Use of miR-342-5p in preparation of a drug for preventing and treating lower limb ischemic diseases

Abstract

The application discloses an application of miR-342-5p in preparation of a medicine for preventing and treating lower limb ischemic diseases, belongs to the technical field of biological medicines, and can significantly improve the function of endothelial cells in muscle tissues, promote the arteriolization level of endothelial cells, thereby promoting the recovery of blood perfusion, improving lower limb ischemia, and enhancing the functional recovery of lower limb muscles; miR-342-5p can reduce the mRNA and protein levels of EYA3, directly targets EYA3 to regulate the phosphorylation level of MYC, arrests the cell cycle, and promotes the arteriolization of endothelial cells; miR-342-5p can reduce the vascular density, increase the levels of endothelial cell arteriolization marker genes GJA4 and alpha-SMA, thereby increasing the arteriolization capacity of endothelial cells.

Description

TECHNICAL FIELD

[0001] The application belongs to the technical field of biological medicine, and particularly relates to application of miR-342-5p in preparation of a medicine for preventing and treating ischemic diseases. BACKGROUND

[0002] Peripheral arterial disease (PAD) refers to a disease that causes intermittent claudication, ulcer or gangrene in lower limbs due to insufficient blood perfusion caused by stenosis or occlusion of lower limb arteries, and further causes hypoxic necrosis of blood supply tissues.

[0003] At present, the pathogenesis of peripheral arterial disease is not clear, so the peripheral arterial disease is mainly treated by surgical intervention and drug treatment. However, the surgery itself has the disadvantages of trauma and incomplete removal, leading to high recurrence and difficulty in treatment. The existing drugs have the disadvantages of slow effect and long cycle, leading to stagnation of the treatment of peripheral arterial disease.

[0004] miRNA is a kind of endogenous, small non-coding RNA with a length of about 21-25 nucleotides, which can participate in various cell behaviors such as cell proliferation, differentiation and apoptosis. As a kind of very important life activity regulating factor, in recent years, it is found that miRNA exists in blood and tissue fluid in the form of exosome wrapping or protein binding, so it can be used as a circulating marker and a therapeutic target to participate in the treatment of various ischemic diseases. However, due to the limitation of current technology and the limitation of understanding of miRNA, how to accurately target and deliver the miRNA capable of treating ischemic diseases to the injury site is still a technical barrier at present, and the mechanism of the influence of miRNA on ischemic diseases is still unclear. SUMMARY

[0005] The purpose of the present application is to overcome the shortcomings of the prior art, and to provide application of miR-342-5p in preparation of a medicine for preventing and treating lower limb ischemic diseases. The miR-342-5p can improve the arterialization level of endothelial cells, promote the recovery of blood perfusion, and improve the lower limb ischemia and enhance the functional recovery of lower limb muscles.

[0006] The first purpose of the present application is to provide application of miR-342-5p in preparation of a medicine for preventing and treating lower limb ischemic diseases.

[0007] Preferably, the medicine is a medicine for reducing lower limb ischemia and blood perfusion loss.

[0008] Preferably, the medicine is a medicine for reducing blood vessel density and increasing the levels of endothelial cell arterial marker gene GJA4 and alpha-SMA.

[0009] Preferably, the drug is a drug that reduces the phosphorylation level of MYC protein, arrests the cell cycle, and promotes the arteriolization of endothelial cells.

[0010] Preferably, the drug is a drug that reduces the mRNA level of EYA3 in endothelial cells.

[0011] A second object of the present application is to provide an application of miR-342-5p as a target in the preparation of a drug for preventing and treating lower limb ischemic diseases.

[0012] Preferably, the drug is a drug that regulates the expression of miR-342-5p at the genetic and / or protein level.

[0013] Preferably, the drug is a drug that synergizes and / or assists miR-342-5p in reducing the vascular density and increasing the levels of endothelial cell arteriolization marker genes GJA4 and alpha-SMA.

[0014] Preferably, the drug is a drug that synergizes and / or assists miR-342-5p in reducing the phosphorylation level of MYC protein.

[0015] Preferably, the drug is a drug that synergizes and / or assists miR-342-5p in reducing the mRNA level of EYA3 in endothelial cells.

[0016] Compared with the prior art, the present application has the following beneficial effects:

[0017] (1) miR-342-5p can significantly improve the function of endothelial cells in muscle tissue, promote the arteriolization level of endothelial cells, thereby promoting the recovery of blood perfusion and improving lower limb ischemia, enhancing the functional recovery of lower limb muscles, and further enriching the types and pathogenesis of therapeutic angiogenesis, so as to provide new ideas and targets for the treatment of peripheral arterial diseases;

[0018] (2) miR-342-5p can reduce the mRNA and protein levels of EYA3, directly target EYA3 to regulate the phosphorylation level of MYC, arrest the cell cycle, and promote the arteriolization of endothelial cells.

[0019] (3) miR-342-5p can reduce the vascular density and increase the levels of endothelial cell arteriolization marker genes GJA4 and alpha-SMA, thereby increasing the arteriolization ability of endothelial cells. BRIEF DESCRIPTION OF DRAWINGS

[0020] Figure 1 Figure 1 is a diagram of the construction of a lower limb ischemia model in the embodiments of the present application; wherein:

[0021] Figure 1(a) is a real object diagram after the right femoral artery of the mouse is ligated after anesthesia; Figure 1 (b) is a blood flow blockage diagram of the fluorescent speckle imaging instrument after the right femoral artery of the mouse is ligated;

[0022] Figure 2 The statistical result diagram of the expression amount of miR-342-5p in the femoral artery group of the mouse ligated with the femoral artery and the control group;

[0023] Figure 3 The quantitative result diagram of miR-342-5p co-localized with endothelial cell marker CD31 in the femoral artery group of the mouse ligated with the femoral artery and the control group;

[0024] Figure 4 The timeline diagram of constructing the hindlimb ischemia model and intramuscular injection of miR-342-5p;

[0025] Figure 5 The blood perfusion diagram of the experimental group and the control group of the mouse at different time periods;

[0026] Figure 6 The quantitative analysis diagram of the expression amount of arterial marker GJA4 in the muscle of different regions of the experimental group and the control group of the mouse;

[0027] Figure 7 The quantitative analysis diagram of the expression amount of α-SMA in the muscle of different regions of the experimental group and the control group of the mouse;

[0028] Figure 8 The detection result diagram of arterial markers of the mimic of miR-342-5p and the blank control group Ctrl by qRT-PCR;

[0029] Figure 9 The detection result diagram of arterial markers of the mimic of miR-342-5p and the blank control group Ctrl by western blotting;

[0030] Figure 10 The observation diagram of the blood vessel density of the experimental group matrix glue and the control group matrix glue under the confocal microscope after masson staining;

[0031] Figure 11 The quantitative analysis diagram of Figure 10 ;

[0032] Figure 12 The detection result diagram of the endothelial cell arteriogenesis ability of the experimental group matrix glue and the control group matrix after immunofluorescence staining;

[0033] Figure 13 The quantitative analysis diagram of Figure 12 ;

[0034] Figure 14 The retinal artery area of ​​the experimental group and the control group after immunofluorescence staining was observed under a confocal microscope;

[0035] Figure 15 for Figure 14 Quantitative analysis chart of

[0036] Figure 16 The figure shows the detection results of MYC mRNA levels in miR-342-5p mimic and blank control group Ctrl;

[0037] Figure 17 The figure shows the detection results of MYC protein levels in miR-342-5p mimic and blank control group Ctrl;

[0038] Figure 18 This is a diagram showing the changes in phosphorylation sites of MYC protein in the mimic of miR-342-5p and the blank control group;

[0039] Figure 19 The diagram shows the changes in each phase of the cell cycle in the mimic of miR-342-5p and the blank control group Ctrl;

[0040] Figure 20 This is a comparison of the results of dual-luciferase reporter gene verification of EYA3 wild type and mutant types;

[0041] Figure 21 Figure 1 is the predicted binding sequence diagram of miR-342-5p and EYA3;

[0042] Figure 22 The graph shows the results of detecting the mRNA level of EYA3 in miR-342-5p mimic and the blank control group Ctrl;

[0043] Figure 23 The graph shows the protein level detection results of EYA3 in miR-342-5p mimic and blank control group Ctrl;

[0044] Figure 24 for Figure 23 Quantitative analysis chart of

[0045] Figure 25 This is a graph showing the results of MYC phosphorylation level detection in human umbilical vein endothelial cells infected with EYA3 overexpression lentivirus group and control lentivirus group;

[0046] Figure 26 Protein expression of arterial markers in human umbilical vein endothelial cells infected with EYA3 overexpression lentivirus group and control lentivirus group. DETAILED DESCRIPTION

[0047] Exemplary embodiments of the present disclosure will be described in more detail below with reference to the accompanying drawings. Although exemplary embodiments of the present disclosure are shown in the accompanying drawings, it should be understood that the present disclosure can be implemented in various forms and should not be limited by the embodiments set forth above. Rather, these embodiments are provided to enable a more thorough understanding of the present disclosure and to fully convey the scope of the present disclosure to those skilled in the art.

[0048] The present invention discloses a novel miR-342-5p, the sequence of which is 5'-CGGAGGGGTGCTATCTGTGATTGAG-3'. ...

[0049] Example 1

[0050] The expression of miR-342-5p was decreased in the established lower limb ischemia model

[0051] (1) Prepare C57BL / 6 wild-type mice (aged 6-8 weeks, weighing approximately 20 g), prepare the skin with depilatory cream and surgical instruments, disinfect with 75% alcohol, incise the skin, fully expose the arteries, veins, and nerves of the right hind limb of the mouse, isolate the femoral artery without damaging the accompanying femoral vein and nerve, ligate the femoral artery at a high position with 0.4 mm surgical sutures, suture the skin and muscularis, and complete the construction of the lower limb ischemia model;

[0052] (2) Observe the ligation and blood perfusion of the mouse's hind limbs using a Rayward fluorescence speckle imaging system to ensure that the blood flow to the lower limbs is completely blocked;

[0053] (3) After the lower limb ischemia model is completed, the construction of the model is observed under the laser Doppler perfusion imaging system, such as Figure 1 As shown, Figure 1 (a) is a photo of anesthetized mice after ligation of the right femoral artery. Figure 1 (b) is a fluorescence speckle imaging image of the femoral artery of the right lower limb of an anesthetized mouse. Figure 1 (b) It can be seen that in mice with ligated right femoral artery, blood perfusion in both hindlimbs was significantly reduced, indicating that the hindlimb ischemia model was successfully established;

[0054] (4) 24 hours after modeling, mice were anesthetized with 10% chloral hydrate at a dose of 100-120 μl / mouse, and PBS was perfused into the heart. The femoral arteries below the ligature on the ligated side and the normal side were isolated under a stereomicroscope, rinsed with physiological saline, and placed in 500 μl of RNA extraction agent TRIZOL. The blood vessels were completely ground in a homogenizer, and the femoral artery RNA was extracted, quantified, and reversed using the conventional method. The femoral artery RNA of the control group (mice were not ligated) was extracted, quantified, and reversed using the same method. The expression of miR-342-5p in the femoral artery on the ligated side and the femoral artery in the control group was detected using qRT-PCR technology. The test results are shown as follows: Figure 2 As shown in the figure, * indicates that P < 0.05 is considered to be statistically different. Figure 2 It can be seen that the expression of miR-342-5p in the femoral artery on the ligated side was significantly lower than that in the control group;

[0055] (5) 24 hours after modeling, mice were anesthetized with 10% chloral hydrate at a dose of 100-120ul / mouse, and PBS was perfused into the heart. The femoral arteries below the ligature on the ligated and normal sides (experimental group) were isolated under a stereomicroscope and fixed with 4% paraformaldehyde for 3-4 hours, fixed and embedded in paraffin. Tissue sections were treated with proteinase K (20μg / mL) and hybridized with miR-342-5p probe at 37°C for 2 hours, then washed with 2×SSC at 37°C for 10 minutes, 1×SSC twice for 5 minutes each, and 0.5×SSC at room temperature for 10 minutes. The tissue was then blocked with 5% BSA at room temperature for 30 minutes and incubated with anti-CD31 antibody (1:200, R&D) at 4°C overnight. After washing, the tissue was incubated with donkey anti-goat IgG conjugated to Alexa Fluor 488 for 2 hours at room temperature, and the control group (mice were not ligated) was treated in the same manner. The quantitative results of the above experiment are shown in Figure 2. Figure 3 As shown, ** indicates that P < 0.001 has statistical significance. Figure 3 It can be seen that compared with the control group, the expression level of miR-342-5p in the ligated side mice was significantly decreased.

[0056] The above studies show that the expression level of miR-342-5p is reduced in the constructed lower limb ischemia model.

[0057] Example 2

[0058] Inhibition of miR-342-5p on lower limb ischemia and blood perfusion injury

[0059] (1) On the 1st day and the 7th day after the establishment of the ischemic model of the lower limbs of Example 1, respectively, 10 μl of a microsyringe was used to inject the in vivo agonist agomir (5 nmol) of miR-342-5p for animals (a double-stranded miRNA agonist specially modified to mimic endogenous miRNA) into the muscles of the posterior limbs of the mice on the side of the ligation as a test group, and the same method was used to inject NC 5 nmol into the muscles of the near and far ends in three points as a blank control group;

[0060] (2) On the 0th day, the 7th day and the 14th day after the ligation of the posterior limbs, the blood perfusion of the posterior limbs of the mice of the test group and the blank control group was observed under a fluorescence speckle imaging system, which was used to compare the blood perfusion at different time periods in the later stage, and the timeline is shown in Figure 4 , and the blood perfusion of the mice of the test group and the blank control group at different time periods is shown in Figure 5 . It can be seen from Figure 5 that the blood flow of the blank control group was basically blocked after the femoral artery of the mice was ligated, and the blood perfusion of the test group was significantly better than that of the blank control group on the 7th day and the 14th day;

[0061] (3) On the 14th day after the ligation of the posterior limbs, the mice of the test group and the blank control group were anesthetized with 10% chloral hydrate, and after the heart was perfused with PBS, the semitendinosus and gastrocnemius muscles of the left and right posterior limbs of the mice were fixed in 4% paraformaldehyde for 3-4 hours, then dehydrated in 30% sucrose overnight, and then embedded in OCT and cut into 0.8 mm thick slices for immunofluorescence staining under a freezing microtome. The cut slices were air-dried at room temperature for 2 hours, and after the circle was drawn with a histological pen, a proper amount of 4% paraformaldehyde was added for fixation for 10 minutes, and then PBS was shaken and washed for 3 times, each for 8 minutes. The blocking solution containing 1% BSA and 0.5% Triton X-100 was added to the slices for blocking at 4°C for 15 minutes. The blocking solution containing the primary antibody (artery marker gene GJA4 and α-SMA recruited around the artery) was added to the slices for overnight incubation at 4°C. PBS was shaken and washed for 3 times, each for 8 minutes, and after the secondary antibody was incubated, the slices were sealed, and then the expression amount of GJA4 (1:200, abcam) and α-SMA (1:200, abcam) in the muscles of the mice of the test group and the blank control group in different regions (semitendinosus and gastrocnemius) was observed under a Nikon confocal microscope. The quantitative analysis results of the expression amount of GJA4 and the expression amount of α-SMA are shown in Figure 6 and Figure 7 , and it can be seen from Figure 6 and Figure 7It can be seen that compared with the blank control group (ctrl group), the expression levels of arterial markers GJA4 and α-SMA in muscles of different regions (semitendinosus and gastrocnemius) of mice increased after injection of the miR-342-5p in vivo agonist agomir, further indicating that the injection of miR-342-5p can promote endothelial cell arterialization and increase blood perfusion.

[0062] Example 3

[0063] Regulation of miR-342-5p on the expression level of arterial marker genes

[0064] 1 mg / mL type I collagenase (ScienCell, San Diego, CA) was added to human umbilical cords, and HUVECs were isolated and maintained in endothelial cell culture medium (ECM, ScienCell, San Diego, CA) containing 5% fetal calf serum (FCS), 1× endothelial cell growth factor (ECGS), 100 U / mL ampicillin, and 100 mg / mL streptomycin. Human umbilical vein endothelial cells were transfected with miR-342-5p mimic and its blank control (50 nM, Guangzhou Ruibo Bio) using Lipofectamine 2000 (Invitrogen, Carlsbad, CA). After 4 hours, the ECM medium was replaced and the cells were cultured in a cell incubator at 37°C for 48 hours. Samples were collected, and RNA and protein were extracted. The use of human samples was approved by the institutional ethics committee. Arterial markers of miR-342-5p mimic and blank control were detected by qRT-PCR and western blotting, respectively. The test results are shown in the figure. Figure 8 and Figure 9 As shown, through Figure 8 It can be seen that compared with the blank control group, overexpression of miR-342-5p in human umbilical vein endothelial cells can significantly increase the mRNA expression of some arterial markers such as HEY2, EFNB2, GJA4 and GJA5, and can actually increase the mRNA expression of markers such as HES1, HEY1, SOX17, etc. Figure 9 As can be seen, compared with the blank control group, western blotting experiments also confirmed that overexpression of miR-342-5p can significantly upregulate the expression of arterial markers EFNB2 and GJA4 at the protein level and downregulate the expression of venous marker EPHB4. Therefore, overexpression of miR-342-5p mimic can significantly increase the expression of arterial markers and downregulate the expression of venous marker EPHB4.

[0065] Example 4

[0066] miR-342-5p regulates vascular density and endothelial cell arterial marker genes (Matrigel plug experiment is an experiment that simulates angiogenesis in animals)

[0067] (1) C57BL / 6 mice (6-8 weeks old) of comparable weight were anesthetized and the skin was prepared with surgical instruments and 75% disinfected;

[0068] (2) Angiogenic factor (VEGF, 400 ng / mL), basic fibroblast growth factor (bFGF, 250 ng / mL) (Sino Biological, Beijing, China) and Matrigel were thoroughly mixed with agomir of miR-342-5p or blank control Ctrl (8 μg / mL) on ice to obtain Matrigel of the experimental group and Matrigel of the control group, respectively.

[0069] (3) After pre-cooling the syringes and surgical instruments used in the experiment at -20°C, 0.3 mL of the experimental group Matrigel and the control group Matrigel were injected subcutaneously along the lateral side of the mouse's ventral midline using a 1 mL syringe. The injection was allowed to rest for more than 1 minute before being slowly withdrawn.

[0070] (4) On day 7, the mice were anesthetized and perfused with PBS into the heart. The Matrigel was carefully removed and immunofluorescence staining was performed in the same manner as in Example 2.

[0071] (5) Observe the matrix gel of the experimental group and the matrix of the control group after immunofluorescence staining under a confocal microscope. The observation results are as follows: Figure 10 As shown, through Figure 10 It can be seen that compared with the control group, the blood vessel density in the mice injected with the miR-342-5p agonist agomir was reduced. Figure 11 for Figure 10 Quantitative analysis chart of Figure 11 Quantitative analysis of vascular area showed that overexpression of miR-342-5p could reduce vascular density compared with the control group. Figure 12 It can be seen that compared with the control group, the arterialization ability of endothelial cells injected with the miR-342-5p agonist agomir was increased (the expression of arterial marker gene GJA4 and the recruitment of α-SMA around the arteries were significantly increased). Figure 13 for Figure 12 The quantitative analysis of the expression of arterial marker GJA4 and α-SMA recruited around the arteries further confirmed that overexpression of miR-342-5p can reduce vascular density and promote the level of endothelial cell arterialization.

[0072] Example 5

[0073] Overexpression of miR-342-5p in the neonatal mouse retina regulates endothelial cell arterialization

[0074] (1) Prepare newborn C57BL / 6 mice on day 3 of life and anesthetize them on ice before proceeding to the following steps;

[0075] (2) 0.5 μl of miR-342-5p in vivo agonist agomir (experimental group) or blank control group (Ctrl group) dissolved in RNase-free phosphate-buffered saline (PBS) was injected into the vitreous of mice under a stereomicroscope using a microinjector. After rewarming, the newborn mice were returned to their nests.

[0076] (3) Retinas were collected on the fifth day after birth and fixed in 4% paraformaldehyde (PFA) for 4 hours. They were then blocked and permeabilized in PBS containing 1% bovine serum albumin (BSA) and 0.5% Triton X-100 at 4°C overnight.

[0077] (4) Then, the immunofluorescence staining operation consistent with the steps of Example 2 was performed, and then the retinas of the test group and the control group after immunofluorescence staining were observed under a confocal microscope. The observation results are as follows: Figure 14 As shown, through Figure 14 It can be seen that compared with the control group, the injection of miR-342-5p can lead to a significant increase in the area of ​​α-SMA+ recruited around the arteries, indicating that miR-342-5p can promote the arterialization of endothelial cells. Figure 15 for Figure 14 Quantitative analysis chart of Figure 15 The results further confirmed that miR-342-5p can promote endothelial cell arterialization.

[0078] Example 6

[0079] Inhibition of MYC protein phosphorylation by miR-342-5p

[0080] (1) Human umbilical vein endothelial cells were transfected with miR-342-5p mimic and its blank control Ctrl (50 nM, Guangzhou Ruibo Biotechnology) using Lipofectamine 2000 (Invitrogen, Carlsbad, CA). After 4 hours, the ECM culture medium was replaced and the cells were cultured in a cell incubator at 37°C for 48 hours. Samples were collected, RNA and protein were extracted, and the use of human samples was approved by the ethics committee. The mRNA and protein levels of MYC in the miR-342-5p mimic and the blank control Ctrl were detected by qRT-PCR and western blotting, respectively. The test results are as follows: Figure 16 and Figure 17As shown in Figure 16 and Figure 17 The results show that miR-342-5p can inhibit the mRNA level and protein level of MYC.

[0081] (2) The same as step (1) above, the protein sample collected after 48 hours of culture of the cells treated with miR-342-5p or blank control group was used to confirm the change of the phosphorylation site of MYC protein in endothelial cells after overexpression of miR-342-5p, increase the degradation of MYC (pT58), reduce the stability of MYC (pS62), inhibit MYC, affect cell proliferation, and cause cell cycle arrest, as shown in Figure 18 .

[0082] (3) The same as step (1) above, the cell sample collected after 48 hours of culture of the cells treated with miR-342-5p or blank control group was used, and the cells were trypsinized and washed with PBS for 3 times at 1300 rpm for 5 minutes. 75% alcohol was used for fixation for 2 hours, and after fixation, the blocking solution containing 1% BSA and 0.5% Triton X-100 was used for blocking at 4°C for 30 minutes. The FACSCalibur flow cytometer (BD immune cell detection system) was used to analyze the cells by fluorescence activated cell sorting, and the Modfit software was used to calculate the cells at each stage, and the results are shown in Figure 19 . Figure 19 It can be seen that overexpression of miR-342-5p in human umbilical vein endothelial cells can significantly affect the cell cycle, increase the cells staying in G0 / G1 and G2 / M phases, and reduce the cells staying in S phase, resulting in cell cycle arrest.

[0083] (4) miR-342-5p directly targets EYA3.

[0084] The dual luciferase reporter gene experiment confirmed that EYA3 is a direct target gene of miR-342-5p. Referring to the reference "Notch activation promotes endothelial quiescence by repressing MYC expression via miR-218. Mol Ther Nucleic Acids 25:554-566", the PGL3-promter EYA3'UTR plasmid and Firefly luciferase&Rinella luciferase dual luciferase reporter gene system were constructed, and the EYA3 mutant plasmid was constructed by Hanheng Biotechnology Co., Ltd.

[0085] HEK293T cells were co-transfected with miR-342-5p mimics or blank control, EYA3-3'UTR-wt (100 ng) or EYA3-3'UTR-mut (100 ng), and phRL-TK (5 ng). Cells were lysed 48 hours after transfection, and luciferase activity was measured and compared using a dual-luciferase reporter gene assay system (Promega). Figure 20 As shown, through Figure 20 It can be confirmed that EYA3 is a direct target gene of miR-342-5p compared with the blank control group, and the binding effect disappears after mutation. Overexpression of miR-342-5p in 293T cells confirmed its direct binding to EYA3. The predicted binding sequence of miR-342-5p and EYA3 is as follows Figure 21 shown.

[0086] (5) The cells were treated as in step (1) above and RNA and protein samples were collected after 48 hours of culture in the overexpression miR-342-5p or blank control group. The mRNA and protein levels of EYA3 in the mimic of miR-342-5p and the blank control group Ctrl were detected by qRT-PCR and western blotting, respectively. The test results were as follows: Figure 22 and Figure 23 As shown, Figure 24 for Figure 23 Quantitative analysis chart of Figure 22- Figure 24 It can be seen that compared with the blank control group, overexpression of miR-342-5p can reduce the mRNA and protein levels of EYA3. MiR-342-5p can directly target EYA3 to regulate the phosphorylation level of MYC, arrest the cell cycle, and promote the arterialization of endothelial cells.

[0087] (6) After 24 hours of infection of human umbilical vein endothelial cells with EYA3 overexpression lentivirus and control lentivirus, the cells were treated as in step (1) above and protein samples (MOI = 10) were collected after 48 hours of culture with overexpression of EYA3 and miR-342-5p. The EYA3 overexpression lentivirus was purchased from Hanheng Biotechnology Co., Ltd. Western blotting confirmed that overexpression of EYA3 could rescue the phosphorylation level of MYC affected by overexpression of miR-342-5p, and had a rescue effect, such as Figure 25 As shown. Figure 26It is proved that overexpression of EYA3 can reduce the protein expression of arterial marker EFNB2, SOX17 and GJA4 which are significantly up-regulated after overexpression of miR-342-5p. The experiment further proves that EYA3 is a direct target gene of miR-342-5p, and miR-342-5p affects the phosphorylation level of MYC protein through EYA3, causes cell cycle arrest, promotes arterialization of endothelial cells, increases recovery of blood perfusion, and thus relieves ischemic diseases.

[0088] In summary, the present application proposes a new miR-342-5p which can significantly improve the function of endothelial cells in muscle tissue, promote the arterialization level of endothelial cells, and thus promote the recovery of blood perfusion, improve lower limb ischemia, and enhance the functional recovery of lower limb muscles, further enriching the types and pathogenesis of therapeutic angiogenesis, so as to provide new ideas and targets for the treatment of peripheral arterial diseases; and miR-342-5p can reduce the mRNA and protein levels of EYA3, directly target EYA3 to regulate the phosphorylation level of MYC, arrest the cell cycle, and promote the arterialization of endothelial cells; miR-342-5p can reduce the vascular density and increase the levels of endothelial arterial marker GJA4 and alpha-SMA, thereby increasing the arterialization ability of endothelial cells.

[0089] Obviously, those skilled in the art can make various modifications and variations to the present application without departing from the spirit and scope of the present application. Thus, if these modifications and variations of the present application fall within the scope of the claims of the present application and their equivalent technologies, the present application also intends to include these modifications and variations.

Claims

1. The use of miR-342-5p in the preparation of a drug for preventing and treating lower limb ischemic diseases, characterized in that, The drug is a drug with the function of reducing lower limb ischemia and blood perfusion loss.

2. Use according to claim 1, characterized in that, The drug is a drug with the function of reducing vascular density and increasing the levels of endothelial cell arterial marker gene GJA4 and α-SMA.

3. Use according to claim 1, characterized in that, The drug is a drug with the function of reducing the phosphorylation level of MYC protein, arresting cell cycle and promoting arterialization of endothelial cells.

4. Use according to claim 1, characterized in that, The drug is a drug with the function of reducing the mRNA level of EYA3 in endothelial cells.

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