Application of miR-301a-5p in preparation of medicine for repairing acute skin wound

Abstract

The invention discloses an application of miR-301a-5p in preparation of a medicine for repairing acute skin wounds. According to the medicine for repairing the acute skin wound, miR-301a-5p is taken as a medicine target spot, and the miR-301a-5p is subjected to overexpression. According to an in-vivo model of whole-cortex skin injury, the miR-301a-5p can be used for reducing inflammation infiltration of an acute skin wound part, so that the healing of a mouse skin wound is accelerated. The miR-301a-5p can be used as a new drug target for promoting acute skin wound repair for the first time, and has important significance on development and screening of skin wound repair drugs.

Description

Technical Field

[0001] The present invention belongs to the field of biomedicine technology, and specifically relates to the application of miR-301a-5p in the preparation of a drug for repairing acute skin trauma. Background Art

[0002] Acute skin trauma is a common clinical pathological injury, and its repair process involves a multi-stage dynamic balance of inflammation regulation, cell proliferation and tissue remodeling. Although traditional treatments (such as growth factors, antibiotics, etc.) can relieve some symptoms, there are still problems such as long repair cycle, high scar formation rate and insufficient targeting. In recent years, microRNAs (miRNAs) have entered our field of vision as an endogenous non-coding small molecule RNA, which has the characteristics of high specificity and high sensitivity. MiRNA can accurately identify and bind to target mRNA, regulate gene expression at the post-transcriptional level, and thus affect the biological function of cells. It is short in length and plays a key role in various biological processes such as cell proliferation, differentiation, and apoptosis. According to previous studies, miRNAs are involved in the regulation of almost every stage of wound healing. At present, there is a lack of research on the treatment and potential drug targets of miRNAs in acute wound healing.

[0003] The present invention aims to provide application of miR-301a-5p in preparing a drug for repairing acute skin trauma. Summary of the invention

[0004] The purpose of the present invention is to provide the use of miR-301a-5p in the preparation of a drug for repairing acute skin trauma.

[0005] The object of the present invention is achieved by using miR-301a-5p in the preparation of a drug for repairing acute skin trauma, wherein the drug for repairing acute skin trauma targets the IKKβ gene and overexpresses miR-301a-5p.

[0006] The beneficial effects of the present invention are as follows: The present invention has experimentally proved that by up-regulating the expression of miR-301a-5p, the proliferation and migration of macrophages can be promoted. Mechanistically, miR-301a-5p targets IKKβ to inhibit the NF-κB signaling pathway, reduce the production of macrophage inflammatory factors, and promote the transformation of macrophages from MI type to MII type. In addition, through the in vivo model of full-thickness skin injury, it can be seen that miR-301a-5p can reduce the inflammatory infiltration of acute skin wound sites, thereby accelerating the healing of mouse skin wounds. The present invention proposes for the first time that miR-301a-5p can be used as a new drug target for promoting the repair of acute skin wounds, which is of great significance for the development and screening of skin wound repair drugs. BRIEF DESCRIPTION OF THE DRAWINGS

[0007] Figure 1 Figure 1 shows differentially expressed miR-301a-5p during wound healing in mouse skin; blue in Figure a represents low expression and red represents high expression. The expression profile of skin MiRs is from wound tissues of normal mouse skin on the first and seventh days; the heat map shows the significant changes in miRs; Figure b is a schematic diagram of C57BL / 6j mouse modeling; Figure c is qRT-PCR detection of miR-301a-5p expression in mouse skin wound tissues at 0h, 24h, and 3days; Figure (d) is in situ hybridization detection of miR-301a-5p expression and localization in skin wound tissues; Figure 2 The effects of miR-301a-5p on the production of inflammatory factors by macrophages: Figures a and b are ELISA detection of the expression of inflammatory factors IL-6 and TNF-α by overexpressing miR-301a-5p; Figures c and d are ELISA detection of the expression of inflammatory factors IL-6 and TNF-α by inhibiting the expression of miR-301a-5p; Figure eg is RT-qPCR detection of the mRNA expression levels of inflammatory factors IL-6, TNF-α, and IL-1β; Figure hj is RT-qPCR detection of the mRNA expression levels of inflammatory factors IL-6, TNF-α, and IL-1β; Figures k and l are WB detection of the protein level expression changes and quantification results of the inflammatory factor IL-6 at the cell level; Figures m and n are WB detection of the protein level expression changes and quantification results of the inflammatory factor TNF-α at the cell level; Figures o and p are WB detection of the protein level expression changes and quantification results of the inflammatory factor IL-1β at the cell level; Figure 3 Figure 1 is the RNA sequencing result and the interaction between miR-301a-5p and IKKβ; Figure a is the number of up- and down-regulated genes of DEGs. Figure b is the signal pathway involved in the differentially expressed mRNA in KEGG enrichment analysis; Figure c is a Veen diagram showing the predicted target number and intersection gene number of miR-301a-5p predicted by the website Targetsca (blue) and MirDB (orange); Figure d is the binding site of miR-301a-5p and IKKβ predicted by the website; Figure e is the quantification of the results of the dual luciferase reporter gene experiment; Figures f and g are WB detection of IKKβ protein expression after overexpression or inhibition of miR-301a-5p; Figure h is RT-qPCR detection of IKKβ mRNA expression level after overexpression of miR-301a-5p for 24 hours; Figures i and j are immunofluorescence detection of the fluorescence expression intensity of IKKβ after overexpression of miR-301a-5p; Figure 4The effect of miR-301a-5p on skin wound healing; Figure a shows the use of Agomir-301a-5p to subcutaneously inject at three points around the mouse skin wound, and the expression of miR-301a-5p was detected by RT-qPCR; Figures b and c are the full-thickness skin wound healing pictures of mice and the quantification of wound healing results; Figures d and e are the Masson staining pictures of skin wound tissues on the 7th day after the injection of miR-301a-5p Agomir and the quantification of the results; Figure 5 These are representative images of H&E staining of skin wound tissues on the 4th and 7th days after injection of Agomir NC and miR-301a-5p Agomir; Figure 6 These are the H&E epidermal thickness quantification results of skin wound tissue on the 4th and 7th days after injection of Agomir NC and miR-301a-5p Agomir. DETAILED DESCRIPTION

[0008] The present invention will be further described in detail below in conjunction with the accompanying drawings and embodiments, but the present invention is not limited in any way. Any changes or improvements made based on the teachings of the present invention fall within the protection scope of the present invention.

[0009] The present invention provides application of miR-301a-5p in preparing a drug for repairing acute skin trauma. The drug for repairing acute skin trauma takes miR-301a-5p as a drug target and overexpresses miR-301a-5p.

[0010] The skin wound repairing drug overexpresses miR-301a-5p and targets IKKβ to exert anti-inflammatory effects, thereby achieving the effect of promoting the repair of acute skin (non-cancerous wound) wounds.

[0011] Example 1 Detection of the expression location of miR-301a-5p in skin tissue Experimental methods: 1. In situ staining experiment Mouse skin wound tissues were obtained at 0h, 24h, and 3d, fixed with RNase Free tissue fixative, dehydrated with 15% sucrose solution for 12h and 30% sucrose solution for 24h, embedded and frozen, and then the miRNA in situ hybridization experiment was performed using the Wuhan Boster Biological in situ hybridization kit according to the instructions to explore the expression location of miR-301a-5p.

[0012] Results: From Figure 1 It can be seen that miR-301a-5p is expressed in both the dermis and epidermis of mice.

[0013] 2. RT-qPCR Mouse skin wound tissues were collected at 0 h, 24 h, 3 d, 5 d, and 7 d, and the tissues were lysed with RNA lysis buffer to extract RNA. The corresponding system was prepared using the Multiplex Gene qPCR Kit, and the changes in miRNA expression levels were detected on a LightCycLe 480 SYBR PCR (Roche Diagnostics, Mannheim, Germany) instrument.

[0014] Results: Compared with 0 h, miR-301a-5p was significantly downregulated in wound tissue at 24 h and significantly upregulated in wound tissue on the 3rd day, suggesting that miR-301a-5p may be related to the inflammatory stage of the healing process.

[0015] Example 2 Effect of miR-301a-5p on the production of inflammatory factors by macrophages Experimental methods: 1. Cell transfection Using the ribo FECT™ CP Transfection Kit, miR-301a-5p Mimic NC (50 nM), miR-301a-5pMimic (50 nM), miR-301a-5p Inhibitor NC (100nM) and miR-301a-5pInhibitor (100nM) were transfected into macrophages according to the instructions. The specific operation is as follows: Taking the final concentration of 50nM as an example, dilute 1.25μL miRNA mimics with 30μL 1X buffer and mix gently. Then add 3μL Reagent, mix well and incubate at room temperature for 15min. Finally, mix well and add to the double antibody-free culture medium of the six-well plate cells and mix well.

[0016] 2. ELISA experiment The cell supernatant was recovered 24 h after transfection, and the ELISA kit (Xinbosheng, China) was used for the experiment according to the instructions. After adding the reaction stop solution, the absorbance at 450 nm was measured at 37°C within 3 min. The standard curve was prepared using the binomial equation, and the concentration was calculated based on the standard curve.

[0017] Results: After overexpression of miR-301a-5p, the secretion of macrophage inflammatory factors IL-6 and TNF-α was reduced.

[0018] 3. WB experiment The cells were lysed with lysis buffer (RIPA:PMSF:Phosphatase Inhibitor=100:1:1), and the collected protein supernatant was quantified using the BCA quantification method of the Beyotime kit. 4X loading buffer (volume ratio 1:4) was added to the protein and heated in a 95℃ metal bath for denaturation for 15min. The denatured protein samples were aliquoted and stored at -80℃ for later use. The target protein was separated by 10% and 12% SDS-PAGE gel electrophoresis. After cutting the gel, the protein was transferred to a PVDF membrane and then blocked with 5% skim milk for 1h or fast blocking solution for 20min. After blocking, the primary antibody was added and incubated overnight at 4℃. After washing the membrane with TBST, the secondary antibody (1:6000) was incubated at room temperature for 1h, the membrane was washed with 1x TBST, and the developer was prepared for exposure.

[0019] Results: After overexpression of miR-301a-5p, the protein levels of IL-6, TNF-α, and IL-1β were downregulated. After inhibition of miR-301a-5p expression, the protein levels of IL-6, TNF-α, and IL-1β were upregulated.

[0020] 4. RT-qPCR The cells were randomly divided into four groups: LPS+Mimic NC (50nM) group, LPS+Mimic (50nM), LPS+InhibitorNC group (100nM), and LPS+Inhibitor group (100nM). RNA was extracted after 24 h of treatment. Total RNA and miRNA were reversed using the reverse transcription procedure of the Fusion Gene Reverse Transcription Kit instruction manual. mRNA was extracted using the Omega kit according to the instructions, and the concentration was measured using a spectrophotometer after extraction. The corresponding system was prepared using the Fusion Gene qPCR Kit, and the changes in mRNA expression levels were detected on the LightCycLe 480SYBR PCR (Roche Diagnostics, Mannheim, Germany) instrument. According to the existing calculation method, the relative expression levels of TNF-α, IL-6, and IL-1β were analyzed using the 2-△△t method with GAPDH or β-actin as the internal reference.

[0021] Results: After overexpression of miR-301a-5p, the mRNA expression levels of inflammatory factors IL-6, TNF-α, and IL-1β were downregulated.

[0022] Example 3 Prediction of target genes of miR-301a-5p 1. Bioinformatics prediction Experimental methods: According to the ceRNA sequencing results, the target genes of miR-301a-5p were analyzed and predicted in combination with the TargetScan algorithm (http: / / www.TargetScan.org) and the miRDB algorithm (https: / / mirdb.org / ), and the binding sites of miR-301a-5p and target genes were predicted using https: / / bibiserv.cebitec.uni-bielefeld.de / rnahybrid / .

[0023] Results: The predicted target gene of miR-301a-5p was IKKβ.

[0024] 2. Dual luciferase reporter assay Experimental method: A dual luciferase reporter assay was used to verify the targeting relationship between miR-301a-5p and the target gene IKKβ. 293T cells were seeded in a 24-well culture plate one day before transfection, so that the cell density was 60-70% during transfection. The culture medium was DMEM + 10% FBS. Plasmid was added to 50 μL serum-free DMEM medium (total plasmid concentration 4 ng / μL), miRNA was diluted to 50 μL serum-free medium in the same tube (total miRNA concentration 50 nM), 4 μL lipo2000 transfection reagent was added to 50 μL serum-free DMEM medium, the above two were mixed and incubated at room temperature for 15 min, and serum-free DMEM medium was added to 500 μL; the medium in the well was aspirated, 500 μL of the transfection complex prepared in the previous step was added, and cultured at 37°C for 4-6 h; the medium was aspirated, 1 mL of complete medium was added, and cultured at 37°C for 48 h; transfection was repeated 5 times; cell lysis: 200 μL of cell lysis buffer (CLB) was added to each well of the 24-well plate, incubated on ice for 5 min, and the cells were fully lysed; after full lysis, the lysate was collected, centrifuged at 10,000 rpm for 5 min, and the supernatant was taken as the test solution; firefly luciferase buffer (LRB) and firefly luciferase substrate (50 ×) (LRS), Renilla luciferase buffer (LRB Ⅱ), Renilla luciferase substrate (50 ×) (LRS Ⅱ), and let it reach room temperature. Dilute to 1× working solution with the corresponding buffer and place in an ice bath for later use. Turn on the chemiluminescence immunoassay analyzer, take another 96-well plate, take 10-20 μL of cell lysate supernatant and add it to the 96-well luminescent plate, then add 100 μL of firefly luciferase detection working solution, shake the plate to mix; measure the luminescence value at 350-700 nm on the machine, and the detection time is 1 sec; after completing the above steps of measuring firefly luciferase, add 100 μL of Renilla luciferase detection working solution, shake the plate to mix, measure the luminescence value at 350-700 nm on the machine, and the detection time is 1 sec; when Renilla luciferase is used as the internal reference, divide the RLU value obtained by the firefly luciferase determination by the RLU value obtained by the Renilla luciferase determination. The activation levels of the target reporter gene between different samples were compared based on the obtained ratio.

[0025] Results: By Figure 2 c As can be seen, a dual luciferase experiment was performed based on the predicted binding site, proving that miR-301a-5p can bind to the 3' UTR end of IKKβ, indicating that miR-301a-5p targets IKKβ.

[0026] Example 4 Effect of upregulated miR-301a-5p expression on full-thickness wound healing in mice 1. Establishment of mouse full-thickness wound model Experimental methods: Male Kunming mice weighing 18-22g at 8 weeks of age were purchased and raised for one week to adapt to the experimental environment. The mice were anesthetized with pentobarbital and the hair on the back was removed. Two full-thickness wounds with a diameter of 8mm were established on both sides of the back of the mice using a biopsy punch. They were divided into the control group (vehicle, PBS), the Agomir NC group (5nmol), and the drug-treated group AgomiR-301a-5p (5nmol). After the wounds were created, the mice were injected subcutaneously at three points, with 50µL injected at each point. Photos were taken and recorded at 0, 3, 5, and 7 days as needed, and pathological staining was performed on the mouse tissues after 4 and 7 days of treatment.

[0027] Results: From Figure 4 ac It can be seen that the expression of miR-301a-5p in the wounds of AgomiR-301a-5p-overexpressing mice can accelerate the healing of full-thickness wounds in mice.

[0028] 2. Masson staining The wound tissue was stained with iron hematoxylin for 30-60 s, differentiated with acidic ethanol differentiation solution for 1-10 s, washed with water, blued with Masson's blue solution for 3-5 min, washed with water for 1 min, stained with Ponceau red solution for 30-1 min, washed with weak acid working solution for 1 min, dehydrated with 95% ethanol and anhydrous ethanol, transparentized with xylene for 5 min, sealed, and quantified by ImageJ.

[0029] Results: After subcutaneous injection of Agomir NC and AgomiR-301a-5p, the AgomiR-301a-5p group promoted collagen deposition, promoted tissue repair, and accelerated the healing of full-thickness wounds in mice ( Figure 4 de).

[0030] 3. H&E staining The skin wound tissue was fixed, dehydrated, transparent, and immersed in wax before paraffin embedding. After embedding, it was cut into 6 μm thick tissue slices and dried. Dewaxed with xylene, hydrated, stained with hematoxylin for 5 min, differentiated with hydrochloric acid and ethanol for 3-5 s, hydrated for 5 min, eosin for 2 min, gradient alcohol, xylene for 5 min, and sealed with neutral gum.

[0031] Results: From Figure 5 and Figure 6 It can be seen that compared with the Agomir NC group, the new epidermis in the Agomir group was significantly thickened and the granulation tissue increased on the 4th day, and the epidermis became thinner and the granulation tissue decreased on the 7th day.

[0032] In summary, by upregulating the expression of miR-301a-5p, IKKβ can be targeted to exert anti-inflammatory effects, promote epidermal regeneration, collagen deposition, accelerate the transition from the inflammatory phase to the proliferative phase, and ultimately promote the repair of acute skin wounds, indicating that miR-301a-5p can be used as a drug target for the treatment of acute skin wound repair.

Claims

1. The use of miR-301a-5p in the preparation of a drug for repairing acute skin trauma, characterized in that: The drug for repairing acute skin trauma takes miR-301a-5p as a drug target and overexpresses miR-301a-5p.

2. The application according to claim 1, characterized in that: The skin wound repairing drug overexpresses miR-301a-5p and targets IKKβ to exert anti-inflammatory effects, thereby achieving the effect of promoting the repair of acute skin wounds.

Images