WO2013032230A2 - Pharmaceutical angiogenic composition including a microrna-382 activator - Google Patents

Abstract

The present invention relates to an angiogenic composition, and more particularly, to a pharmaceutical angiogenic composition including a microRNA-382 activator. The inventors of the present invention have confirmed that microRNA-382, the expression of which is elevated in stomach cancer cells in a low oxygen environment, affects the promotion of angiogenesis. Therefore, provided in the present invention is the pharmaceutical angiogenic composition which includes the microRNA-382 activator, which is angiogenic and thus promotes cell proliferation, and can be valuably used in treating injuries, ischemic myocardial infarctions, or foot ischemia.

Description

Pharmaceutical composition for promoting angiogenesis comprising an activator of micro-AL-382

The present invention relates to an angiogenic composition, and more particularly to an angiogenic pharmaceutical composition comprising an activator of microRNA-382.

Angiogenesis refers to the process by which new blood vessels are formed, which rarely occurs in normal living conditions, but is an essential process in embryogenesis, corpus luteum formation or wound healing. Angiogenesis process is generally caused by the stimulation of angiogenesis factors, blood vessels are reconstructed by the formation of lumen due to the breakdown of the basal membrane, proliferation, proliferation, and differentiation of vascular endothelial cells due to proteases. Consists of being generated. Angiogenesis processes are known to be tightly controlled by several types of promoters and inhibitors, including growth factors, cytokines, lipid metabolites and latent fragments of hemostatic proteins.

On the other hand, microRNAs are small non-coding RNAs that inhibit gene expression in post-transcriptional regulation. microRNA consists of an average of 18 to 25 nucleotides and forms a hairpin structure. Complementary binding to the 3 ′ UTR region of the target gene sequence inhibits mRNA degradation and translation into proteins, and more than 5,000 human genes have been identified as targets of microRNAs. As a result, depending on which target genes are regulated, the functions of microRNAs in vivo are diversified into cell differentiation and proliferation, control of developmental stage and metabolism, angiogenesis and cell death, and the importance of microRNA role is emphasized more. The trend is falling.

Therefore, the present inventors intend to use microRNA, which is a factor regulating angiogenesis mechanism, to promote angiogenesis.

Accordingly, the present inventors have discovered a specific microRNA involved in angiogenesis in a hypoxic environment without any side effects, and as a result of intensive research on an effective angiogenic composition using the same, confirmed the angiogenesis promoting effect when activating microRNA-382 Thus, the present invention has been completed.

Accordingly, an object of the present invention is to provide a pharmaceutical composition for promoting angiogenesis through activation of microRNA-382 and a method for promoting angiogenesis using the pharmaceutical composition.

However, the technical problem to be achieved by the present invention is not limited to the above-mentioned problem, another task that is not mentioned will be clearly understood by those skilled in the art from the following description.

The present invention provides a pharmaceutical composition for promoting angiogenesis comprising a microRNA-382 activator. The angiogenic pharmaceutical composition is used for the treatment of wound healing, ischemic myocardial infarction, or foot ischemia.

The present invention also provides a method for promoting angiogenesis comprising administering to the individual a pharmaceutically effective amount of the pharmaceutical composition.

The pharmaceutical composition of the present invention can promote angiogenesis by activating microRNA-382, and is expected to be usefully used for the treatment of wound healing, ischemic myocardial infarction, or foot ischemia.

Figure 1 shows the results of comparing the expression pattern of the microRNAs changed compared to normal oxygen conditions when cultured gastric cancer cell line MKN1 cells in the hypoxic condition through a microarray experiment. It was found that there was a change in the amount of expression of a plurality of microRNAs under two conditions, among which the expression of microRNA-382 increased under hypoxic conditions.

Figure 2 shows the expression level of microRNA-382 through real-time PCR after incubating the gastric cancer cell line MKN1 cells in hypoxic conditions by time. It was confirmed that the expression of microRNA-382 was increased in the hypoxic condition compared to normal oxygen, and it was found that the microRNA-382 was most expressed in the culture for 24 hours.

Figure 3 shows the result of suppressing the proliferation of the cells when cultured vascular endothelial cells in the conditioned medium in which the expression of the microRNA-382 increased in hypoxic conditions reduced by treatment with the inhibitor.

Figure 4 shows the results of suppression of cell migration ability when vascular endothelial cells were cultured in a condition medium in which microRNA-382 with increased expression in hypoxic conditions was reduced by treatment with an inhibitor.

FIG. 5 shows the results of suppressing angiogenesis of cells when vascular endothelial cells were cultured in a condition medium in which microRNA-382 with increased expression in hypoxic condition was reduced by treatment with an inhibitor.

Figure 6 shows the results of improved cell proliferation when cultured vascular endothelial cells in a condition medium that increased the expression of microRNA-382 under normal oxygen conditions.

Figure 7 shows the results of improved cell mobility when cultured vascular endothelial cells in a condition medium that increased the expression of microRNA-382 under normal oxygen conditions.

Figure 8 shows the results of improved cell vascular formation ability when cultured vascular endothelial cells in a condition medium that increased the expression of microRNA-382 under normal oxygen conditions.

Figure 9 shows the results confirmed that the expression level of PTEN is reduced in cancer cells of hypoxic conditions.

10 is a diagram showing a binding site of 3'UTR of microRNA-382 and PTEN.

FIG. 11 illustrates a recombinant vector fused with luciferase protein containing 3'UTR of PTEN to confirm microRNA-382 binding to PTEN 3'-UTR, and microRNA- into a cell line transduced with the vector. After treating 382, a graph showing the activity of luciferase expressed in cells.

12 shows the results of confirming the change in the expression level of PTEN protein by miroRNA-382.

Figure 13 is a result of performing a CAM assay, to confirm the angiogenic effect of microRNA-382 (scale bar = 2 mm).

14 is a result of performing a tube formation assay to confirm whether the angiogenic effect of microRNA-382 is inhibited by PTEN.

 15 is a result of performing a CAM assay to confirm whether PTEN inhibits angiogenic effects of microRNA-382.

The present invention provides a pharmaceutical composition for promoting angiogenesis comprising a microRNA-382 activator. The present inventors confirmed through an experiment the regulation of angiogenesis and proliferation of vascular endothelial cells of microRNA-382 in the hypoxic environment of the cell. In other words, the present inventors confirmed the different microRNA expression in the normal oxygen state and hypoxic state of the cell, in particular, it was confirmed that the expression of microRNA-382 is increased in the hypoxic state. In addition, when the microRNA-382 activator was activated using a microRNA-382 activator, it was confirmed through experiments that the proliferative capacity, mobility, and angiogenesis of the surrounding vascular endothelial cells increased.

This suggests that angiogenesis-related factors are regulated by microRNA-382, which regulates gene expression in the body, which may be useful for the treatment of hypoxic sites such as wound healing, ischemic myocardial infarction, and foot ischemia. .

Accordingly, the present invention provides a pharmaceutical composition, which is used to treat wound healing, ischemic myocardial infarction, or foot ischemia by activating microRNA-382 to promote angiogenesis and proliferation of vascular endothelial cells in the hypoxic region. do.

In one embodiment of the present invention, microRNAs showing different expression levels when hypoxic in gastric cancer cell line MKN1 cells were confirmed through microarray experiments (see FIG. 1), and microRNA-382 was 24 hours under hypoxic conditions. The highest expression was confirmed by real-time PCR (see FIG. 2).

In another embodiment of the present invention, the expression of microRNA-382, whose expression is increased in hypoxic gastric cancer cells, was reduced by inhibitors. When vascular endothelial cells were cultured in the conditioned medium extracted after 6 hours, 12 hours, and 24 hours, cell proliferation was reduced (see FIG. 3). It was confirmed to decrease (see FIGS. 4 and 5).

In addition, it was confirmed that cell proliferation was improved when vascular endothelial cells were cultured in the extracted medium after overexpressing microRNA-382 in normal oxygen gastric cancer cells (see FIG. 6). It was confirmed that the improved by a similar value to the hypoxic state (see FIGS. 7 and 8).

In addition, it was confirmed that the target gene of the microRNA-382 is PTEN, and the microRNA-382 binds to the 3'-UTR of the PTEN (see FIGS. 9 to 12). The CAM assay and the tube formation assay were performed to in vivo. In addition, it was confirmed that the angiogenic effect was shown by microRNA-382 (see FIGS. 13 to 15).

From the above, the inventors confirmed that microRNA-382 plays a role in promoting angiogenesis and proliferation of vascular endothelial cells. Ultimately, the results suggest that activating microRNA-382 may promote the proliferation of vascular endothelial cells, angiogenesis, and may be effectively used for the treatment of wound healing, ischemic myocardial infarction, or foot ischemia.

The pharmaceutical composition of the present invention may comprise a pharmaceutically acceptable carrier. The pharmaceutically acceptable carrier may include physiological saline, polyethylene glycol, ethanol, vegetable oil, isopropyl myristate, and the like, but is not limited thereto.

The pharmaceutical compositions of the present invention may be formulated in ointments or creams for topical application and may be formulated with common saline, water-soluble solvents such as 5% dextrose, or vegetable oils, synthetic fatty acid glycerides, higher fatty acid esters, or propylene glycol. The compounds may be formulated into injections by dissolving, suspending, or emulsifying the compound in the same non-aqueous solvent. Formulations of the present invention may include conventional additives such as solubilizers, isotonic agents, suspending agents, emulsifiers, stabilizers, and preservatives.

The present invention also provides a method for treating wounds, ischemic myocardial infarction, or foot ischemia by administering to a subject a pharmaceutically effective amount of an angiogenic composition comprising a microRNA-382 activator. As used herein, "individual" means a subject in need of treatment for a disease, and more specifically human, or non-human primates, mice, rats, dogs, cats, horses, and cattle Means such mammals. In addition, in the present invention, the "pharmaceutically effective amount" may be adjusted in various ways depending on the weight, age, sex, health condition, diet, administration time, administration method, excretion rate, and severity of the disease of the patient. It is obvious to

The preferred dosage of the pharmaceutical composition of the present invention depends on the condition and weight of the patient, the extent of the disease, the form of the drug, the route of administration, and the duration, and may be appropriately selected by those skilled in the art. However, preferably, it is administered at 0.001 to 100 mg / kg body weight per day, more preferably 0.01 to 30 mg / kg body weight. Administration may be administered once a day or may be divided several times. The microRNA-382 activator of the present invention may be present in an amount of 0.0001 to 10% by weight, preferably 0.001 to 1% by weight, based on the total weight of the total composition.

The pharmaceutical composition of the present invention can be administered to mammals such as mice, mice, livestock, humans, and the like by various routes. The method of administration is not limited, and may be administered by oral, rectal, or intravenous, intramuscular, subcutaneous, intrauterine dural, or intra cerbroventricular injection.

Hereinafter, preferred examples are provided to aid in understanding the present invention. However, the following examples are merely provided to more easily understand the present invention, and the contents of the present invention are not limited by the following examples.

EXAMPLE

Example 1. Cell Culture

BAEC cells (bovine aortic endothelial cells) were put together in DMEM medium containing 10% fetal calf serum and incubated in a 37 ° C thermostat with 5% carbon dioxide. Human gastric cancer cells were put together in RPMI-1640 medium containing 10% fetal calf serum and cultured in a 37 ° C thermostat with 5% carbon dioxide. When the cells were sufficiently grown, the cells were cultured in a 10 cm 2 dish for RNA extraction and protein isolation. When two culture vessels were separated, adherent cells were removed from the culture vessel using trypsin-EDTA, centrifuged at 1000 rpm for 5 minutes, and then transferred to the new culture vessel in the same manner as the adherent cells. When the hypoxic state was maintained, the culture was carried out in an incubator maintaining 1% O ₂ concentration.

Example 2. Comparative experiment of microRNA-382 expression level in hypoxic state

The hypoxic state is formed around the cancer, and in such an environment, changes in the expression level of various factors that control mechanisms such as cell proliferation and angiogenesis occur. Among them, we compared microRNAs with different expression levels when given hypoxic conditions in MKN1 cells, gastric cancer cell lines.

Specifically, MKN1 cells were incubated for 24 hours at 1% oxygen concentration. After 24 hours, the medium was rapidly removed and the RNA extract was processed to separate RNA. In comparison with RNA isolated from MKN1 cells cultured under normal oxygen conditions, microarray and real-time PCR were performed to confirm that expression of microRNA-382 was increased in hypoxic conditions among microRNAs with varying expression levels (FIG. 1). And FIG. 2). Real-time PCR experiment method is to extract the total RNA of MKN1 cells using Purelink miRNA kit (Invitrogen) according to TRIzol reagent and manufacturer's protocol, and then total using GenoExplorer ™ miRNA First-Strand cDNA Core Kit (Genosensor) Complementary DNA (cDNA) was generated with 500 ng of RNA. Real-time PCR was performed with the Biosystems 7300 Real-Time PCR system using SYBR Green PCR Master Mix (Applied Biosystems) and GenoExplorer ™ miRNA qPCR Primer Sets (Genosensor). Mature microRNAs in U6 RNA (internal control MKN1 cells) were calculated.

Figure 1 shows the results of comparing the expression pattern of the microRNAs changed compared to normal oxygen conditions when cultured gastric cancer cell line MKN1 cells in the hypoxic condition through a microarray experiment. As shown in FIG. 1, it was found that there was a change in the amount of expression of a plurality of microRNAs under two conditions, among which the expression of microRNA-382 increased under hypoxic conditions.

Figure 2 shows the expression level of microRNA-382 through real-time PCR after incubating the gastric cancer cell line MKN1 cells in hypoxic conditions by time. As shown in Figure 2, it was confirmed by real-time PCR that the microRNA-382 shows the highest expression at 24 hours in the hypoxic state. That is, it was confirmed that the expression of microRNA-382 was increased in the hypoxic condition compared to normal oxygen, and it was found that the microRNA-382 was most expressed in the culture for 24 hours.

The results suggest that microRNA-382 may play a role in controlling the phenomena that occur in the hypoxic state of cells.

Example 3. Confirmation of vascular endothelial cell proliferation according to the expression of microRNA-382

In the hypoxic microenvironment that is formed during the unlimited proliferation of cancer cells, proliferation may be accelerated by surrounding vascular endothelial cells affected by various growth factors secreted by cancer cells.

Therefore, the present inventors performed the following experiment because it is thought that microRNA-382, which is increased in hypoxic cancer cells, will also affect peripheral vascular endothelial cells.

Specifically, MKN1 cells, which are gastric cancer cell lines, were cultured in an antibiotic-free medium for one day, and then transfected with PNAs ^™ microRNA-382 inhibitor (PANAGENE). After 20 hours of incubation in normal oxygen, the cells were transferred to 1% oxygen and incubated for 6 hours, 12 hours, and 24 hours, respectively. After culturing each time, the culture medium was extracted and concentrated, and then treated with vascular endothelial cells to observe the proliferative capacity.

In order to observe the proliferative capacity of vascular endothelial cells, vascular endothelial cells were cultured in DMEM medium containing 10% FBS in a 48-well plate for about 24 hours, and then cultured for 20 hours after changing to medium containing only 1% FBS. Incubated for 24 hours. [³ H] -cymidine was treated with 1 uCi per well for 4 hours and then washed three times with PBS. After fixing for 5 minutes in methanol at 4 ℃, washed three times with distilled water. After 10 minutes treatment with 5% TCA (trichloroacetic acid), washed three times with distilled water, dissolved with 0.3N sodium hydroxide, and then measured the radioactivity using a liquid scintillation counter (Perkin Elmer) to determine the proliferation of the cells, The results are shown in FIG.

As shown in FIG. 3, it was confirmed that the vascular endothelial cells reduced the proliferative capacity of vascular endothelial cells by treating the vascular endothelial cells with reduced expression of microRNA-382 with increased inhibitory activity in hypoxic cancer cells.

From these results, microRNA-382 could be expected to play a role in promoting the proliferative capacity of vascular endothelial cells, and to reconfirm this, the effect of overexpression of microRNA-382 in normal oxygen state on the proliferative capacity of vascular endothelial cells was examined. .

Specifically, mature microRNA-382 cloned into pENTR ^™ / H1 / T0 vector was transfected into gastric cancer cell line MKN1 cells to induce overexpression. After culturing for 24 hours, the medium was extracted, concentrated, and treated with vascular endothelial cells to observe proliferative activity. The results are shown in FIG. 6.

As shown in Figure 6, it was confirmed that increased expression of microRNA-382 improves the proliferative capacity of vascular endothelial cells.

Example 4. Confirmation of vascular endothelial cell migration according to expression of microRNA-382

In addition to Example 3, the effect of microRNA-382 on the mobility of vascular endothelial cells was observed.

Specifically, MKN1 cells, which are gastric cancer cell lines, were cultured in an antibiotic-free medium for one day, and then transfected with PNAs ^TM microRNA-382 inhibitor (PANAGENE). After 20 hours of incubation in normal oxygen, the cells were transferred to 1% oxygen and incubated for 6 hours, 12 hours, and 24 hours, respectively. After culturing each time, the culture medium was extracted and concentrated, and then treated with vascular endothelial cells.

In order to observe the vascular endothelial cell migration, 24-well transwells with 8 um porosity polycarbonate filters were coated with type 1 collagen and dried for 1 hour at room temperature. Under the chamber, the extracted medium was put in the same number of cells with a serum-free medium on the top of the chamber and incubated for 20 hours, and the number of cells passing through the membrane was counted.

The separated cells were separated by methanol fixation, hematoxylin 10-minute staining, eosin 1-minute staining, and the cells on the unmoved membrane were removed with a cotton swab, and the cells stained under a microscope were counted. The results are shown in FIG. 4. It was.

As shown in FIG. 4, it was confirmed that the vascular endothelial cell migration ability was reduced by treating the vascular endothelial cells with reduced conditions of microRNA-382 with increased expression in hypoxic cancer cells through inhibitors.

From these results, microRNA-382 was expected to play a role in promoting vascular endothelial cell migration, and to reconfirm this, the effects of overexpression of microRNA-382 under normal oxygen on the vascular endothelial cell migration were discussed. .

Specifically, mature microRNA-382 cloned into pENTR ^™ / H1 / T0 vector was transfected into gastric cancer cell line MKN1 cells to induce overexpression. After culturing for 24 hours, the medium was extracted, concentrated, and treated with vascular endothelial cells to observe the migration ability, and the results are shown in FIG. 7.

As shown in Figure 7, it was confirmed that increased expression of microRNA-382 improves the ability of vascular endothelial cells.

Example 5. Confirmation of vascular endothelial cell production capacity according to expression of microRNA-382

The present inventors observed the effect of microRNA-382 on the blood vessel formation ability of vascular endothelial cells.

In order to observe the vascular endothelial ability of vascular endothelial cells, Matrigel was first coated on a 48-well plate at 37 ° C. for 30 minutes, and then the microRNA-382 having increased expression in the hypoxic state was extracted from the medium of cancer cells in which the inhibitor was reduced through an inhibitor. After culturing vascular endothelial cells together with the medium, the tube formation was confirmed under a microscope 12 hours later, and the results are shown in FIG. 5.

As shown in FIG. 5, it was confirmed that when vascular endothelial cells were treated with a condition medium in which microRNA-382 with increased expression in hypoxic cancer cells was reduced through an inhibitor, vascular endothelial cell vascular formation ability was reduced.

From the above results, microRNA-382 was expected to play a role in promoting angiogenesis, and to reconfirm this, the effect on the angiogenesis when overexpression of microRNA-382 in normal oxygen state was considered.

It was confirmed that the tube formation was increased when the blood vessel formation ability of the cells put together with the extracted medium after overexpressing the microRNA-382 in the same manner, the results are shown in FIG.

As shown in Figure 8, it was confirmed that increased expression of microRNA-382 improves the vascular endothelial ability of vascular endothelial cells.

Example 6 Confirmation of angiogenesis-promoting effect of microRNA-382 in vivo

6-1.Reduced PTEN Expression in Hypoxic Conditions

The present inventors performed the following experiment to identify the target gene of the microRNA-382 is increased expression in cancer cells of hypoxic conditions.

Specifically, PTEN was selected from target genes through Target Scan, miRanda, and Sanger miRbase Target, which are programs for predicting target genes and binding sites of microRNAs. First, PTEN expression levels in cancer cells under hypoxic conditions were confirmed. .

As a result, it was confirmed that the expression level of PTEN in cancer cells under hypoxic conditions was reduced than in normal oxygen conditions (see FIG. 9).

6-2. Confirmation of PTEN with microRNA-382

Through this, the present inventors predicted that PTEN, which decreases expression in hypoxic cancer cells, is a target gene of microRNA-382. First, miR-130a and miR-495 bind to 3'-UTR portion of PTEN through miRanda program. The site was found. And it was confirmed that the binding site with the microRNA-382 in the 3'-UTR of PTEN mRNA is preserved in various species (see Fig. 10).

In order to confirm that microRNA-382 directly binds to the predicted binding site of PTEN, the 3'-UTR portion of PTEN including the binding site of microRNA-382 was cloned into a luciferase reporter vector, and pGL3-luciferase-PTEN 3 '-UTR-vector was constructed. Thereafter, the prepared vector was transduced into cells, microRNA-382 was treated to cells transfected with the vector, and luciferase activity in the cells was measured.

As a result, it was confirmed that luciferase activity was significantly reduced by microRNA-382 treatment in cells transduced with the vector including the 3′-UTR portion of PTEN (see FIG. 11). Through this, it can be seen that microRNA-382 binds directly to the 3'UTR of the target gene PTEN.

6-3. Decreased expression of PTEN by miR-382

In order to confirm the effect of microRNA-382 on the protein expression of PTEN, the present inventors have identified miRNA mimic (MSY0000737) that can overexpress microRNA in gastric cancer cell line MKN1 cells and miRNA inhibitor that can inhibit microRNA (Qiagen miScript miRNAs). Was treated, and the expression level of PTEN protein was confirmed.

Specifically, when the mimic of microRNA-382 was treated in cancer cells under normal oxygen conditions, the expression level of PTEN protein was decreased in a concentration-dependent manner, and even when the oxygen cells were treated with microRNA-382 inhibitors in a hypoxic condition. , It was confirmed that the expression level of the PTEN protein was recovered (see FIG. 12).

6-4. Confirmation of angiogenic effects of microRNA-382 by chick chorioallantoic membrane assay

The present inventors performed chick chorioallantoic membrane (CAM) assay to confirm the angiogenic effect of microRNA-382 in vivo.

Specifically, CAM assay was performed as follows.

The fertilized eggs were incubated in an incubator maintained at a temperature of 37-38 ° C. and a humidity of 90% or higher. The cut ends of the fertilized eggs were injured with a knife, then the holes were sealed and incubated again with the holes facing down. Thereafter, a circular window having a diameter of 2-3 cm toward the air sac of the fertilized egg (the opposite side of the syringe hole) was cut out, and only the one identified as the fertilized egg was blocked with a wide glass tape and cultured again to induce the production of CAM.

Subsequently, for the treatment of microRNA mimic, the negative control and microRNA-382 mimic were transduced into cells under normal oxygen conditions, and the conditioned medium obtained after 24 hr was concentrated 30 times, mixed with matrigel in a 1: 1 ratio to form a mixture. This was processed on CAM.

In addition, for the treatment of the microRNA inhibitor, the negative control and the microRNA-382 inhibitor were transduced into the cells and subjected to hypoxic condition after 16 hr, and after 24 hr, the medium was separated / concentrated and mixed with matrigel in a 1: 1 ratio. A mixture was made, which was processed on CAM and photographed close by camera after 4 days.

As a result, CAM treated with the conditioned medium mixture containing microRNA-382 increased the number of branches of microvessels compared to the negative control, whereas treated conditioned medium containing microRNA-382 inhibitors under hypoxic conditions. In one CAM, it was confirmed that the number of branches of microvessels was reduced compared to that of the hypoxic state (see FIG. 13). Through this, it can be seen that mirroRNA-382 has an effect of promoting angiogenesis in vivo.

6-5. Confirmation of inhibition of angiogenic effects of microRNA-382 by PTEN treatment

The present inventors confirmed whether angiogenesis-promoting effect can be suppressed by treatment with PTEN protein, a target gene of miroRNA-382.

Specifically, tube formation assay was performed using a condition medium obtained from cells transduced with PTEN so that the PTEN protein was overexpressed. As a result, tube formation induced by microRNA-382 was inhibited by PTEN treatment. It was confirmed (see FIG. 14).

In addition, CAM assay confirmed that angiogenesis increased by microRNA-382 was decreased by PTEN, and angiogenesis was reduced by treatment with microRNA-382 inhibitor in hypoxic condition than in hypoxic state (see FIG. 15). ).

Thus, overexpression of PTEN inhibits the angiogenic effects induced by microRNA-382.

The foregoing description of the present invention is intended for illustration, and it will be understood by those skilled in the art that the present invention may be easily modified in other specific forms without changing the technical spirit or essential features of the present invention. will be. Therefore, it should be understood that the embodiments described above are exemplary in all respects and not limiting.

Claims (3)

1. Pharmaceutical composition for promoting angiogenesis comprising a microRNA-382 activator.
2. The method of claim 1,

   The pharmaceutical composition for promoting angiogenesis is characterized in that it is used for the treatment of wound healing, ischemic myocardial infarction, or foot ischemia.
3. A method for promoting angiogenesis comprising administering to a subject a pharmaceutically effective amount of the pharmaceutical composition of claim 1.

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