| Background:Wound healing has long been one of the topics in the medical field.For burn orthopedic surgeons,it is necessary to understand the mechanism and process of skin wound healing,as well as the relevant treatment methods.Wound repair undergoes complex biological processes,including inflammatory cell infiltration,granulation tissue filling,epidermal cell proliferation,and scar tissue remodeling,all of which occur in succession and overlap.It also requires the participation of various functional cells,such as neutrophils,fibroblasts,macrophages,epidermal cells,etc.Under the chemotaxis of cytokines,they migrate into the wound bed in the blood vessels of self-created peripheral tissues,remove tissue debris,produce matrix such as collagen,and complete wound closure.If the biological behavior of the above cells is abnormal,the wound repair will be delayed or even difficult,including excessive repair such as scar hyperplasia.Epithelial tissue reconstruction is essential in the wound healing process,which requires keratinocytes to repair the wound through cell proliferation and migration from the edge of the wound.Therefore,the study on the regulation mechanism of keratinocytes proliferation and migration can help us understand the wound healing process and provide theoretical basis for clinical treatment.During wound healing,a series of genes and signaling pathways are involved in keratinocytes migration and wound tissue reconstruction.Bcl2-related protein 3(BNIP3),a bcl-2 family protein,has recently been shown to promote the movement and metastasis of keratinocytes in hypoxic conditions.In addition,BNIP3 is involved in the repair of hypoxic induced neuronal injury.Long-chain non-coding RNA can promote the ultraviolet irradiation of keratinocytes by up-regulating the expression of BNIP3,and it has also been reported that the overexpression of BNIP3 can protect keratinocytes from apoptotic injury caused by ultraviolet irradiation.The above studies indicate that BNIP3 can be used as a potential molecular target in the clinical treatment of wound healing.However,the mechanism of BNIP3 gene in wound healing is still unclearMicroRNAs(miRNAs)have been reported to play a target role in a variety of diseases,including wound healing.MiRNA is a kind of endogenous non-coding RNA with a length of 22 nucleotides,which regulates gene expression through the 3'-non-translation region(UTR)of the target gene and plays a role of transcriptional inhibition.MiRNA can regulate the expression of multiple target mrnas to complete the regulation of a series of cell biological functions such as cell proliferation,apoptosis and differentiation.In addition,numerous studies have shown that mirnas are involved in regulating cell proliferation,release of immune factors,reconstruction of extracellular matrix and angiogenesis during wound healing.According to TargetScan,mir-96-5p was predicted to be the upstream regulated miRNA of BNIP3,and the 3'-utr segment of BNIP3 had interaction sites with mir-96-5p,and mir-96-5p was reported to be involved in the regulation of proliferation and differentiation of fibroblasts.In view of these findings,our study attempted to explore the regulatory mechanism of mir-96-5p on BNIP3 and investigate the mechanism of mir-96-5p promoting wound healing through regulating BNIP3 gene expression,providing theoretical basis for clinical application.Focal kinase(FAK),also known as pp125FAK,is a non-receptor cytotyrosine kinase,which plays an important role in integrin signal transduction.Study found that FAK(can interact with cellular signal transduction protein,forming local gelling spot,participate in regulating cell adhesion,migration,proliferation,apoptosis,such as process,its expression level is related to cell apoptosis,FAK(expression can prevent apoptosis.A large number of literature shows that FAK(involved in regulating cell migration in wound healing skin.The application of specific integrin monoclonal antibody can down-regulate the activation level of FAK and inhibit the migration of epidermal cells.The wound healing of FAK-deficient mice was significantly delayed.In a variety of tumors,FAK signaling pathway and its downstream signal are involved in regulating actin-cytoskeleton polymerization,depolymerization and rearrangement,which are key links related to cell migration,and BNIP3 can regulate the remodeling of actin-cytoskeleton.Therefore,FAK may be involved in the regulation of epidermal cell migration by BNIP3.Objectives:The purpose of this paper is to study miR-96-5p to regulate skin wound healing by inhibiting BNIP3-mediated FAK signaling pathways.This project aims to solve the following problems through experimental research:1.How the expression levels of mir-96-5p and BNIP3 change in the healing process of skin injury;2.Whether BNIP3 could promote the proliferation and metastasis of keratinocytes;3.Whether BNIP3 is a direct downstream target gene of miR-96-5p,and how miR-96-5p regulates the expression of BNIP3;4.Whether miR-96-5p plays a regulatory role in the proliferation and migration of human keratinocytes;5.Whether mir-96-5p regulates skin wound healing through the bnip3-mediated FAK signaling pathway.Methods:1.Build the skin damage model:randomly selected 15 only Sprague wrote-Dawley skin injury model in rats,rats back hair removal,after anesthesia with 3 mm skin perforator in hair removal parts caused by trauma,1 day,3 days after trauma,5 days and 7 days with 4 mm skin perforator position in traumatic tissue damage,at the same time take skin tissues in healthy rats the same location;2.Protein in the wound and healthy tissue was extracted,and the protein expression difference of BNIP3 between the injured tissue and healthy tissue was detected by Western blot;3.RNA was extracted from the wound surface and healthy tissues,and the differential expression of mir-96-5p in damaged tissues and healthy tissues was detected by qRT-PCR;4.Human primary keratinocytes(HPK)were purchased from the American model culture repository for culture and passage.5.BNIP3 overexpression plasmid pcDNA3.1-BNIP3 was constructed;6.Cultured HPK were divided into four groups:pcDNA3.1-BNIP3 transfected group,BNIP-siRNA transfected group,negative control group and blank control group.The expression level of BNIP3 was detected by Western blot;7.The survival rate of HPK transfected with pcDNA3.1-BNIP3 was detected by MTT colorimetry,and the migration ability was detected by cell scratch assay;8.The survival rate of HPK transfected with BNIP-siRNA was detected by MTT colorimetry,and the migration ability was detected by cell scratch assay;9.Prediction of miRNA target genes using TargetScan bioinformatics analysis and target prediction website;10.According to the TargetScan prediction miR-96-5 p for BNIP3 upstream regulation of micrornas,will first BNIP3 3 'UTR-piece of the mutations,the wild type or mutant BNIP3 3' UTR cloning to containing luciferase reporter gene vector pmirGLO downstream,and miR-96-5 p mimics were transfection to cutin cell formation,72 hours after calculating relative luciferase activity value,detection in miR-96-5 p mimics the presence of can lead to the change of the luciferase activity;11.HPK were transfected with mir-96-5p mimic and mir-96-5p inhibitor,respectively,and compared with cells in the negative groups;12.MTT assay was used to detect the survival rate of mir-96-5p mimics or mir-96-5p inhibitor cells;13.Cell migration after transfection with mir-96-5p mimics or mir-96-5p inhibitor was measured by cell scratch assay;14.miRNA transfected with miR-96-5p mimics or mir-96-5p inhibitor cells and control cells was extracted and the expression level of mir-96-5p was detected by qRT-PCT;15.Cell proteins transfected with mir-96-5p mimics or mir-96-5p inhibitor cells and control cells were extracted and the expression level of BNIP3 and phosphorylated protein(p-FAK)of FAK,a key gene in FAK signaling pathway,were detected by Western blot;16.Statistical treatment:all results were expressed as mean ± standard deviation of three repeated measurements.The difference between the two groups was evaluated by SPSS 15.0 statistical software and t test.Univariate analysis of variance(ANVOA)was used to evaluate the comparison between two or more groups.Paired t test was used to determine the statistical significance of paired tissue comparison.If P<0.05,the difference was statistically significant.Results:1.Western blot results showed that the protein expression of BNIP3 in the injured skin tissues of rats was significantly increased compared with the healthy tissues of rats,and with the increase of healing days,the protein expression of BNIP3 was significantly increased(P<0.05);2.qRT-PCR results showed that the expression of mir-96-5p decreased gradually with the increase of wound healing days(P<0.05),which was contrary to the expression level of BNIP3;3.Western blot detection of BNIP3 expression level in HPK transfected with pcDNA3.1-BNIP3 or BNIP3-siRNA showed that the expression level of BNIP3 was significantly increased in pcDNA3.1-BNIP3 transfected cells(P<0.05),and significantly decreased in BNIP3-siRNA group(P<0.05);4.MTT assay for cell viability:compared with the control group,cell viability of HPK transfected with pcDNA3.1-BNIP3 plasmid was significantly enhanced(P<0.05),while cell viability of BNIP3-siRNA transfected was decreased(P<0.05);5.The cell scratch test showed that the migration ability of HPK after transfection with the over-expressed BNIP3 plasmid was significantly increased(P<0.05).The migration ability of BNIP3-siRNA transfected cells was significantly reduced(P<0.05).BNIP3 has the effect of promoting proliferation and migration of keratinocytes;6.The co-transfection of wild-type BNIP3 and miR-96-5p mimics resulted in a significant decrease in luciferase activity(P<0.05),indicating that miR-96-5p could lead to a decrease in the expression of BNIP3,while the co-transfection of mutant BNIP3 and miR-96-5p mimics did not result in a decrease in luciferase activity;7.The protein expression of BNIP3 was significantly decreased due to transfection of miR-96-5p mimics(P<0.05),while the protein expression of BNIP3 was significantly increased due to miR-96-5p inhibitor(P<0.05);8.MTT experiment showed that when miR-96-5p was over-expressed alone,the proliferation rate of HPK after transfection with miR-96-5p mimics was significantly inhibited compared with the negative control group(P<0.05),and the proliferation activity of HPK after miR-96-5p inhibitor treatment was increased(P<0.05);9.Cell scratch test showed that the migration ability of miR-96-5p mimic was significantly inhibited in the transfected group(P<0.05).However,the migration of miR-96-5p cells after inhibitor treatment was increased(P<0.05);10.qRT-PCR test showed that HPK transfected with miR-96-5p mimic could induce the overexpression of miR-96-5p(P<0.05),and HPK transfected with miR-96-5p inhibitor inhibited the expression of miR-96-5p(P<0.05);11.The expression of p-FAK decreased after transfection of HPKwith miR-96-5p mimics(p<0.05),while the expression of p-FAK increased after transfection with miR-96-5p inhibitor(p<0.05).It indicated that miR-96-5p could inhibit wound healing by inhibiting the FAK signaling pathway.Conclusions:1.The expression of BNIP3 protein and miR-96-5p in the injured tissue of the rat showed an opposite trend with the extension of wound healing time,suggesting that the two were negatively correlated;2.Overexpression of BNIP3 had the effect of promoting proliferation and migration of keratinocytes,while low expression of BNIP3 inhibited proliferation and migration of keratinocytes;3.BNIP3 is a direct downstream target gene of miR-96-5p in HPK and is negatively regulated by miR-96-5p,which can inhibit the expression of BNIP3 through direct binding;4.MiR-96-5p negatively regulates the proliferation and migration of HPK5.The negative regulation of miR-96-5p was achieved by inhibiting the expression of BNIP3 and the associated FAK signaling pathway.In conclusion,our experiment explored the new function of BNIP3 to accelerate skin healing,and the possible molecular mechanism was that miR-96-5p regulated its downstream target gene BNIP3 and FAK pathway,which provided a molecular basis for clinical promotion of wound healing. |
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