KR101209129B1 - Composition for proliferation, differentiation, and anti-aging of mesenchymal stem cell using miRNA control - Google Patents

Abstract

The present invention relates to a composition for promoting proliferation and differentiation of mesenchymal stem cells using miRNA regulation and inhibiting aging, and more specifically, proliferation and differentiation of stem cells containing an inhibitor of expression of miR-486 or miR-598 as an active ingredient. It relates to a composition for promoting and inhibiting aging.
According to the present invention, by regulating the expression of specific miRNA, the expression of SIRT1, which plays an important role in the aging of human stromal cells (including MSC), can regulate cell growth, differentiation, inhibit cell aging, and in diabetic patients. Angiogenesis of mesenchymal stem cells with reduced function can be increased. Therefore, it can be used in the development of technology to control the proliferation, differentiation and therapeutic effect of mesenchymal stem cells using miRNA to control the expression of SIRT1.

Description

Composition for proliferation, differentiation promotion and anti-aging of mesenchymal stem cells using miRNA control {Composition for proliferation, differentiation, and anti-aging of mesenchymal stem cell using miRNA control}

The present invention relates to a composition for promoting proliferation and differentiation of mesenchymal stem cells using miRNA regulation and inhibiting aging, and more specifically, proliferation and differentiation of stem cells containing an inhibitor of expression of miR-486 or miR-598 as an active ingredient. It relates to a composition for promoting and inhibiting aging.

Aging is a phenomenon in which the body's physical functions deteriorate over time. Aging of the cell appears to lose its ability to divide. Stem cells are also a cell population, and gradually lose their function. In general, the aging of cells depends on the Rb (retinoblastoma) protein-dependent aging mechanism [Kiyono T et al., Nature. 1998 Nov 5; 396 (6706): 84-8] and aging caused by the loss of telomeres, which play a role in protecting chromosomes [Karlseder J et al., Science. 2002 Mar 29; 295 (5564): 2446-9], Aging by Antioxidant Stress [Sohal RS et al., Science. 1996 Jul 5; 273 (5271): 59-63, Aging due to DNA damage [de Boer J et al., Science. 2002 May 17; 296 (5571): 1276-9. These DNA damage signaling and stress signaling mechanisms share much of the tumor suppression signaling that inhibits cell deformation that may be caused by external environmental factors, and many related genes are being studied.

In particular, Sirtuin1 (SIRT1) belongs to genes that regulate chronic inflammation, cancer and aging.In 2000, MIT professor Leonard Guarente said that a yeast gene called SIR2 acts as a master switch to regulate energy processing. Found and published in Nature [Imai S et al., Nature. 2000 Feb 17; 403 (6771): 795-800]. The paper demonstrated that cells with SIR2 gene activation live longer than average life, whereas cells lacking SIR2 gene have short life spans. Subsequently, over-expression of SIR2 has been shown to extend lifespan in small nematodes and fruit flies [Guarente L et al., Nature. 2000 Nov 9; 408 (6809): 255-262; Tissenbaum HA et al., Nature. 2001 Mar 8; 410 (6825): 227-230], Dr. Pere Puigserver of Spain, who was studying at John Hopkins Medical School in 2005, reported to Nature that the mammalian SIRT1 gene is equivalent to SIR2 and has the same effect. Rodgers JT et al., Nature. 2005 Mar 3; 434 (7029): 113-118. Human SIRT1 is a NAD ⁺ dependent protein / histone deacetylase that deacetylates histones and nonhistone proteins to regulate inflammatory mediators and is known as an anti-aging and anti-inflammatory protein [Imai S et al., Nature. 2000 Feb 17; 403 (6771) 795-800. Recently, studies have shown that SIRT1 plays a positive role in stress, metabolism, apoptosis and premature ejaculation [Alcendor RR et al., Circ Res. 2007 May 25; 100 (10): 1512-1521; Kennedy BK et al., Cell. 2005 Nov 18; 123 (4): 548-550.

Meanwhile, miRNA (microRNA) is a 21-25 nt single-stranded RNA molecule, which binds to 3'-UTR of mRNA (messengerRNA) and controls eukaryotic gene expression [Bartel DP et al., Cell. 2004 Jan 23; 116 (2): 281-297]. The production of miRNA is made by Drosha (RNase III type enzyme) into a pre-miRNA having a stem-loop structure, migrated into the cytoplasm, cleaved by Dicer, and made into miRNA [Kim VN et al., Nat Rev Mol Cell Biol. 2005 May; 6 (5): 376-385. The miRNAs thus produced are involved in development, cell proliferation and death, fat metabolism, and tumorigenesis by regulating the expression of target proteins [Wienholds E et al., Science. 2005 Jul 8; 309 (5732): 310-311; Nelson P et al., Trends Biochem Sci. 2003 28; 534-540; Lee RC et al., Cell. 1993 75: 843-854; Esquela-Kerscher A et al., Nat Rev Cancer. 2006 6: 259-269.

Mesenchymal Stem Cells (MSCs) are easily proliferated in vitro and can be differentiated into various cell types (fat cells, chondrocytes, muscle cells, bone cells) [Caplan, A.I. et al., J. Orthop. Res. 1991 9: 641-650; Beresford J.N. et al., J. Cell Sci. 1992 102: 341-351; Pittenger M.F. et al., Science. 1999 284: 143-147; Patricia A.Z. et al., Tissue engineering. 2001 7: 211-227. Therefore, mesenchymal stem cells can be used as a useful target in gene therapy and cell therapy, and understanding of the growth mechanism and aging regulation of miRNA in mesenchymal stem cells is important for cell therapy using them.

However, the function of miRNA on the proliferation and senescence of mesenchymal stem cells has not been known so far.

Accordingly, the present inventors have made a thorough research to overcome the problems of the prior art, when inhibiting the expression of miR-486 or miR-598 in mesenchymal stem cells, can control the senescence and proliferation of mesenchymal stem cells It was confirmed that the present invention was completed.

Therefore, a main object of the present invention is to provide a composition for promoting proliferation, differentiation and senescence of mesenchymal stem cells having a miRNA inhibitory effect.

According to an aspect of the present invention, the present invention provides a composition for inhibiting proliferation, differentiation and senescence of stem cells comprising an expression inhibitor of miR-486 or miR-598 as an active ingredient.

miRNA (microRNA) is a single stranded RNA molecule of 21-25 nucleotides (nt) and is a regulator that controls gene expression in eukaryotes. This miRNA consists of two stages of processing. The first miRNA transcript (primary miRNA) is made into a 70-90 nt stem-loop structure, or pre-miRNA, by an RNaseIII type enzyme called Drosha in the nucleus, which then migrates into the cytoplasm and is cleaved by an enzyme called Dicer. Made of 21-25 nt mature miRNA. The miRNA thus produced complementarily binds to the target mRNA and acts as a post-transcriptional gene suppressor, inducing translation inhibition and mRNA destabilization.

In the composition of the present invention, the numbering of the miRNA is a number numbered according to the order of discovery of the small RNA, miR-486 is the miRNA [Fu H, Tie Y et al FEBS Lett. 579: 3849-3854 (2005), Landgraf P, Rusu M et al. Cell. 129: 1401-1414 (2007)], miR-598 is the miRNA found at 598 [Cummins JM, He Y et al Proc Natl Acad Sci US A. 103: 3687-3692 (2006), Landgraf P, Rusu M et. al Cell. 129: 1401-1414 (2007). It is apparent to those skilled in the art what miR-486 and miR-598 mean [ http://www.mirbase.org ].

Preferably, miR-486 is located at 8 of the human chromosome and has the nucleotide sequences of SEQ ID NOs: 1 and 2. In addition, miR-598 is located at 8 of the human chromosome and is characterized by having the nucleotide sequences of SEQ ID NOs: 3 and 4.

The base sequences are mature forms of each miRNA, and the mature miRNA form of miR-486 is derived from different strands of precursor miRNA (precusor microRNA) of hairpin structure, wherein SEQ ID NO: 1 represents mmu-miR-486 of mouse and its The sequence is the same.

In the composition of the present invention, the inhibitor of miR-486 or miR-598 can be used any inhibitor that can inhibit miRNA, but preferably antagomir, antisense oligonucleotide, or RNA It is characterized by being an inhibitory molecule (inhibitory RNA molecule). In the present invention, specifically miRNA hairpin inhibitor (hairpin inhibitor) was used.

The antagomir or antisense oligonucleotide is characterized by having a sequence complementary to the nucleotide sequence of miR-486 or miR-598. Inhibitors having sequences complementary to each of these miRNAs bind to miR-486 or miR-598 and serve to prevent these miRNAs from working.

The miRNA hairpin inhibitor is a single RNA strand of hairpin structure comprising an antisense sequence complementary to the miRNA and binds to a complete miRNA finally made in Dicer, thereby preventing the miRNA from functioning. Here, RNA used as an inhibitor is a chemical modification to increase the efficiency, using a technique such as attaching another strand, such as a hairpin structure next to the complementary strand. Chemical modifications are made to make the inhibitor less degraded, since the inhibitor itself is also RNA, which is susceptible to degradation. There are three types of chemical modifications. First, there is a method of binding a methyl group to an oxygen group attached to carbon number 2 of the sugar structure of the RNA structure, and the second is more efficient than 2'-O-methyl-group. The carbon-oxygen group is attached to the methoxyethyl group (Methoxyethyl group). Third is the locked nucleic acid (LNA) -modified structure, in which the carbon-oxygen group 2 is linked to carbon 4 (J Weiler, J Hunziker and J Hall Gene Therapy (2006) 13, 496.502). ].

Attaching the hairpin structure to both sides of the complementary strand (antisense) is considered to increase efficiency by blocking the complementary strand itself because it may be a siRNA and react to the other. The actual effect is reported to be better inhibition of miRNA when hairpin.

The miRNA inhibitor of the present invention produced by the above method is an RNA oligo, which binds various chemicals to the antisense of each mature miRNA and serves to inhibit specific miRNA present in the cell. That is, the single-stranded RNA having a complementary sequence of the miRNA serves to inhibit the function of the miRNA by chemically modifying the RNA base.

Preferably, the miR-486 inhibitor is characterized by having the nucleotide sequence of SEQ ID NO: 5, the inhibitor of miR-598 is characterized by having the nucleotide sequence of SEQ ID NO: 6.

In the composition of the present invention, the stem cells are characterized in that the mesenchymal stem cells (Mesenchymal Stem Cell; MSC).

The stem cells are characterized in that the mesenchymal stem cells isolated from diabetic patients.

In the composition of the present invention, the expression inhibitor of miR-486 or miR-598 is characterized in that to promote the regeneration of mesenchymal stem cells.

Specifically, as confirmed in the embodiment of the present invention, when the mesenchymal stem cells derived from diabetic patients who suppressed the expression of miR-486 or miR-598, blood flow increased similarly to normal people.

In addition, the expression inhibitor of miR-486 or miR-598 is characterized by increasing the expression of SIRT1, an aging inhibitory gene by interfering with the binding of miR-486 or miR-598 and 3'UTR of SIRT1.

As confirmed in the Examples of the present invention, luciferase activity was reduced in cells overexpressing miR-486 or miR-598, but luciferase was inhibited in cells inhibiting the expression of miR-486 or miR-598. It was confirmed that the activity was increased.

According to another aspect of the present invention, the present invention provides a method for screening proliferation, differentiation promotion and aging inhibitor of stem cells, comprising the following steps:

(a) treating any agent with an animal cell expressing miR-486 or miR-598; And

(b) determining whether the expression of miR-486 or miR-598 is reduced compared to the control group not treated with the agent in the animal cells.

According to another aspect of the present invention, there is provided a method for screening proliferation, differentiation promotion and aging inhibitor of stem cells, comprising the following steps of the present invention:

(a) treating any agent with an animal cell expressing miR-486 or miR-598; And

(b) determining whether complementary binding of miR-486 or miR-598 to the 3'UTR of SIRT1 is inhibited in said animal cell.

As described above, according to the present invention, by regulating the expression of specific miRNAs, the expression of SIRT1, which plays an important role in aging of human stromal cells (including MSCs), can regulate cell growth and differentiation and inhibit cell aging. In addition, it can increase angiogenesis of mesenchymal stem cells with reduced function in diabetic patients. Therefore, it can be used in the development of technology to control the proliferation, differentiation and therapeutic effect of mesenchymal stem cells using miRNA to control the expression of SIRT1.

Figure 1a is a result of confirming the senescent mesenchymal stem cells through the aging SA-β-Gal staining, Figures 1b and 1c is the Northern blot and RT-PCR that increased miR-486 or miR-598 in aged cells This is the confirmed result.
Figure 2a is the result of confirming the progression of aging of mesenchymal stem cells overexpressed miR-486 or miR-598 through SA-β-Gal staining, Figure 2b is a medium in which the expression of miR-486 or miR-598 is suppressed It was confirmed by SA-β-Gal staining that senescence is inhibited in the leaf stem cells.
Figure 3 is a result of confirming the osteogenic and adipose differentiation ability according to the expression of miR-486 or miR-598 in mesenchymal stem cells through SA-β-Gal staining.
Figure 4a is a result of measuring the proliferative capacity of the mesenchymal stem cells overexpressed miR-486 or miR-598, Figure 4b is a result of measuring the proliferative capacity of mesenchymal stem cells suppressed miR-486 or miR-598 expression to be.
5a shows the results of Western blot measurement of SIRT1 expression according to senescence of mesenchymal stem cells, and FIG. 5b shows luciferase activity of mesenchymal stem cells in which the expression of miR-486 or miR-598 is controlled. The result of the measurement is shown.
Figure 6 shows the results of measuring the expression changes of miR-486 with the progression of aging of NSC derived from the brain of fetus and adult mice.
7 is a result of measuring the proliferative capacity according to the expression of miR-486 or miR-598 in mesenchymal stem cells isolated from diabetic patients.
Figure 8 is the result of measuring the blood flow recovery ability when transplanting mesenchymal stem cells of diabetic patients with controlled expression of miR-486.

Hereinafter, the present invention will be described in more detail with reference to Examples. These examples are only for illustrating the present invention, and the scope of the present invention is not to be construed as being limited by these examples.

Example  1. from fat Mesenchymal stem cells  detach

Stromal stem cells were isolated from fats obtained from patients who underwent liposuction at Pusan National University Hospital.

More specifically, in order to separate the stromal stem cells from the adipose tissue, after obtaining the adipose tissue from a patient who underwent subcutaneous fat removal surgery in plastic surgery, the isolated adipose tissue was washed with HBSS and then using tweezers and scissors Cut adipose tissue thinly. Cut adipose tissue was treated with 0.075% collagenase at 37 ℃ for 30 minutes. Subsequently, the supernatant is removed by centrifugation, and the remaining pellet is added with a medium consisting of α-MEM and 10% FBS (Fetal Bovine Serum) to resuspend, followed by passage through a 100 μm nylon mesh. I got them. The detached cells were plated in a dish and the unattached cells were removed after 24 hours.

Example 2. Confirmation of miR-486 and miR-598 expression of senescent mesenchymal stem cells

When the stem cells isolated in Example 1 grew 70-80%, the cells were separated into single cells by treatment with trypsin / EDTA, and subcultured by adding them at a density of 2000 / cm ² . After repeated passages several times, the degree of aging of the cells was measured using SA-β-Gal (Senescence-Associated β-Galactosidase). To this end, cells were first fixed with PBS fixative (2% (v / v) formaldehyde, 0.2% (w / v) glutaraldehyde), followed by SA-β-Gal dye (5 mM K ₃ Fe (CN) ₆ , 2 mM _{MgCl 2, 150 mM NaCl, 30} mM Citric acid / Phosphate buffer (pH 6), 1 mg / ml X-gal solution) by reacting at 37 ℃ for 16 hours, it was confirmed the aging degree through the dyeing degree, 20 times Aging models of mesenchymal stem cells were obtained at subculture. Total RNA was isolated from aging cells, and microarray was performed using a human-miRNA-V1 Bead Chip (illumina). The measurement result is shown in FIG.

As shown in FIG. 1, the expression of miR-486 and miR-598 in aging cells (P20 cells) compared to P2 normal cells (cells capable of maintaining differentiation and proliferation, ie, stem cells) are respectively expressed. 66.35 times, 3.07 times increased.

And, by performing Northern blot (RT) and RT-PCR it was confirmed that the expression of miR-486 and miR-598 significantly increased in aging cells compared to normal cells (Fig. 1b).

Example  3. In mesenchymal stem cells miR -486 and miR Induction of aging by -598

In order to confirm the induction of aging according to the expression of miR-486 and miR-598, the degree of aging was measured in cells that regulate the expression of miRNA.

First, in order to overexpress miRNA, the cloned clone of the mature miRNA sequence of has-miR-486 and miR-598 into pcDNATM6.2-GW / EmGFP vector expressing GFP (Green Fluorescent Protein) Then, the final vector, the lentivirus expression vector, pLenti6 / V5, was cloned using LR clonase (LV-miR-486, LV-miR-598). As a control, LV-miLacZ targeting β-galactosidase was used. Then, a virus was made using a lentiviral vector and treated with human mesenchymal stem cells for 6 to 12 hours to overexpress each miRNA.

Induction of miRNA expression reduction induced transfection of miR-486 and miR-598 inhibitors (microRNA hairpin inhibitor from Thermo Scientific) having sequences complementary to mesenchymal stem cell miRNAs, respectively, to induce inhibition of each miRNA.

The degree of senescence of mesenchymal stem cells was measured as in Example 2 while continuously passaged each cell in which miRNA expression was controlled. As a result, as shown in Figure 2, it was confirmed through the SA-β-Gal staining that the aging of cells overexpressing miR-486 and miR-598 significantly progressed (Fig. 2a), miR-486 and miR It was confirmed that the progression of aging is significantly reduced in the cells reduced in -598 (Fig. 2b).

Example  4. In mesenchymal stem cells miR -486 and miR By -598 Eruption  Confirm

 Osteogenic differentiation and adiogenic differentiation of mesenchymal stem cells according to the expression levels of miR-486 and miR-598 were confirmed. At this time, the cells of the miRNA expression is controlled was used the cells of Example 3.

Induction of bone differentiation was induced using differentiation-inducing media (10% fetal bovine serum, 50 μg / ml ascorbic acid, 1.5 mg / ml β-glycerophosphate, 10-8 M dexamethasone in α-MEM) for 10 days. , alizarin red S staining was confirmed. Induction of adipose differentiation induces differentiation in adipose differentiation-inducing medium (10% fetal bovine serum, 1 μM dexamethasone, 0.5 mM 3-isobutyl-1-methylxanthine, 10 μM insulin, 200 μM indomethacin in α-MEM), After staining with 2% Oil Red O for 1 hour at room temperature, the degree of differentiation was observed under a microscope. The differentiation observation result is shown in FIG.

As shown in Figure 3, the cells overexpressing miR-486 and miR-598 significantly reduced the differentiation capacity as the aging of the cell, but in the cells with reduced expression of miR-486 and miR-598 reduced the extent of differentiation capacity decreases It can be confirmed.

Example  5. In mesenchymal stem cells miR -486 and miR By -598 Proliferative capacity  Confirm

The proliferation of mesenchymal stem cells according to the expression level of miR-486 and miR-598 was confirmed. At this time, the miRNA expression-controlled cells were used as the cells used in Example 3, each cell was cultured 1 per cm ² to confirm the cell colonization form. The results are shown in FIG.

As a result, it was confirmed that the cells overexpressing miR-486 and miR-598 had a low rate of forming a colony, and the number of cells constituting each colony was also significantly smaller (FIG. 4A). However, in the cells with reduced expression of miR-486 and miR-598, the rate of forming a colony was high, and the number of cells constituting each colony was also remarkably high (FIG. 4B).

In addition, 5 × 10 ³ cells of each mesenchymal stem cell were added to 6-wells, and the number of cells was measured after 3 days. As a result, it was confirmed that the number of cells overexpressing miR-486 and miR-598 was significantly reduced (FIG. 4A), and the number of cells whose expression of miR-486 and miR-598 was inhibited was significantly increased. ((C) of FIG. 4B).

Example  6. miR -486 and miR According to the expression of -598 Luciferase ( luciferase ) Active measurement

The target proteins of miR-486 and miR-598 were found through Targetscan 5.1 and Sanger database, and SIRT1, an aging gene, was selected as a candidate gene. First, it was confirmed that SIRT1 was decreased as the aging of the cells through western blot (FIG. 5A).

Then, in order to confirm the interaction between each miRNA and SIRT1, the T4 ligase to the pMIR-reporter vector cleaved the HindIII and SpeI restriction enzymes at sites where each miRNA is expected to target in the 3'UTR of SIRT1. Cloning was performed using. Each vector was transfected into cells that controlled miRNA (cells used in Example 3) and luciferase activity was measured. The measurement results are shown in FIG. 5.

As a result, the cells overexpressing miR-486 and miR-598 decreased luciferase activity, but the luciferase activity was increased in cells in which miR-486 and miR-598 expression was reduced. Therefore, inhibiting the binding of miR-486 and miR-598 to the 3'UTR of SIRT1 may increase the expression of SIRT1, an aging inhibitory gene (FIG. 5B).

Example 7. Mice Development and Mice Cell Aging Control by miR-486

To investigate the development and cellular senescence of mice following expression of miR-486, brains were isolated from 15-day-old fetuses and 7-week-old adult rats, and miRNAs were isolated from each brain. Confirmed. In addition, experiments were carried out using rat neural stem cells.

The separation method of neural stem cells is described in detail as follows. One day after birth, the subventricular zone (SVZ) of the rat was isolated, washed with HBSS, and the tissue was thinly cut using tweezers and scissors. The cut tissues were treated with 0.25% trypsin and 0.04% DNase at 37 ° C. for 30 minutes. Subsequently, the supernatant is removed by centrifugation and resuspended by adding a medium consisting of neurobasal media and 10% FBS (Fetal Bovine Serum) to the remaining pellets, and then passing the cells through a 100 μm nylon mesh. Got it. The separated cells were plated in a dish coated with poly-D-lysin, and after 24 hours, the attached cells were placed in a dish coated with laminin on neurobasal media, 1 mM L-. Culture was performed using a medium consisting of glutamine, B27 wuplemented, 20ng / ml FGF2, 20ng EGF, and 1:50 B27. When 70-80% of the neural stem cells isolated above were grown, the cells were separated into single cells by treatment with trypsin / EDTA, and subcultured by adding them at a density of 2000 / cm ² , and then in P1 and P10 cells. miRNA was isolated and the expression rate of miR-486 was confirmed. The measurement results are shown in FIG. 6.

As a result, the expression rate of miR-486 was significantly higher in the brains of adult rats than the brains of mouse embryos, and the expression rate of miR-486 was also increased with passage of neural stem cells.

Example  8. In diabetics  Originated Mesenchymal stem cells miR -486 and miR Growth control by -598

To investigate the proliferative changes of mesenchymal stem cells according to the expression of miR-486 and miR-598, the miRNAs of the mesenchymal stem cells isolated from diabetic patients were reduced using miR-486 and miR-598 inhibitors. After the proliferation of stem cells was confirmed. The measurement result is shown in FIG.

Stem cells isolated from diabetic patients showed significantly decreased proliferative capacity compared with cells isolated from normal subjects. However, when miR-486 and miR-598 were reduced, it was confirmed that the proliferative capacity of the stem cells of diabetic patients recovered to the extent similar to that of normal persons.

Example  9. In diabetics  Detached Mesenchymal stem cells miR By -486 Angiogenesis  control

In order to confirm the angiogenesis of mesenchymal stem cells according to the expression of miR-486, the femoral artery was ligated from immunodeficient mice, and 10 ⁶ adipose tissue stromal cells were transplanted into the muscle one day after ligation. Blood flow was measured at 1 and 2 weeks after implantation with a Laser Doppler. The measurement results are shown in FIG. 8.

As a result, blood flow was significantly decreased when stem cells derived from diabetic patients were transplanted compared with that of normal mesenchymal stem cells. However, when the mesenchymal stem cells derived from diabetic patients with reduced miR-486 were transplanted, the blood flow increased similarly to that of normal persons.

Attach an electronic file to a sequence list

Claims (11)

Composition for inhibiting proliferation, differentiation and senescence of stem cells comprising the expression inhibitor of miR-486 having the nucleotide sequence of SEQ ID NO: 1 and 2 as an active ingredient.
delete The method of claim 1, wherein the inhibitor of miR-486 is antagomir (antiagomir), antisense oligonucleotide (antisense oligonucleotide), or RNA inhibitory molecule (inhibitoy RNA molecule) characterized in that to promote the proliferation, differentiation of stem cells and aging Inhibitory composition.
The composition of claim 3, wherein the antagomir or antisense oligonucleotide has a sequence complementary to the nucleotide sequence of miR-486.
The composition of claim 4, wherein the inhibitor of miR-486 has a nucleotide sequence of SEQ ID NO: 5, wherein the stem cell proliferates, promotes differentiation, and inhibits aging.
The composition of claim 1, wherein the stem cells are mesenchymal stem cells.
The composition of claim 6, wherein the mesenchymal stem cells are mesenchymal stem cells isolated from diabetic patients.
The composition for inhibiting proliferation, differentiation and senescence of stem cells according to claim 6, wherein the miR-486 expression inhibitor promotes vascular regeneration of mesenchymal stem cells.
The method of claim 1, wherein the miR-486 expression inhibitor increases the expression of Sirt1, an aging inhibitory gene, by inhibiting the binding of miR-486 and 3'UTR of Sutuin 1 (SIRT1). Promotes proliferation, differentiation of stem cells and composition for inhibiting aging.
Screening method of stem cell proliferation, differentiation promotion and aging inhibitor comprising the following steps:
(a) treating any agent with an animal cell expressing miR-486 having the nucleotide sequences of SEQ ID NOs: 1 and 2; And
(b) determining whether the expression of miR-486 is reduced compared to the control group not treated with the agent in the animal cells.
Screening method of stem cell proliferation, differentiation promotion and aging inhibitor comprising the following steps:
(a) treating any agent with an animal cell expressing miR-486 having the nucleotide sequences of SEQ ID NOs: 1 and 2; And
(b) determining whether complementary binding of miR-486 to the 3'UTR of SIRT1 is inhibited in said animal cell.

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