The Effect Of MiR-194-5pp On Renal Tubular Epithelial Cells Injury Through Thrombospondin-1(TSP-1) At High Glucose Concentrations

ObjectiveTo investigate the expression changes of miR-194-5p and its target gene TSP-1 in renal tubular epithelial cells cultured in a high glucose environment;observe that renal tubular epithelial cells overexpress miR-194-5p in a high glucose environment,and through Western Blot detects the changes in the expression of TSP-1,Bcl-2,and Bax proteins,explores the role and mechanism of miR-194-5p in renal tubular epithelial cell injury,and provides a theoretical basis for finding new therapeutic targets for diabetic nephropathy.MethodsHuman renal tubular epithelial cells were cultured in vitro and divided into groups as follows:(1)Divided into normal group and high glucose group.After culturing for 48 hours at 37°C and 5% CO2,real-time fluorescent quantitative polymerase chain reaction(qRT-PCR)method to detect the expression changes of miR-194-5p in the two groups of cells and Western Blot to detect the expression changes of TPS-1 protein;(2)miR-194-5p mimic was transfected to construct miR-194-5p overexpression,divided into four groups: normal group(5.6mmol/L),high glucose group(30mmol/L),high glucose+miR-NC transfection group,miR-194-5p mimic transfection The group was cultured for 48 hours,the transfection efficiency of miR-194-5p was detected by qRT-PCR,and TSP-1,B-lymphoma-2(Bcl-2),and B-lymphoma-2 in each group were detected by Western Blot.Related protein(Bax)protein expression changes,using enzyme-linked immunosorbent assay(ELISA)to detect the levels of interleukin 8(IL-8)and tumor necrosis factor-a(TNF-a);(3)The binding site of miR-194-5p and TSP-1 was predicted and mutated by Target Scan bioinformatics,which was confirmed by dual luciferase report experiment.Results1.The expression levels of miR-194-5p and TSP-1 in HK-2 cells induced by high glucose concentrations changed as follows:After being cultured at high glucose concentrations,the expression of miR-194-5p decreased,and the expression level of TSP-1 protein increased.It was presumed that miR-194-5p and TSP-1 might be involved in the pathogenesis of DKD.2.Overexpression of miR-194-5p in a high-glucose environment rescued renal tubular epithelial cell damage by regulating the target gene TSP-1.The miR-194 transfected with the renal tubular epithelial cells in the miR-194-5p mimic group was detected by qRT-PCR The expression of-5p increased,and the expression of TSP-1 and Bax protein was decreased by western Blot,and the expression of Bcl-2 increased.3.According to Target Scan bioinformatics software,it is predicted that miR-194-5p has a binding site with the 3’UTR of TSP-1.The dual luciferase vector reporter gene is constructed and verified that the target gene of miR-194-5p is TSP-1.This study found that the expression level of miR-194-5p decreased in renal tubular epithelia at high glucose concentrations.Through Target Scan bioinformatics prediction,qRT-PCR and Western Blot detection,it was presumed that TSP-1 was the target gene of miR-194-5p.The dual luciferase reporter gene test verified the direct binding of TSP-1 and miR-194-5p.ConclusionThe miR-194-5p can rescue renal tubular epithelia from damage by down-regulating the expression of target gene TSP-1 at the post-transcriptional level at high glucose concentrations.