CN112522268A

CN112522268A - mRNA (messenger ribonucleic acid) related to diabetic vascular injury, target gene and application

Info

Publication number: CN112522268A
Application number: CN202011496927.0A
Authority: CN
Inventors: 莫朝晖; 万新星; 罗寓芳; 赵立玲; 梁芳; 郭梓; 曾萍玉; 蒋斯惟; 莫嘉可
Original assignee: Third Xiangya Hospital of Central South University
Current assignee: Third Xiangya Hospital of Central South University
Priority date: 2020-12-17
Filing date: 2020-12-17
Publication date: 2021-03-19

Abstract

The invention discloses mRNA (messenger ribonucleic acid) related to diabetic vascular injury, a target gene and application thereof, wherein the nucleotide sequence of the mRNA is shown as SEQ ID No.1, the nucleotide sequence of the target gene is shown as SEQ ID No.2, miR-139-5p mediates the injury of diabetic vascular endothelial cells and endothelial progenitor cells through the target gene c-jun, and miR-139-5p or c-jun is regulated to promote the regeneration of diabetic ischemic limb vessels and the wound repair. The invention improves the pathogenesis of the diabetes blood vessel injury and the regeneration disorder and provides a new target point for preventing and treating the diabetes blood vessel complication.

Description

mRNA (messenger ribonucleic acid) related to diabetic vascular injury, target gene and application

Technical Field

The invention relates to the technical field of treatment of diabetic vascular complications, in particular to mRNA (messenger ribonucleic acid) related to diabetic vascular injury, a target gene and application.

Background

At present, diabetes becomes one of the most common diseases threatening the health of people, the prevalence rate of diabetes in China reaches 10.9 percent, and the proportion of diabetes in the early stage is higher by 35.7 percent. With the prolongation of the course of diabetes, endothelial dysfunction, the damage of the self-repair ability and the regeneration function of blood vessels are increased, and the complications of the blood vessels are increased. Diabetic vascular complications are the main cause of disability and death of diabetics, wherein lower limb angiopathy and blocked neovascularization are the main causes of non-healing of diabetic foot ulcers and even foot gangrene and amputation. Endothelial Cell (EC) dysfunction includes impaired endothelial cell migration ability, proliferative ability, angiogenic properties, as well as vasodilatory function and vascular integrity. Currently, mechanisms for diabetic endothelial cell dysfunction and angiogenesis disorders may be due in part to reduced vascular endothelial growth factor-a (VEGF-a) signaling, and other pathways include accumulation of advanced glycosylation endproducts, protein kinase c (pkc) activation, sorbitol-inositol imbalance, NF-kB mediated vascular inflammation, and the like. However, the exact mechanism of vascular dysfunction and poor angiogenic response in diabetes remains unclear. Furthermore, progenitor cells belonging to the endothelial lineage play a key role in vascular integrity and postnatal angiogenesis. Vascular complications and angiogenesis disorders in diabetic patients are also promoted by the reduced number of Endothelial Progenitor Cells (EPCs) in diabetes mellitus, which impairs angiogenic function. Therefore, the research on the mechanism of the dysfunction of the diabetic endothelial cells and the EPCs is helpful for searching for effective targets for improving the function of the endothelial cells of the diabetic endothelial cells and providing a new strategy for the prevention and treatment of the diabetic vascular diseases.

microRNAs (microRNAs) are a class of non-coding RNAs that inhibit the post-transcriptional translation of a target gene mRNA by specifically binding to the 3' untranslated region of the target gene sequence or destroy the target gene mRNA. It has been found that various microRNAs can influence the function of endothelial cells and participate in the regulation of angiogenesis. Due to the wide range and importance of gene regulation, the microRNA target molecules are concerned about diabetic vascular complications, and the more the microRNA target molecules are found, so that bright prospects are provided for diagnosis and treatment of related diseases. At present, a large amount of microRNA enters clinical tests for disease diagnosis and treatment or is about to become a new clinical treatment drug.

Disclosure of Invention

The invention provides microRNA and a target gene related to the damage of diabetic vascular endothelial cells and endothelial progenitor cells, and application thereof in endothelial repair and vascular regeneration in the prevention and treatment of diabetic vascular complications, perfects the pathogenesis of diabetic vascular damage and dysgenesis, and provides a new target for the prevention and treatment of diabetic vascular complications.

In order to achieve the purpose, the invention adopts the following technical scheme:

the mRNA related to diabetic vascular injury is miR-139-5p, and the sequence of the mRNA is as follows: the nucleotide sequence is shown as SEQ ID No. 1.

Furthermore, the target gene of the mRNA is a c-jun sequence, and the nucleotide sequence of the target gene is shown as SEQ ID No. 2.

Furthermore, diabetes induces the expression of vascular endothelial cells and endothelial clone cells miR-139-5p to be up-regulated, the expression of miR-139-5p inhibits the proliferation, migration and tube formation functions of the endothelial cells and diabetic endothelial clone cells, and the expression of miR-139-5p is down-regulated to improve the proliferation, migration and angiogenesis capacities of the diabetic endothelial cells.

Further, the application of the antagonist of miR-139-5p in preparing the medicine for repairing the wound surface of the diabetic patient.

Furthermore, the administration sites are the bottom and the edge of the wound surface, the administration mode is injection, and the concentration of the miR-139-5p antagonist in the injection is 0.2 mg/mL.

Further, the application of the inhibitor of miR-139-5p in preparing products or medicines for preventing or treating vascular injury complications caused by diabetes mellitus.

Further, the vascular injury disease is selected from one or more of vascular endothelial cell injury, endothelial progenitor cell injury and vascular regeneration function injury.

The composition contains an inhibitor of miR-139-5p and a target gene c-jun, wherein the miR-139-5p is combined with mRNA molecules of the c-jun, and the composition is used for preparing a product or a medicament for preventing or treating vascular injury complications caused by diabetes.

Further, the inhibitor of miR-139-5p inhibits the expression of miR-139-5p in endothelial cells and ECPC cells, and improves the proliferation, migration and tube forming capability of the cells; the composition is prepared by combining an antagonist of miR-139-5p with ECPC (endothelial cell activator protein) and is applied to in-vivo angiogenesis; by up-regulating the expression of c-jun, the proliferation, migration and tube forming capability of endothelial cells and ECPC cells are promoted.

The invention has the following beneficial effects:

(1) the invention determines the action of miR-139-5p/c-jun in mediating the injury of diabetic endothelial cells, and provides a new target for regulating and controlling miR-139-5p/c-jun to prevent and treat diabetic vascular complications and ischemic diseases

(2) The miR-139-5p inhibitor promotes the proliferation, migration and tube formation of vascular endothelial cells and ECFC, the 139-5p antagonist combined with the ECFC can obviously improve the lower limb ischemia of diabetic nude mice compared with the single ECFC injection, and the miR-139-5p antagonist provides a new product or a new idea for optimizing the ECFC transplantation in vivo to treat ischemic diseases

(3) The miR-139-5 antagonist preparation is locally injected to accelerate the healing of diabetic wounds and can be developed into a new product for treating chronic wounds such as diabetic feet

(4) The c-jun has obvious regulation effect on the proliferation, migration and angiogenesis of the ECFCs, has the function of regulating the ECFCs and provides a new target point for promoting the regeneration of the blood vessels of the diabetic ischemic lesions by up-regulating the expression of the c-jun

Drawings

FIG. 1 expression levels of miR-139-5p in isolated ECFC, murine EC and HUVEC, and in high culture experimental and control groups;

FIG. 2 expression of miR-139-5p affects the function of ECFCs and HUVECs cultured in vitro; wherein, A, the scratch test evaluates the influence of the miR-139-5p simulator or inhibitor on the migration of ECFC; B. CC8K detects the effect of miR-139-5p on ECFC proliferation; C. effect of miR-139-5p mimetics or inhibitors on ECFC vascularization; D. evaluating the influence of the miR-139-5p simulator or inhibitor on HUVEC migration by a scratch test; E. effect of miR-139-5p mimetics or inhibitors on HUVE vascularization;

FIG. 3c-jun is the target gene of miR-139-5 p; wherein, the A, the B and the target gene of the prediction miR-139-5p are verified by bifluorescent reporter enzyme; C. detecting the expression of C-jun in normal ECFCs and diabetic ECFCs transfected with miR-139-5p mimics and miR-139-5p inhibitors respectively by using western blot;

FIG. 4c-jun effect on ECFCs function; wherein: A. the scratch test detects the influence of c-jun siRNA on the migration of ECFCs; B. c, Hoechst and EdU staining and CCK8 to evaluate the effect of C-jun siRNA or up-regulated expression on the proliferation of ECFCs; D. the angiogenesis assay shows the effect of c-jun siRNA or up-regulation on ECFCs angiogenesis; (N-3) P <0.05 compared to control; data shown in the figure represent mean ± standard deviation;

FIG. 5miR-139-5p regulates the function of ECFCs through c-jun; wherein: the effect of co-transfection of miR-139-5p mimics and c-jun plasmids on migration (A) and angiogenesis (B) of normal ECFCs, or the effect of co-transfection of miR-139-5p inhibitors and c-jun siRNA on migration (A) and angiogenesis (B) of diabetic ECFCs (40-fold fluorescence microscopy); (N-3) P <0.05 compared to control; data shown in the figure represent mean ± standard deviation;

FIG. 6 Effect of miR-139-5p on endothelial cell function by c-jun regulation of VEGF and PDGF-B; wherein: A. detecting the expression of PDGF-B and VEGF of healthy and diabetic patients by a Western blot method; B. the miR-139-5p simulant and the C-jun plasmid transfect the normal ECFC, or the expression of C-jun, VEGF and PDGF-B after the miR-139-5p inhibitor and the C-jun siRNA transfect the diabetic ECFC is detected by a Western blot method; C. carrying out immunofluorescence staining to detect the expression of C-jun, VEGF and PDGF-B after the miR-139-5p simulant and C-jun plasmid transfect normal ECFC or after miR-139-5p inhibitor and C-jun siRNA transfect diabetic ECFC, wherein red represents Cy 3-labeled C-jun, and green represents FITC-labeled VEGF; D. detecting the expression of PDGF-B, VEGF after HUVEC is treated by the miR-139-5p simulant by a Western blot method; E. expression of PDGF-B, VEGF following transfection of HUVEC with C-jun plasmid or C-jun siRNA;

FIG. 7 Effect of miR-139-5p on ECFC angiogenic function following treatment with KDR and PDGF-B antibodies: observing tubule formation in diabetic ECFC transfected with miR-139-5p inhibitor in the presence or absence of anti-KDR/anti-PDGF treatment;

FIG. 8 in vivo matrigel assay; wherein: A. representative photographs, (N-4) P <0.05 compared to normal or diabetic groups; B. CD31 histological images under high power field of view (200X) of normal light microscope; (N-3) P <0.05 compared to normal or diabetic group; data shown in the figure represent mean ± standard deviation;

FIG. 9 downregulation of miR-139-5p expression in vivo promotes ECFC-mediated angiogenesis and blood flow perfusion in diabetic HLI mice;

wherein: A. 5 groups of representative ischemic photographs taken 14 days after treatment sequentially comprise a sham operation group, a PBS treatment group, an antagonist-139 treatment group, an EGFC treatment group and an ECFC + miR-139-5p antagonist treatment group from left to right;

B. the transplantation of ECFC and antagonist 139-5p improves blood flow perfusion in the ischemic limb of diabetic mice; images and quantitative analysis of HLI post-surgery with or without ECFC transplantation and/or antagomiR-139-5p show the time course of blood perfusion; the blood perfusion is the ratio of ischemic to non-ischemic limb perfusion measured by a PeriCam perfusion speckle imager;

C. time-dependent effects of ECFC and/or antagomiR-139 on skin temperature in lower limbs of ischemic mice; (N-3) × P <0.05 and # P <0.01, both compared to PBS;

D. effect of ECFC transplantation and/or antagomiR-139 on lower limb vascular density in ischemic mice; the number of CD31 stained vessels (arrows) was counted under a normal light microscope at 5 high power fields (200 ×) to determine the vessel density; (N-3) × P <0.05 and # P <0.01, both compared to PBS; data shown in the figure represent mean ± standard deviation;

E. representative fluorescence images and quantification of CM-DiI labeled ECFCs (red, white arrows) and α -smooth muscle actin (α SMA) positive arterioles (green) in ischemic adductor tissues and merged images of CM-DiI, α -SMA and DAPI, 5 high power fields (200 ×). (N-3) P <0.05 compared to the transplant only group;

FIG. 10 change of wound healing of diabetic rat.

Detailed Description

The technical solution of the present invention will be clearly and completely described by the following detailed description.

Example 1:

(1) ECFC was isolated from peripheral blood of healthy volunteers and type 2 diabetic patients, respectively, and the ECFC was isolated by collecting peripheral blood (20 ml) in heparinized solution. The separated mononuclear cells were resuspended in EGM-2 medium (EGM-2(Lonza # cc-3162) containing 10% FBS, double antibody), adjusted to a cell mass concentration of 5X 107/mL, and plated in 6-well plates coated with rat tail type I collagen for culture. Isolated ECFC was characterized and isolated cultured cells 3-5 were used for analysis and transplantation

(2) Endothelial cells were isolated from the aorta of diabetic rats and diabetic nude mice, and the aorta of diabetic animals was dissected from the aortic arch to the abdominal aorta and immersed in 20% fetal bovine serum medium (FBS-DMEM) containing 1000U/mL heparin. After removal of fat or connective tissue and ligation, the aorta was filled with collagenase type II solution and incubated at 37 ℃ for 45 minutes. Washed with 5 ml of DMEM containing 20% FBS and collected by centrifugation at 1200rpm for 5 minutes. The pellet was then resuspended with a pipette and 2mL DMEM containing 20% FBS and cultured in a 35mm collagen type 1 coated petri dish. After incubation at 37 ℃ for 2 hours, the medium was removed, the cells were washed with warm Phosphate Buffered Saline (PBS), and the medium was added. One week later, fused endothelial cells were harvested for detection.

(3) The expression level of miR-139-5p in endothelial cells of diabetic ECFC and rodent is detected by qRT-PCR, and the expression level of HUVEC after high-sugar (30mmol/l) culture is detected.

Comparing the expression level of miR-139-5p in high-glucose and high-lipid culture and control groups of endothelial clonal cells (ECFC) isolated from peripheral blood of diabetic patients and aortic endothelial cells or umbilical vein endothelial cells (HUVEC) of diabetic animal models, it can be seen from FIG. 1 that the expression of miR-139-5p is up-regulated in four experimental groups.

Example 2:

(1) isolated ECFCs were cultured to passage 5-7 for cell function assays and analyzed for in vitro tubule formation. ECFCs or HUVECs were seeded onto 96-well tissue culture plates coated with 30. mu.L matrix gel (BD Biosciences, San Jose, Calif., USA) at cell densities of 5000 to 20000 cells/well. Cells were observed every 2 hours with an inverted microscope using a 40-fold mirror to observe capillary-like formation. CCK-8 was used to detect cell proliferation, 10 cells⁴The density of individual cells/well was seeded in 96-well plates and cultured at 37 ℃ for 24 hours. Then 10. mu.L of CCK-8 solution was added to the plate. After incubation for 1-4 hours in an incubator, the absorbance at 450nm was measured. The cell migration ability was evaluated by the scratch test.

(2) miR-139-5p mimetics (minics), inhibitors (inhibitors) were transfected with liposomes 3000 when ECFCs were approximately 70% -80% confluent.

As can be seen from FIG. 2, the up-regulation of miR-139-5p can inhibit the proliferation, migration and tube formation functions of ECFC and HUVEC, and the transfection of miR-139-5p inhibitor into ECFC from diabetes can improve the proliferation, migration and tube formation functions of ECFC from diabetes.

This example confirms that miR-139-5p can affect the proliferation, migration and angiogenesis functions of ECFCs and HUVECs cultured in vitro.

Example 3:

the target gene for miR-139-5p was predicted using TargetScan 6.2(http:// www.targetscan.org), PicTa r (http:// picture. mdc-berlin. de), and PITA (genie. weizmann. a c.il/pubs/miR07/miR07_ prediction. html) software, as confirmed by the dual-fluorescein reporter enzyme.

The 3' -UTR of c-jun was predicted by the software described above to contain the conserved site ACTGTAG to which miR-139-5p binds (FIG. 3A); and C-jun expression is respectively down-regulated or up-regulated after the ECFC is transfected by miR-139-5p minics and inhibitors (FIG. 3C); the prediction was subsequently validated by dual luciferase reporter analysis of whether miR-139-5p could recognize c-jun3' -UTR. The pmiR-RB-REPORTTM vector was selected for detection in the assay. Transfection efficiency was normalized by co-transfection with the reporter plasmid lipofectamine 2000 reagent (Invitrogen, Carlsbad, Calif., USA) with the miR-139-5p mimic and the synthetic luciferase-expressing vector, phrG TK (Promega). Luciferase activity was measured on an LB 960Centro XS3 luminometer using dual luciferase reporter assay reagent (Promega). Each experiment was performed in triplicate and the data are presented as mean ± standard deviation of three independent experiments.

The results of prediction and validation are shown in FIGS. 3 (A) and (B), and it can be confirmed that c-jun is the target gene of miR-139-5 p.

And cloning a wild type (Luc-c-jun-3'-UTR) or a mutant sequence (Luc-c-jun-mut3' -UTR) in the predicted binding site to the c end of the luciferase gene to construct a report vector taking RLuc as report luciferase and Luc as control luciferase. And the cloning mutant or wild c-jun3' -UTR vector was treated with a miR-139-5p mimic. The results are shown in FIG. 3 (B).

It can be seen that the relative RLuc/Luc ratios of ECFCs transfected with miR-139-5P and wild-type c-jun3'-UTR were significantly reduced (P <0.05), while the relative RLuc/Luc ratios of ECFCs transfected with miR-139-5P and Mut-c-jun3' -UTR were not significantly changed. This indicates that miR-139-5p can directly target and reduce the expression of c-jun.

Example 4:

as shown in FIG. 4, c-jun siRNA and c-jun plasmid were transfected by liposome 3000 (Invitrogen, Carlsbad, Calif., USA) when ECFCs were approximately 70% -80% fused. After 72h, the cells were subjected to an induced differentiation process. C-jun is silenced or up-regulated, and is shown to have a remarkable regulating effect on the proliferation, migration and angiogenesis of ECFCs by CCK8 detection, Hoechst and EdU staining, a scratch test and an angiogenesis test. The c-jun has the function of regulating the ECFCs.

Example 5:

and co-transfecting the miR-139-5p simulant and c-jun plasmid or the miR-139-5p inhibitor and si-c-jun. As shown in FIG. 5, up-regulation of c-jun can relieve the decrease of migration and tube forming ability of the ECFCs induced by the miR-139-5p mimics, and silencing of c-jun can block the increase of migration and tube forming ability of the ECFCs induced by the miR-139-5p inhibitor. In conclusion, miR-139-5p can regulate the functions of ECFCs through c-jun.

Example 6:

VEGF and PDGF-B levels were determined when ECFCs and HUVECs were transfected with miR-139-5p mimetics or inhibitors.

From fig. 6, it can be found that: the miR-139-5p mimetics or inhibitors modulate the expression of VEGF and PDGF-B consistent with the miR-139-5p mimetics or inhibitors modulating c-jun. And transfection of c-jun expression can reverse miR-139-5p mimetics to inhibit VEGF and PDGF-B expression, while transfection of si-c-jun blocks miR-139-5p inhibitor from enhancing VEGF and PDGF-B expression. That is, miR-139-5p regulates the function of VEGF and PDGF-B on endothelial cells through c-jun.

Example 7:

when ECFCs were pooled to about 70% -80%, the ECFCs were treated with KDR antibody (1:100, BD Biosciences, Bedford, MA, USA) and PDGF-B antibody (1:100, BD Biosciences, Bedford, MA, USA), and incubated for 48h with or without miR-139-5p inhibitor, pancreatically digested cells, seeded onto a matrix, and subjected to a tube formation experiment.

As can be seen in FIG. 7, in KDR and PDGF-B antibody experiments, c-jun was able to exert the function of regulating vascular endothelial cell function via VEGF and PDGF-B.

Example 8: matrix gel plug assays were performed in animals.

Will contain normal/diabetic ECFC (2X 10)⁶Individual cells/gel) were transfected with miR-139-5p mimetics/inhibitors or PBS (500 μ L) mixed and the mixed matrix gel was injected subcutaneously (n ═ 4) into the abdomen of nude mice. After two weeks, the matrigel was isolated from the euthanized mice and paraffin sections were prepared. After preparation, the vessels were stained with CD31 and examined microscopically under a 200-fold magnification.

As can be seen in FIG. 8, immunohistochemical CD31 staining showed that the miR-139-5p mimic inhibits the angiogenesis of ECFCs, while the inhibitor improves the angiogenesis of diabetic ECFCs, suggesting that miR-139-5p modulates the neovascular capacity of ECFCs and is associated with diabetic ECFC injury.

Example 9:

in the present example, SPF-grade nude mice (all purchased from SJA laboratory animals ltd, hannan, province) were selected and the animals were bred under specific conditions according to the regulations of "guidance on ethical treatment of animals", which was approved by the ethical committee of animals in yasan hospital, xiang, university, south China.

(1) Establishing diabetic nude mouse lower limb ischemia model (HLI)

40 male nude mice, 10 weeks old in weight, were intraperitoneally injected with STZ (150mg/kg body weight) after fasting for 12 hours, and blood glucose levels were measured on days 3 and 7 after STZ injection, respectively. Blood glucose levels above 16.7mmol/L, mice showing symptoms of polyuria and polydipsia are considered to be diabetic.

Diabetic nude mice were used to establish HLI models. After anesthesia by intraperitoneal injection of 1% pentobarbital, the entire right superficial femoral artery and vein (from just below the deep femoral artery to the popliteal artery and vein) were ligated with 8-0 silk thread, excised by an electric condenser, and the skin was sutured with 5-0 silk thread. After the mice were awakened, feeding was not restricted and returned to the cage.

(2) After surgery, 30 mice with HLI model were randomly divided into 5 groups. Adopting pseudo operation in group A, injecting PBS in group B, injecting 139-5p antagonist in group C, and injecting 1 × 10 in group D⁶DiI-labeled (CellTracker C7000, Invitrogen, Carlsbad, Calif., USA) ECFC, group E injection antagonist 139-5p, and CM-DiI-labeled ECFCA compound (I) is provided.

To assess limb perfusion rates (ischemic limb [ right ]/normal limb [ left ]), real-time microcirculation imaging analysis was performed at 0, 7, 14 and 21 days post-ischemia using a PeriCam Perfusion Speckle Imager (PSI) based on laser speckle contrast analysis technique. At the same time, the skin temperature was measured with a digital thermometer.

Visual observations at 7, 14, and 21 days after implantation indicated that the implantation resulted in a gradual reddening of the skin color of the ischemic lower limb and improved the appearance of a darkened toenail. Mice treated with a combination of ECFCs and 139-5p antagonist showed the fastest ischemic limb recovery and almost complete reversal of skin darkening and toe gangrene (fig. 10A). At all time points from day 7 to day 21, mice in the ECFC + antagonist 1395P treated group had a significant increase in skin temperature and blood perfusion (P <0.05) over the other groups, as shown in fig. 10(B, C).

Vascular density of ischemic hind limbs was assessed by CD31 expression. The combination of ECFC and 139-5P antagonists (P <0.05) significantly increased vascular density compared to the PBS treated group (P <0.01), fig. 9D.

Using CM-DiI labeling and ECFC tracking, it was further determined whether miR-139 downregulation promoted ECFC entry into ischemic tissue and involvement in angiogenesis. CM-DiI labeled ECFCs were visible around gastrocnemius and blood vessels on day 14 post-transplantation (fig. 9E), and higher CM-DiI labeled cells were observed in the ECFC + antagonist-139-5P combination group than in the ECFC group (P <0.05), which also increased α -SMA + labeled arteries (fig. 9E). These results indicate that ECFC is a potential participant in angiogenesis and that miR-139 down-regulation promotes the involvement of ECFC in angiogenesis.

Example 10:

after 1 week of adaptive feeding of SD rats, high-sugar, high-fat diet was given for 2 weeks. The diet included 0.5% sodium cholate, 2% cholesterol, 4% milk powder, 10% fat, 20% sugar, and 63.5% regular diet. Next, 35mg/kg streptozotocin (Sigma) (100mM citrate buffer solution, pH 4.5) was administered to rats by intraperitoneal injection. The induction of diabetes was confirmed by conducting blood glucose tests on

days

1, 3 and 7 (greater than 16.7mmol/L) after STZ injection. Diabetic rats were anesthetized with isoflurane and the skin of the back was shaved to form a full-thickness circular wound 1.0 cm in diameter.

The rats were then randomized into 4 groups (4 per group) (a) normal negative control group, (b) normal rat miR-139-5p inhibitor subcutaneous injection group, (c) diabetic control group, and (d) DM miR-139-5p antagomiR subcutaneous injection group. The wound was divided into four quadrants, and miR-139-5p antagonist (20 μ g in 100ul saline) was injected directly into the dermis 4 surrounding the wound at

days

0, 2, 4, and 7 post-wound, with 25ul injection at the bottom and edge of the wound for each quadrant. The control group was given an equal amount of inhibitor at the wound side.

The wound and the camera are photographed every day, and as can be seen from fig. 10(A), the expression of miR-139-5p is inhibited by using the inhibitor of miR-139-5p, so that the diabetic wound repair can be promoted.

Obtaining the area of the wound area by using the ImageJ software function, and calculating the wound closure rate by adopting the following formula: wound closure rate (%) (1-non-healed wound area/original area) × 100% (fig. 10 (B)).

This example demonstrates that miR-139-5p antagonists can promote diabetic wound healing, enhance c-jun, VEGF, PDGFA expression in wound tissue, and increase tissue angiogenesis.

The above-mentioned embodiments are merely descriptions of the preferred embodiments of the present invention, and do not limit the concept and scope of the present invention, and various modifications and improvements made to the technical solutions of the present invention by those skilled in the art should fall into the protection scope of the present invention without departing from the design concept of the present invention, and the technical contents of the present invention as claimed are all described in the technical claims.

Sequence listing

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agttggactg ggttgcgtcc tgacggcgcc cccagtgtgc acgagtggga aggacttggc 2160

gcgccctccc ttggcgtgga gccagggagc ggccgcctgc gggctgcccc gctttgcgga 2220

cgggctgtcc ccgcgcgaac ggaacgttgg acttttcgtt aacattgacc aagaactgca 2280

tggacctaac attcgatctc attcagtatt aaagggggga gggggagggg gttacaaact 2340

gcaatagaga ctgtagattg cttctgtagt actccttaag aacacaaagc ggggggaggg 2400

ttggggaggg gcggcaggag ggaggtttgt gagagcgagg ctgagcctac agatgaactc 2460

tttctggcct gccttcgtta actgtgtatg tacatatata tattttttaa tttgatgaaa 2520

gctgattact gtcaataaac agcttcatgc ctttgtaagt tatttcttgt ttgtttgttt 2580

gggtatcctg cccagtgttg tttgtaaata agagatttgg agcactctga gtttaccatt 2640

tgtaataaag tatataattt ttttatgttt tgtttctgaa aattccagaa aggatattta 2700

agaaaataca ataaactatt ggaaagtact cccctaacct cttttctgca tcatctgtag 2760

atactagcta tctaggtgga gttgaaagag ttaagaatgt cgattaaaat cactctcagt 2820

gcttcttact attaagcagt aaaaactgtt ctctattaga ctttagaaat aaatgtacct 2880

gatgtacctg atgctatggt caggttatac tcctcctccc ccagctatct atatggaatt 2940

gcttaccaaa ggatagtgcg atgtttcagg aggctggagg aaggggggtt gcagtggaga 3000

gggacagccc actgagaagt caaacatttc aaagtttgga ttgtatcaag tggcatgtgc 3060

tgtgaccatt tataatgtta gtagaaattt tacaataggt gcttattctc aaagcaggaa 3120

ttggtggcag attttacaaa agatgtatcc ttccaatttg gaatcttctc tttgacaatt 3180

cctagataaa aagatggcct ttgcttatga atatttataa cagcattctt gtcacaataa 3240

atgtattcaa ataccaa 3257

Claims

1. The mRNA related to diabetic vascular injury is characterized in that the microRNA is miR-139-5p, and the sequence of the mRNA is as follows: the nucleotide sequence is shown as SEQ ID No.1_。

2. A target gene of mRNA according to claim 1, wherein the target gene is c-jun sequence, and the nucleotide sequence thereof is shown in SEQ ID No. 2.

3. The mRNA related to diabetic vascular injury according to claim 1, wherein diabetes induces up-regulation of miR-139-5p expression of vascular endothelial cells and endothelial clonal cells, miR-139-5p expression up-regulation inhibits proliferation, migration and tube formation of endothelial cells and diabetic endothelial clonal cells, and miR-139-5p expression down-regulation improves proliferation, migration and angiogenesis capacity of diabetic endothelial cells.

Use of an antagonist of miR-139-5p in preparation of a medicament for repairing a wound of a diabetic patient.

5. The use of claim 4, wherein the administration sites are the bottom and the edge of a wound surface, the administration mode is injection, and the concentration of the miR-139-5p antagonist in the injection is 0.2 mg/mL.

Application of the inhibitor of miR-139-5p in preparation of products or medicines for preventing or treating vascular injury complications caused by diabetes.

7. The use according to claim 6, wherein the vascular damaging condition is selected from one or more of vascular endothelial cell damage, endothelial progenitor cell damage, impairment of the regenerative function of blood vessels.

8. The composition contains an inhibitor of miR-139-5p and a target gene c-jun, wherein the miR-139-5p is combined with mRNA molecules of the c-jun, and the composition is used for preparing a product or a medicament for preventing or treating vascular injury complications caused by diabetes.

9. The use according to claim 8, wherein the vascular injury disorder is selected from one or more of vascular endothelial cell injury, endothelial progenitor cell injury, impairment of the regenerative function of blood vessels.

10. The use of claim 9, wherein the inhibitor of miR-139-5p inhibits the expression of miR-139-5p in endothelial cells and ECPC cells, improving cell proliferation, migration, and tube formation ability; the composition is prepared by combining an antagonist of miR-139-5p with ECPC (endothelial cell activator protein) and is applied to in-vivo angiogenesis; by up-regulating the expression of c-jun, the proliferation, migration and tube forming capability of endothelial cells and ECPC cells are promoted.