The Relationship Between MicroRNA194 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). Peritoneal fibrosis (PF) is almost the final result of all peritoneal pathological changes. 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). 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. EMT is an early event in fibrosis and UFF during PD, but the mechanism by which effects induce EMT in PD is not well understood. MicroRNAs (miRNAs) are short noncoding RNAs of 18-26 nt that recently have been shown to play important roles in mammalian gene expression. Evidence suggests that miRNA is an essential modulator for TGF-β1 induced EMT. However, the role of miRNAs in peritoneal EMT and fibrosis during PD are largely unknown.Objective:Determine the miRNA194 expression in HPMC from effluent derived of patients undergoing CAPD and analysis the relationship between miRAN194 and EMT related proteins.Methods:(1)We randomly selected patients with PD from The 2^nd xiangya hospital and The 3^rd 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 miRNA 194 expression was assessed by real time PCR with a TaqMan miRNA 194 probe (ABI) following the manufacturer's protocols. (3) The proteins or mRNAs expression of E-cadherin, α-smooth muscle actin(α-SMA) 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,miRNA194 was up-regulated expression in HPMCs from effluent derived of patients undergoing CAPD over a year, compared with those undergoing CAPD start (P<0.05). (4) The results of Western blot and quantitative real time PCR showed that there was a markedly lower level of E-cadherin expression in HPMCs from effluent derived of patients undergoing CAPD start than those undergoing CAPD over a year (P<0.05) . The level ofα-SMA in HPMCs from effluent derived of patients undergoing CAPD over a year was higher than those undergoing CAPD start by Western blot and quantitative real time PCR (P<0.05) . (5)There was a negative correlation between the expressions of miRNA194 and E-cadherin mRNA of HPMCs from effluent derived of patients undergoing CAPD (r=-0.952, P<0.05). There was a positive correlation between the expressions of miRNA194 andα-SMA mRNA of HPMCs from effluent derived of patients undergoing CAPD (r=0.760, P<0.05).Conclusion: 1. MiRNA194 was expressed in HPMCs from effluent derieved of patients undergoing CAPD, and the expression increased 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, EMT related proteins such as E-cadherin andα-SMA were expressed obviously abnormally, which indicated that the EMT level aggravated with the prolongation of PD 3. MiRNA194 had relations with the abnormal expression of HPMCs EMT related genes, showing that it related to HPMCs EMT of PD patients.