Research On The Roles And Mechanisms Of N~6-Methyladenosine Aggravating Renal Fibrosis Through ZEB2

Renal fibrosis(RF)is the ultimate common outcome of many renal diseases.Renal tubular epithelial cells are the most abundant and vulnerable intrinsic cells in the kidney,and their transformation to mesenchymal phenotype(EMT)is an important factor promoting renal fibrosis.However,the specific mechanism leading to renal tubular epithelial EMT has not been fully elucidated.In recent years,more and more studies have shown that N6-methyladenosine(m6A)plays an important role in human diseases and pathological processes.However,the specific role and mechanism of m6A in renal fibrosis have not been fully elucidated.In the study,m6A positively regulates zinc finger E-box binding homeobox 2(ZEB2)to promote the progression of renal fibrosis.Through comprehensive analysis of m6A sequencing,mRNA sequencing and functional studies,it was confirmed that m6A regulates ZEB2 to promote RF and EMT processes.Specifically,the ZEB2 mRNA coding region(CDS)m6A2137 binds to the YTH domain m6A binding protein 1/Translation Extension factor 2(YTHDF1/eEF-2)complex and insulin-like growth factor 2-mRNA binding protein 3(IGF2BP3),respectively.Upregulates the translation activity of ZEB2 and promotes the stability of ZEB2 mRNA.In addition,specific demethylation of ZEB2 and m6A using the dm6ACRISPR system significantly decreased the expression of ZEB2 and delayed the EMT process in renal tubular epithelial cells.In vivo and clinical data provide further supporting evidence for the positive effect of the m6A/ZEB2 axis on RF progression.The study shows that m6A regulates the progression of renal fibrosis through ZEB2,which provides new therapeutic strategies for targeting the m6A/ZEB2 axis to inhibit renal fibrosis.Research objective:The study intends to further analyze the role and molecular mechanism of m6A in promoting RF.Focusing on the role of m6A in the occurrence and development of RF,it was confirmed that m6A positively regulated ZEB2 to promote the process of renal fibrosis,clarified the molecular mechanism of m6A regulating the EMT of renal tubular epithelial cells,and revealed the role of m6A modification in renal fibrosis by combining animal models and clinical samples.In order to explore the strategy and feasibility analysis of targeting m6A/ZEB2 axis to inhibit renal fibrosis,and provide a new therapeutic strategy with great potential for inhibiting the occurrence and development of renal fibrosis.Research methods:Wound healing assay;Co-Immunoprecipitation;Double-luciferase assay;RNA Binding Protein Immunoprecipitation;RNA stability and protein stability assays;RTqPCR;Polysome profiling assay;LC-MS;Northern blot;Western blot;Immunohistochemistry staining;Mouse UUO model and Mouse UIRI model,etc.Research results:1.m6A promotes EMT generation in renal tubular epithelial cells2.m6A positive regulation ZEB2 EMT process promotes renal tubular epithelial cells3.YTHDF1 recognition ZEB2 mRNA CDS m6A2137 raises ZEB2 translation activity4.IGF2BP3 recognition ZEB2 mRNA CDS m6A2137 promotes ZEB2 mRNA stability5.m6A methylation is positively correlated with the process of renal fibrosisConclusion and significance:1.The study expounded a possible mechanism of EMT in the renal tubular epithelium and the possible mechanism of renal fibrosis via RNA epigenetics2.The study elucidated that m6A modification positively affected renal fibrosis,which might provide a potential therapeutic strategy for this disease...