The Role And Mechanism Of ETS2 On Epithelial-to-mesenchymal Transition Of Renal Tubular Epithelial Cells And Renal Fibrosis

Background and aimAt present,more than 13%of the populations in the worldwide suffer from various chronic kidney diseases,which has been a serious threat to human health.Tubulointerstitial fibrosis plays a leading role in the outcome of kidney disease.The severity of renal fibrosis is an important indicator for evaluating the degree of renal dysfunctuion and the risk for renal failure progression.A number of studies have shown that endothelial-mesenchymal transition(EMT)plays an important role in the process of renal injury repair and organ fibrosis.However,the molecular mechanism of EMT in renal tubular epithelial cells has not been fully elucidated.To explore the molecular mechanism of EMT in renal tubular epithelial cells is of great significance in preventing renal fibrosis.We used microarray to screen the genes that were differentially expressed in HK2 cells during EMT induced by TGFβ1.Then we selected ETS2 and JUNB for candidate genes that may regulate EMT in HK2 cells by bioinformatics analysis.No studies have been report on the role of ETS2 and JUNB in chronic renal fibrosis.In this study,we explore the role of ETS2 and JUNB in renal tubular epithelial cell transdifferentiation and renal fibrosis.We also investigate whether JUNB is regulated by ETS2,which provide a theoretical basis for a new therapeutic target of renal fibrosis.Methods and results1.A total of 587 genes were differentially expressed in HK2 cells stimulated by TGFβ1 using gene expression microarray.There were 248 genes up-regulated and 339 down-regulated genes.Bioinformatics analysis of these genes showed that 12 genes may be closely related to TGFβ1-induced EMT in HK2 cells.2.Real-time quantitative PCR method verified that:at the cellular level,the expression trends of these 12 genes are consistent with the results of gene expression microarray;in the mouse renal fibrosis model,there are only 6 genes expression trends are consistent with the results of gene expression microarray.Finally,ETS2 was selected for further study through bioinformatics analysis and literature search methods3.ETS2 was mainly expressed in the nucleus of renal tubular epithelial cells.In the UUO group,ETS2 expression was significantly higher than the sham group.In TGFβ1-stimulated HK2 cells,ETS2 expression was increased in a concentration-and time-dependent manner.4.Knockdown of ETS2 expression in HK2 cells by siRNA significantly inhibited TGFβ1-induced EMT in HK2 cells.Knockdown of ETS2 prevented the increased expression of a-SMA,Vimentin,N-cadherin,Collagen I and FN proteins in HK2 cells induced by TGFβ1.Furthermore,ETS2 depletion significantly inhibited the expression of CCL2,IL18,TNFa and IL6 mRNA in HK2 cells.5.Bioinformatics analysis predicts the interaction between ETS2 and JUNB.We used luciferase assays to verify that ETS2 directly regulates the transcription of the JUNB promoter.Furthermore,we identify specific sites of ETS2 bind to JUNB by means of segmented truncation and site mutation.ETS2 depletion can significantly inhibit TGFβ1-induced the expression of JUNB in HK2 cells.6.JUNB is mainly distributed in the nucleus of renal tubular epithelial cells,which was significantly higher in UUO group than in sham group.In the TGFβ1-stimulated HK2 cells,the expression of JUNB was increased in a concentration-and time-dependent manner.7.The knockdown of JUNB expression significantly inhibited the process of EMT in HK2 cells induced by TGFβ1,with down-regulated the expression of Vimentin,N-cadherin,Collagen I and FN proteins.However,overexpression of JUNB significantly promoted the expression of Vimentin,N-cadherin and FN protein in HK2 cells induced by TGFβ1.Conclusion1.In the mouse model of renal fibrosis,ETS2 and JUNB expression significantly increased in UUO group than sham group.The expression of ETS2 and JUNB in HK2 cells were increased in a concentration-and time-dependent manner induced by TGFβ1.2.Both ETS2 and JUNB can promote the morphological changes of EMT and the expression of fibrosis-related proteins in HK2 cells induced by TGFβ1.3.ETS2 directly regulates the transcription of JUNB,which may promote TGFβ1-induced EMT in HK2 cells.4.This study investigated the roles and mechanisms of ETS2 and JUNB in the transdifferentiation of renal tubular epithelial cells,providing a theoretical basis for the search for new therapeutic targets for renal fibrosis.