogenin and myoD, which are used as main markers at the time of differentiation of muscle cells, were increased, it was found that the differentiation into muscle cells proceeded normally.

In addition, NF-κB phosphorylation level increased to 1.5-fold at 60 min when treated with 2 ng / ml TNF-α in differentiated cells (5 days), and the level of IκB-α protein (Fig. 1B). At this time, the level of p-NF-κB was not significantly increased even when the amount of TNF-α treated was increased to 20 ng / ml.

In addition, protein levels of NF-κB did not change with time, but levels of IκB-α protein were reduced by TNF-α treatment for 10 or 30 minutes, and 60 minutes after treatment with different treatments And recovered to the original amount of the control (Fig. 1B).

On the other hand, to study the chronic effect of TNF-a on inflammatory signals, the inventors treated TNF-a for more than 4 days. As a result, phosphorylation of NF-kB increased early (10 and 30 min) and gradually decreased to the basal level according to the time of TNF-a treatment (Figs. 1C and 1D). In addition, phosphorylation of p38 and ERK-1/2 was altered during TNF-a treatment in root canal during the test period.

Example  2: Root canal  About Insulin Resistance TNF Effect of -α

Since TNF- [alpha] is a major mediator of inflammation that is considered to be a trigger for insulin resistance, the present inventors have studied whether insulin signaling in C ₂ C ₁₂ root canal is affected by TNF- [alpha].

As shown in FIG. 1 (e), phosphorylation of Akt at the Ser473 site was significantly increased in the insulin-stimulated C ₂ C ₁₂ root canal compared to the control group. In the case of pretreatment with 2 ng / ml TNF-α for 2 hours, . In the case of treatment with 2 ng / ml of TNF-α for 4 days, Akt phosphorylation was further reduced at the Ser473 site, which was about 45% of that of the control (FIGS. 1e and 1f). Furthermore, treatment of TNF-a with a high level (20 ng / ml) confirmed that p-Akt levels were further reduced at the Ser473 and Thr308 sites.

As shown in the results of Examples 1 and 2, it was confirmed that the phosphorylation level of NF-κB and the phosphorylation level of Akt contained in the inflammatory signal pathway were different from each other. , It was found that insulin resistance induced by acute and chronic inflammation is regulated by other regulators that affect the phosphorylation level of Akt, not NF-κB.

Example  3: TNF -α involved in inducible inflammation miRNA

In order to study the mechanism of action of inflammatory mediating insulin resistance, the present inventors investigated miRNAs that were not regulated in inflammatory conditions induced by TNF- [alpha], in order to imitate low levels of chronic inflammatory conditions, 2 ng / ml TNF-α-treated C ₂ C ₁₂ root canal was subjected to miRNA microarray analysis (miRbase v9.0).

Specifically, five days after differentiation, supplemented medium containing 2 ng / ml TNF-α was treated every 2 days for 4 days in C ₂ C ₁₂ root canal. Total RNA was then obtained in C ₂ C ₁₂ canaliculus cells using TRIzol.

The obtained total RNA was subjected to a reverse transcription reaction using a TaqMan 占 MicroRNA Reverse Transcription Kit (Invitrogen) to obtain cDNA, and the obtained cDNA was amplified using TaqMan 占 MicroRNA Assays (Invitrogen) using Mx3005P qPCR System (Agilent Technologies, USA) ) And GeneChip (R) miRNA Array (Affymetrix) to determine the expression levels of the respective miRNAs contained in the total RNA. At this time, snoRNA202 and U6 snRNA were used as internal controls to regulate the amount of miRNA expression, and the relative expression levels of miRNAs were compared using the 2- ^{DELTA Ct} method. The obtained results were scanned using Genechip Array scanner 3000 7G (Affymetrix), and the data obtained by scanning were analyzed using R software (FIG. 2). As a result, as shown in FIG. 2, it was confirmed by qRT-PCR analysis that miR-494 was doubled after treatment with 20 ng / ml of TNF-α for 2 hours or 4 days (FIG. In addition, the expression levels of miR-467b (FIG. 2b) and miR-101b (FIG. 2c) were also increased by treatment of TNF-α, but miR-331 expression levels were not changed (FIG.

Example  4: About insulin action miR Effects of -494

Since TNF-α is to induce insulin resistance and miR-494 expression, the inventors have found that miR-494 is to determine whether or not to adjust the insulin action, miR-494 is a transition of C ₂ C for ₁₂ Muscle Cell 10, or 100 nM insulin. At this time, the transfer efficiency of miR-494 was confirmed to be about 80%.

In miR-494 overexpressed cells, the phosphorylation of the downstream targets of AS160 and p70S6K of Akt was significantly down regulated (Fig. 3a) and the levels of insulin-stimulated Akt phosphorylation at the Ser473 and Thr308 sites were reduced to the lowest levels, The level of p-GSK-3? /? was not changed statistically (Fig. 3b). Also, tyrosine phosphorylation of IRS-1 was not altered at the 608 site (FIG. 3C).

Next, the effect of miR-494 on insulin signaling in CHOIR / IRS1 cells was examined. As a result, phosphorylation of AS160 and p70S6K was significantly down-regulated by overexpression of miR-494 in the same manner as C ₂ C ₁₂ source cells, and miR-494 was found to be decreased in the transfected CHOIR / IRS1 cells The p-Akt level of GSK-3? /? And T308 sites was significantly decreased.

Taken together, these results indicate that miR-494 attenuates insulin action by downregulating Akt activity.

Example  5: TNF -α inducibility NF -KB activity miR Effects of -494

In order to evaluate whether miR-494 affects TNF-α induced inflammation, C ₂ C ₁₂ myoblasts were grown to 50% saturation and Lipofectamine RNAiMAX reagent and Lipofectamine 2000 (Invitrogen) Were used to transduce miR-494 mimic polynucleotides, followed by stopping feeding overnight, and then treated with 2 or 20 ng / ml TNF-a for 30 min.

As shown in FIGS. 4A and 4B, the TNF-α induced phosphorylation level of NF-κB and the protein level of IκB were not changed by overexpression of miR-494 compared to the control.

Thus, it was found that miR-494 had no significant effect on TNF-a induced inflammation.

<110> Gachon University of Industry-Academic cooperation Foundation <120> Composition for diagnosing muscle inflammation and uses thereof <130> PA130474 / KR <160> 2 <170> Kopatentin 2.0 <210> 1 <211> 22 <212> RNA <213> Artificial Sequence <220> <223> miR-494 <400> 1 ugaaacauac acgggaaacc uc 22 <210> 2 <211> 81 <212> RNA <213> Artificial Sequence <220> <223> pre-miR-494 <400> 2 gauacucgaa ggagagguug uccguguugu cuucucuuua uuuaugauga aacauacacg 60 ggaaaccucu uuuuuaguau c 81

Claims (8)

A composition for diagnosing muscle inflammation, comprising an agent that measures the level of miR-494.
The method according to claim 1,
Wherein the agent for measuring the level of miR-494 comprises a primer or a probe specifically binding to the miR-494.
The method according to claim 1,
Wherein said miR-494 induces insulin resistance following muscle inflammation.
A kit for diagnosing muscle inflammation, comprising the composition of any one of claims 1 to 3.
5. The method of claim 4,
Wherein the kit is an RT-PCR kit or a DNA chip kit.
(a) measuring the level of miR-494 from a biological sample isolated from a patient suspected of having a muscle inflammation; And
(b) comparing the level of the measured miR-494 to a level measured from a sample of a normal control, and determining that the muscle inflammation is increased if increased, a method for providing information for diagnosis of muscle inflammation .
The method according to claim 6,
The level of miR-494 was determined by RT-PCR, competitive RT-PCR, real-time quantitative RT-PCR, RNase protection assay RNase protection method, Northern blotting or DNA chip technology assay.
(a) measuring the level of miR-494 from a sample of an experimental animal in which muscle inflammation is a control;
(b) administering a candidate agent expected to be able to treat muscle inflammation in said experimental animal;
(c) measuring the level of miR-494 from a sample of an experimental animal to which the candidate agent is administered; And
(d) when the level of miR-494 measured in the control group is lower than that measured in the test group, as measured in the test group, compared to that measured in the test group, the candidate substance may be used as a preventive measure against the onset of muscle inflammation Or a pharmaceutically acceptable salt, solvate, or prodrug thereof, as a therapeutic agent for the treatment of inflammatory diseases or inflammation.

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