The Molecular Mechanism Of MiR-509-3p In Regulating Renal Interstitial Fibrosis Through Targeting YAP

Objective:Renal interstitial fibrosis is a typical pathological process for various chronic kidney diseases,eventually leading to end-stage kidney disease(ESRD).There is no effective treatment to block the process of renal fibrosis completely in clinical practice.Renal interstitial fibrosis is characterized by the proliferation of myofibroblasts and the massive deposition of extracellular matrix(ECM).The myofibroblasts are derived from fibroblasts produced by epithelial-mesenchymal transition or endothelial-mesenchymal transition,and its Continuous activation is an essential biological event in the progression of fibrosis.Recent studies have shown that Yes-associated protein(YAP)and its downstream factors are abnormally activated in fibrosis,promoting the increase of myofibroblasts,and lead to excessive ECM deposition.Therefore,inhibiting YAP activity will be a new therapeutic strategy to prevent renal fibrosis.Studies have confirmed that miRNAs,as critical post-transcriptional regulators,are related to fibrosis caused by various kidney diseases.This study found that miR-509-3p was down-regulated in mouse renal fibrotic tissue through bioinformatics analysis.Another study found that miR-509-3p uses YAP as a target to inhibit the production of ECM in ovarian cancer.It is speculated that miR-509-3p may play a role in renal fibrosis.This study intends to use renal fibrosis animal models and cytological experiments to analyze the relationship between miR-509-3p and YAP and its downstream genes in the formation of renal interstitial fibrosis through overexpression and knockdown of miR-509-3p in order to improve renal fibrosis treatment and provide new clinical treatment ideas.Methods1.Use the GEO database to analyze the differentially expressed miRNAs in mouse fibrotic kidneys,and screen out significantly down-regulated miRNAs.2.Use real-time fluorescent quantitative PCR(qPCR)to detect the expression level of miR-509-3p in the serum of healthy people and patients with chronic kidney disease and CKD renal fibrosis.3.In order to study the role of miR-509-3p in renal fibrosis,a mouse model of nephropathy induced by high-dose folic acid(FA)was established.Observe the changes in kidney morphology and evaluate renal filtration function through tissue staining(PAS,Masson)and serum biochemical analysis(creatinine,urea nitrogen)to confirm the successful establishment of the model,and observe Aquaporin1(Aquaporin1,AQP1)by immunohistochemistry and aquaporin 2(Aquaporin2,AQP2)expression.4.Use qPCR to verify the expression of miR-509-3p in the kidney tissue and serum of model mice.5.Use immunohistochemistry,qPCR,and Western blotting to locate and quantify YAP expression in the kidney tissues of fibrotic mice and patients.6.Construct a renal fibrotic cell model,detect miR-509-3p by qPCR,detect the expression of YAP and fibrosis indicators by qPCR and Western blotting: Fibronectin(Fn),collagen IV(Collagen IV,Col IV),?-Smooth muscle actin(?-Smooth muscle actin,?-SMA).7.Overexpression and inhibition of miR-509-3p in cell models,detection of YAP expression changes,and detection of Fibronectin,Collagen IV,?-SMA;content luciferase reporter gene method to determine miR-509-3p and YAP The binding site.8.In order to verify that miR-509-3p regulates the process of renal fibrosis in mice by regulating YAP and its downstream genes,a FA nephropathy model was constructed to overexpress miR-509-3p.The mice with FA renal fibrosis were injected with specially labeled and chemically modified double-stranded small RNA(agomir)through the tail vein.Western blotting,qPCR,and immunohistochemical staining were used to detect YAP expression and its transcriptional regulation on downstream target genes CTGF(Connective tissue growth factor)and CYR61(Cysteinerich 61).9.Using tissue staining(PAS,Masson,immunohistochemistry),qPCR,Western blotting to analyze the pathological changes in the kidneys of mice overexpressing miR-509-3p,and to detect the fibrosis-related proteins ?-SMA,Collagen IV and Fibronectin,and cadherin Changes in the expression of E-Cadherin and Vimentin,and serum biochemical tests to evaluate renal function.Results:1.GEO database screening showed that miR-509-3p was down-regulated in mouse kidney fibrotic tissue.Simultaneously,it was verified that the expression of miR-509-3p in serum samples of patients with renal fibrosis decreased,and its expression level was closely related to the glomerular filtration rate and creatinine of patients.2.14 days after the intraperitoneal injection of FA,the renal tubules atrophy,a large number of inflammatory cells infiltration and collagen fiber deposition in the renal interstitium could be observed;serum creatinine and blood urea nitrogen(BUN)were detected significantly increased;AQP1 and AQP2 expression in severely damaged renal tubules were significantly reduced.3.Mi R-509-3p is low expressed in kidney tissue and serum of mice with FA nephropathy.Simultaneously,the expression of YAP was up-regulated in the kidney tissue of FA mice,and the number of YAP nucleus-positive cells increased.The number of YAP nucleus-positive cells in human fibrotic kidney tissue also increased significantly.4.In a renal fibrotic cell model,miR-509-3p is low expressed and negatively correlated with YAP.Overexpression of miR-509-3p can down-regulate the expression level of YAP and inhibit the expression of fibrosis-related proteins ?-SMA,Fibronectin,Collagen IV.5.The luciferase reporter gene experiment shows that miR-509-3p binds to the 3'-UTR region of the YAP gene.6.After FA mice overexpressed miR-509-3p,YAP expression and its downstream factors CTGF and CYR61,decreased.PAS and Masson staining showed that renal tubules' pathological damage in each group of mice was reduced,and the collagen fibers in the renal interstitium decreased;Serum biochemical test indicates that renal function was restored;the expression of fibrosis-related proteins ?-SMA,Collagen IV,and Fibronectin decreased.The expression level of E-cadherin protein increased,and the expression level of Vimentin protein decreased.Conclusion: Mi R-509-3p is significantly low-expressed in human and mouse renal fibrotic tissues and negatively correlates with the YAP level.Overexpression of miR-509-3p inhibits YAP expression while improving renal interstitial fibrosis and renal function in mice folic acid nephropathy models,Suggesting that miR-509-3p slows down the process of renal fibrosis and through YAP,thereby protecting renal function.