| BackgroundPeritoneal dialysis (PD) has the advantages of simple operation, hemodynamic stability and the protection of residual renal function, so it is widely used in end-stage renal disease (ESRD) patients. PD is the main treatment for patients with end-stage renal disease. However, peritoneal fibrosis (PF) caused by long-term PD would eventually lead to ultrafiltration failure(UFF), which limits the application of PD. Transforming growth factor beta (TGF-β) and high glucose mediated peritoneal fibrosis has been a research focus in the field of PD. Recent studies have found that epithelial mesenchymal transdifferentiation (EMT) plays a key role in the process of peritoneal fibrosis, it is considered to be the initial step and reversible phase of peritoneal fibrosis.Small RNA (microRNA, miRNA, miR) are21～25nucleotide small non-coding RNAs. They regulate gene expression by base-pairing to3’ untranslated region(UTR) of target mRNAs. resulting in protein translation or target mRNA degradation is inhibited and affecting the expression of post transcriptional gene regulation. MicroRNAs have been implicated in various biological processes. They are also related to pathological processes such as EMT. The miRNA-200family is a research hotspot in recent years. Especially the miRNA-200family is closely related with EMT.miRNA-200is also plays an important role in the process of invasion and metastasis of tumor and development of organ fibrosis.The expression of miRNA-200c in peritoneal mesothelial cells has not been reported at home and abroad. It is unknown the relationship between miR-200c and EMT in peritoneal mesothelial cells.Based on the above analysis, we think, microRNA-200c may be involved in the regulation of peritoneal mesothelial cells of EMT and peritoneal fibrosis, and the expression levels of miRNA-200c may influence the EMT of peritoneal mesothelial cells. Chapter Ⅰ.The expression of microRNA-200c in human peritoneal mesothelial cells isolated from effluents in dialysis fluid and its relationship with epithelial-mesenchymal transitionObjective:To detect the expression of microRNA-200c in HPMC from effluents, and to investigate the relationship between microRNA-200c and EMT in peritoneal dialysis.Methods:27patients undergoning continuous ambulatory peritoneal dialysis (CAPD) were enrolled into this study,13patients undergoing PD start while14patients undergoing PD over1year. The isolated cells from effluents in dialysis fluid were cultured and identified by morphology and Immunofluorescence. The expressions of E-cadherin,Col-I, vimentin and FN were tested by realtime PCR and western blot. The correlation analysis was done between microRNA-200c and E-cadherin, vimentin, FN, Col-1.Results:Cells isolated from effluents of dialysis fluid were identified as HPMC. The morphology varied according to how long patients undergoing PD. Compared to patients undergoing PD start, The expression of microRNA-200c and E-cadherin in HPMCs derived from dialysate is decreased in PD patients longer than1year, while the expression of vimentin and fibronectin both in mRNA and protein level was remarkable increased in PD over1year group, compared to PD start group. The expression of microRNA-200c is positively related to the level of E-cadherin,while negatively related to the level of vimentin, FN, Col-1.Conclusion:Cells isolated from effluents of dialysis fluid were identified as HPMC, and the expression of microRNA-200c in HPMCs were confirmed.With the prolongation of PD time, microRNA-200c expression was significantly reduced and associated with EMT, suggesting that microRNA-200c may be associated with peritoneal fibrosis. Chapter II.Expression of microRNA-200c and its role in the process of high glucose induced EMT in HMrSV5cell linesObjective:To investigate the effect of miRNA-200c in the process of high glucose induced EMT in HMrSV5cell lines.Methods Methods:HMrSV5cells were exposed respectively in different concentrations of D-glucose (30,60,90mM) for24hours and90mM D-glucose with different durations (0,12,24,48h). The morphological change of cells was detected by microscope. The expression of miRNA-200c was examined by real-time PCR, and the expression of E-cadherin、Vimentin、Col-Ⅰ and FN were examined by Western blot and real-time PCR. miRNA-200c mimics and miRNA-200c mimics negative control were transfected into HMrSV5cells using lipofectamine2000, then cells were stimulated in90mM D-glucose. The expression of miRNA-200c, E-cadherin、Vimentin、Col-Ⅰ and FN were examined as above, besides the expression of E-cadherin and Vimentin、 were also measured by fluorescence microscopy.Results:Compared to control group, high glucose decreased the expression of E-cadherin but increased the expression of Vimentin Col-Ⅰ and FN in HMrSV5cells in dose and time-dependent manner. Compared to control group, high glucose decreased the expression of miRNA-200c in HMrSV5cells in dose and time-dependent manner. Overexpression of miRNA-200c increased the mRNA and protein expression of E-cadherin, and decreased the mRNA and protein expression of Vimentin、Col-Ⅰ and FN in HMrSV5cells stimulated with high glucose.Conclusion:High glucose induces EMT in HMrSV5cells; High glucose decreases the expression of miRNA-200c, while Overexpression of miRNA-200c inhibits EMT in HMrSV5cells, which suggests that miRNA-200c could regulate and reverse the EMT of peritoneal mesothelial cells. Chapter Ⅲ. microRNA-200c regulates high glucose induced EMT via modulation of ZEB1in HMrSV5cell linesObjective:To detect the expression of ZEB1both in HPMCs from PD patients with different dialysis durations and HMrSV5cell lines. And to investigate the mechanism by which miRNA-200c modulates the process of high glucose induced EMT in HMrSV5cell lines.Methods:The expression of ZEB1in HPMCs derived from PD patients and HMrSV5stimulated with high glucose with different concentrations (30,60,90mM D-glucose) and with different durations (0,12,24,48h) were examined by Western blot and real-time PCR. The expression of ZEB1in HMrSV5cells stimulated with high glucose was also examined by Western blot, real-time PCR and fluorescence microscopy after overexpression of miRNA-200c by transfecting with miRNA-200c mimics and negative control.Results:Comparing to PD start patients, the expression of ZEB1in HPMCs derived from dialysate effluent was increased in PD≥1year patients and high glucose increased the expression of ZEB1in dose and time-manner. Overexpression of miRNA-200c decreased mRNA and protein expression of ZEB-1in HMrSV5cells stimulated with high glucose.Conclusion:The expression of miRNA-200c in HPMCs derived from dialysate effluent is significantly decreased in the long term PD patients; High glucose increases the expression of ZEB1, while overexpression of miRNA-200c decreases the mRNA and protein expression of ZEB1in HMrSV5cells stimulated with high glucose, which suggests that miRNA-200c may regulate the EMT of peritoneal mesothelial cells mediated by ZEB1. |
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