Study On The Mechanism Of MiR-199a-5p In Wound Healing Of Diabetic Foot Ulcer

PurposeDiabetes mellitus(DM)is a growing health problem.As a common complication of DM,diabetic foot ulcer(DFU)results in delayed wound healing and is a leading cause of non-traumatic amputation.This study is based on clinical analysis of changes in miR-199a-5p expression in DFU wound tissues and aims to exploring the effects of miR-199a-5p on wound healing and its possible molecular mechanism,and also the therapeutic effect of regulating miR-199a-5p in vitro and in vivo,so as to provide theoretical basis for enriching the � arsenal� against DFU.Methods1.We collected DFU samples from patients,and established diabetic rat models and high glucose(HG)-induced cells.Expression of miR-199a-5p and downstream pathway genes in the tissues and cells mentioned above were detected by qRT-PCR.2.In vitro,we enhanced or inhibited expression of miR-199a-5p,VEGFA or ROCK1 via miRNA or si RNA transfection.In normal or HG-induced cells,cell proliferation and migration was detected by CCK-8,scratch test and Transwell test.Gene expression in different groups of cells were detected by qRT-PCR and Western Blot at mRNA and protein levels,respectively.3.We established full-thickness skin defect diabetic rat model,and induced enhanced or inhibited expression of miR-199a-5p via local transfection.The impact on gene expression,wound healing rate,granulation tissue histology and thickness,angiogenesis were observed by H&E staining,IHC,IF,qRT-PCR?Western Blot,and the roles VEGFA,ROCK1 played in diabetic wound healing were analyzed.Results1.The expression of miR-199a-5p was significantly increased in DFU wound tissues,skin tissues of diabetic rats,and HG-induced cells.The expression of VEGFA and ROCK1 was significantly reduced in the tissues and cells mentioned above.2.In normal cells,over-expression of miR-199a-5p inhibited the expression of VEGFA and ROCK1 significantly,and inhibited cell proliferation and migration of fibroblasts and endothelial cells(ECs),resulting in a cell phenotype similar to that of the HG-induced cells.On the contrary,in HG-induced cells,inhibiting the expression of miR-199a-5p alleviated the inhibition of VEGFA and ROCK1,thereby rescued impaired proliferation and migration of HG-induced cells,and restored the normal function of the cells to some extent.3.In non-diabetic rats,over-expression of miR-199a-5p significantly inhibited the expression of VEGFA and ROCK1 and impeded wound healing.On the contrary,in diabetic rats,inhibition of miR-199a-5p significantly increased the expression of VEGFA and ROCK1,significantly promoted wound healing,and rescued impaired wound healing to some extent.ConclusionOver-expression of miR-199a-5p induced by diabetic stimuli inhibited the expression of VEGFA and ROCK1,impaired normal function of fibroblasts and ECs and impeded granulation tissue formation and angiogenesis,resulting in delayed wound healing.Inhibition of miR-199a-5p showed therapeutic effect on diabetic wounds.