The Relationship Between MicroRNA302c Expression And Epithelial To Mesenchymal Transition In Human Peritoneal Mesothelial Cells Of Patients With PD

Background Continuous ambulatory peritoneal dialysis (CAPD) is an alternative to hemodialysis for the treatment of end-stage renal disease (ESRD). Long-term exposure to the hyperosmotic, hyperglycemic, and acidic solutions used in dialysis often causes low-grade, chronic inflammation and injury of the peritoneum, which progressively results in the denudation of mesothelial cells, leading to the PF and Ultrafiltration failure (UFF), EMT is an early event in fibrosis and UFF during PD. UFF is the main reason out of peritoneal dialysis. Peritoneal mesothelial cells have historically been considered to be the primary cells involved in the development of PF. Recent studies have shown that epithelial-to-mesenchymal transition (EMT) may be a major source of myofibroblast.,and TGF-β1 signaling pathway plays a decisive role in peritoneal fibrosis, Connective tissue growth factor (CTGF) is a major downstream factor which by TGF-β1 induced fibrosis. microRNA302c belong to microRNA302 family. From the analysis of biological model program by mir302,found that play a key role in the regulation of CTGF expression, however there is no relevant reports at home and abroad.Objective:Determine the microRNA302c,CTGF expression in HPMC from effluent derived of patients undergoing CAPD and analysis the relationship between microRNA302c and EMT related proteins.Methods:(1)We randomly selected patients with PD from The 2nd xiangya hospital and The 3rd xiangya hospital of Central South University. Patients will be divided into two groups:Group A:CAPD start; Group B:CAPD over a year. Human peritoneal mesothelial cells(HPMCs) were cultured from effluents in dialysis fluid from patients undergoing CAPD. After 14 days, the cultured cells were identified by invert microscope and immunohistochemistry of vimentin and cytokeratin antibodies. The primary cells were used in our study.(2). The miRNA302c expression was assessed by real time PCR with a TaqMan miRNA194 probe (ABI) following the manufacturer's protocols. (3) The proteins or mRNAs expression of CTGF,vimentin and zo-1 were detected by western blot and quantitative real time PCR respectively.Results:(1) 95%of the cultured cells from effluent derived of patients undergoing CAPD were identificated as HPMCs. (2) HPMCs from effluent derived of patients undergoing CAPD had markedly varied morphologic features, ranging from a cobblestone-like appearance similar to that of mesothelium derived from omentum to fibroblast-like cells or mixed cell populations. (3) By real time PCR with a TaqMan, miRNA302c was down-regulated expression in HPMCs from effluent derived of patients undergoing Group B, compared with those undergoing Group A (P<0.05). (4) The results of Western blot and quantitative real time PCR showed that there was a markedly lower level of Zo-1 expression in HPMCs from effluent derived of patients undergoing Group A than those undergoing Group B(P＜0.05). The level of CTGF,Vimentin in HPMCs from effluent derived of patients undergoing Group B was higher than those undergoing CAPD start by Western blot and quantitative real time PCR (P<0.05). (5)There was a negative correlation which isolated from patients undergoing Group A and Group B (r=-0.54, P＜0.05). The expressions of microRNA302c and Vimentin mRNA of HPMCs were in a negative correlation which isolated from patients undergoing Group A and Group B (r=-0.884, P<0.05). The expressions of microRNA302c and Zo-1 mRNA of HPMCs were in a positive correlation which isolated from patients undergoing Group A and Group B (r= 0.86, P<0.05). The expressions of CTGF mRNA and Vimentin mRNA of HPMCs were in a positive correlation which isolated from patients undergoing Group A and Group B (r= 0.83, P＜0.05). The expressions of CTGF mRNA and Zo-1 mRNA of HPMCs were in a negative correlation which isolated from patients undergoing CAPD start and over a year (r=-0.72, P＜0.05)Conclusion:1.MicroRNA302c was expressed in HPMCs from effluent derieved of patients undergoing CAPD, and the expression droped appearantly with the prolongation of PD.2. In the HPMCs from effluent derieved of patients undergoing CAPD over a year compared with those undergoing CAPD start, CTGF and EMT related proteins such as vimentin and zo-1 were expressed obviously abnormally, which indicated that the EMT level aggravated with the prolongation of PD.3. MiRNA302c had relations with the abnormal expression of HPMCs CTGF and EMT related genes, showing that it related to HPMCs EMT of PD patients.