The Effect And Mechanism Of MiR-302c-3p In Cell Injury Of HRMCs Treated With High-glucose

Diabetes nephropathy(DN)is one of the most important microvascular complications of diabetes and an important cause of chronic kidney disease and end-stage kidney disease.The pathogenesis of DN is complex and regulated by multiple factors,and glomerular injury is one of the most significant pathological manifestations.Glomerular mesangial cells(HRMC)are the main cellular components of the mesangium,which have functions such as phagocytosis of macromolecular complexes and control of glomerular blood flow.HRMC are damaged,it will lead to glomerular dysfunction.micro RNAs regulate gene expression at the post-transcriptional level and are closely related to various biological processes.In previous study,we found that cell proliferation was inhibited and the expression of miR-302c-3p was significantly increased in HRMC treated with high glucose.However,it is unclear whether miR-302c-3p is involved in cell injury of HRMC treated byhigh glucose.In this study,we investigate the phenotypic changes of HRMC after high glucose treatment and transfection with miR-302c-3p mimic,analyze the functions and signaling pathways of differentially expressed genes,and identify potential target genes.The aim of this study is to reveal the changes of HRMC treated with high glucose and the regulatory role of miR-302c-3p,providing new ideas for elucidating the involvement of micro RNA in the pathogenesis of DN.In this study,we first investigated the effect of high glucose treatment in cell injury of HRMC.By detecting oxidative stress indicators,it was found that SOD and CAT levels decreased,while MDA levels and ROS productionincreased after treatment with high glucose.The addition of antioxidants(NAC)reversed the above phenomenon.We used flow cytometry and probe method,and found that high glucose treatment resulted in the increase in the apoptosis rate,while the mitochondrial membrane potential decreased,and the expression of apoptosis related genes changed including the upregulation of p27,BAX,Caspase-3,APAF-1,p53,and the downregulation of Bcl-2 and Survivin.The expression of ECM related genes LN and FN was downregulated,while the expression of MMP2 was upregulated.The expression of inflammatory factor TNF-α was downregulated and IL-18 was upregulated.Secondly,we explored the effect and mechanism of miR-302c-3p mimic on HRMCs.Compared with the control group,miR-302c-3p mimic caused an increase in oxidative stress levels and induced cell apoptosis.The expression trends of ECM and inflammation related genes are consistent with those under high glucose conditions Finally,we investigated the effects of high glucose treatment and transfection with miR-302c-3p mimic on the gene expression profile in HRMC cells.The transcriptome sequencing results showed that 25 differentially expressed genes were down-regulated in both high glucose group and miR-302c-3p mimic group,involving NF-?appa B signaling pathway and cytokine-cytokine receptor interaction pathway.We predicted that high glucose treatment and transfection with miR-302c-3p mimic were associated with urinary system diseases,cancer,nutritional and metabolic diseases;The bioinformatics analysis showed that RGL1 and TIMP3 might be potential target genes of miR-302c-3p.Luciferase reporter assay confirmed that TIMP3 is a direct target gene of miR-302c-3p,having negative targeting regulation relationship.Transfection of si-TIMP3 caused an increase in oxidative stress levels,induced cell apoptosis,and resulted in gene expression changes in ECM and inflammation,which are consistent with those changes after high glucose treatment and miR-302c-3p mimic transfection.In summary,high glucose treatment leaded to an increase in oxidative stress levels,which promoted cell apoptosis through the p53-mediated mitochondrial pathway,induced ECM dysregulation and increased secretion of inflammatory factors.miR-302c-3p can negatively regulate TIMP3 to participate in cell injury induced by high glucose treatment.These results of this study are greatly significant for understanding the regulatory role of miR-302c-3p in the pathogenesis of DN.