Effect Of MicroRNA-29c On Epithelial-mesenchymal Transition In Human Peritoneal Mesothelial Cells And Its Possible Mechanism

BackgroundPeritoneal dialysis (PD) is a very important alternative renal replacement therapy for patients with end stage renal disease. Compared to hemodialysis, PD is relatively economy, easily operating and better for protecting residual renal function, which may be more realistic for our China. Peritoneal fibrosis is a common complication of long term peritoneal dialysis (PD), this alteration of peritoneal structure would ultimately lead to ultrafiltration failure of peritoneum, which restricts the widely application of PD. However, the mechanism and regulation of peritoneal fibrosis are incompletely understood. It is believed that the epithelial-mesenchymal transition (EMT) of peritoneal mesothelial cells, plays a pivotal role in the pathogenesis of peritoneal fibrosis.microRNAs (miRNAs) are21-25nucleotide non-coding RNA molecules. miRNAs are involved in multiple cell activities, by regulating at least one-third of protein coding transcripts in the mammalian genome. Recent studies demonstrated that miRNA could regulate EMT in different kind of cells and it was shown effective to inhibit organ fibrosis, such as cardiac, liver, lung, renal fibrosis as well as systemic sclerosis, by targeting miRNAs to reverse EMT. It seems that miRNAs could potentially serve as therapeutic targets and novel disease biomarkers. However, the role of miRNA in EMT of peritoneal mesothelial cell and peritoneal fibrosis is largely unknown.miRNA-29family consists of miRNA-29a/b/c, which is closely related to a variety of fibrotic diseases. Previous miRNA microarray studies by our group revealed that the expression of miRNA-29c in peritoneal mesothelial cell are upregulated in patients undergoing PD over6months compared to those newly started, and peritoneal mesothelial cells derived from long term PD patients underwent EMT process. Hence, we hypothesized that miRNA-29c may be involved in the process of EMT in peritoneal mesothelial cell. Therefore, experimental research is carried out as follow: Chapter I. The expression of miRNA-29c in peritoneal mesothelial cells from PD patients and its relationship with EMTObjective:To detect the expression of miRNA-29c in peritoneal mesothelial cells in PD patients with different dialysis durations, and to investigate the relationship between miRNA-29c and EMT in peritoneal mesothelial cell.Methods:24PD in and outpatients in our department were enrolled into the study and were divided into newly started group (PD<30days) and PD≥1year group. Their overnight dialysate effulent were collected to obtain human peritoneal mesothelial cells (HMPCs) after centrifugation and cultivation. HMPCs were identified by microscope and flow cytometry (FCM).The expression of miRNA-29c in HMPCs was detected by real-time PCR. And the expression of E-cadherin, a-smooth actin(a-SMA), Collagen-Ⅰ(Col-1) and fibronectin (FN) in HMPCs were tested by western-blot and real-time PCR.Results:1. The HPMCs derived from dialysate effluent in PD patients longer than1year showed fibroblast-like appearance but cobblestone-like appearance in newly started PD patients;2. The proportion of CD45-/CD68-/cytokeratin+cells in the entire cultivated cells is93.61±2.52%;3. Comparing to newly starters, the expression of miRNA-29c in HPMCs derived from dialysate effluent is increased in PD patients longer than1year;4. Comparing to newly starters, the expression of E-cadherin in HPMCs is increased, while a-SMA, Col-Ⅰ and FN decreased in PD patients longer than1year;5. The expression of miRNA-29c is negatively related to the level of E-cadherin, while positively related to the level of a-SMA,Col-Ⅰ and FN in HPMCs.Conclusion:The expression of miRNA-29c in HPMCs is significantly increased and accompanied with EMT change in the long term PD patients, which suggestes that the expression of miRNA-29c maybe related to the EMT process of peritoneal mesothelial cells. Chapter Ⅱ. The expression of miRNA-29c and its role in the high glucose induced EMT in human peritoneal meosthelial cellsObjective:To detect the expression of miRNA-29c in human peritoneal mesothelial cell lines (HMrSV5cells) and investigate the role of miRNA-29c in the development of high glucose induced EMT in HMrSV5cells.Methods:HMrSV5cells were exposed respectively in different concentrations of D-glucose (30,60,90mM) for24hours and60mM D-glucose with different durations (0,12,24,48h). The morphological change of cells was detected by microscope. The expression of miRNA-29c was examined by real-time PCR, and the expression of E-cadherin,α-SMA,Col-Ⅰ and FN were examined by Western blot and real-time PCR. miRNA-29c inhibitor and miRNA-29c inhibitor negative control were transfected into HMrSV5cells using lipofectamine2000, then cells were stimulated in60mM D-glucose. The expression of miRNA-29c, E-cadherin,α-SMA,Col-Ⅰ and FN were examined as above, besides the expression of E-cadherin and a-SMA were also measured by fluorescence microscopy.Results:1. Compared to control group, cobblestone-like HMrSV5cells transformed to fibroblast-like cells, after stimulation with high glucose for24hs, obvious being seen in60,90mM D-glucose. 2. Compared to control group, high glucose decreased the expression of E-cadherin but increased the expression of a-SMA,Col-Ⅰ and FN in HMrSV5cells in dose and time-dependent manner.3. Compared to control group, high glucose increased the expression of miRNA-29c in HMrSV5cells in dose and time-dependent manner.4. Downregulation of miRNA-29c increased the mRNA and protein expression of E-cadherin, and decreased the mRNA and protein expression of α-SMA,Col-Ⅰ and FN in HMrSV5cells stimulated with high glucose.Conclusion:High glucose induces EMT in HMrSV5cells; High glucose increases the expression of miRNA-29c, while downregulation of miRNA-29c inhibits EMT in HMrSV5cells, which suggests that miRNA-29c could regulate the EMT of peritoneal mesothelial cells ChapterⅢ. miRNA-29c regulates Spry-1expression and its relationship with high glucose induced EMT in human mesothelial cellsObjective:To detect the expression of Spry-1both in HPMCs from PD patients with different dialysis durations and HMrSV5cells and investigate the underlying mechanism of miRNA-29c in regulation of EMT.Methods:The expression of Spry-1in 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 Spry-1in HMrSV5cells stimulated with high glucose was also examined by Western blot, real-time PCR and fluorescence microscopy after downregulation of miRNA-29c by transfecting with miRNA-29c inhibitor and negative control.Results:1. Comparing to newly starters, the expression of Spry-1in HPMCs derived from dialysate effluent was decreased in PD patients longer than1year;2. Compared to control group, high glucose decreased the expression of Spry-1in dose and time-manner;3. Downregulation of miRNA-29c increased the protein expression not mRNA expression of Spry-1in HMrSV5cells stimulated with high glucose.Conclusion:The expression of miRNA-29c in HPMCs derived from dialysate effluent is significantly decreased in the long term PD patients; High glucose decreases the expression of Spry-1, while downregulation of miRNA-29c increases the protein expression of Spry-1in HMrSV5cells stimulated with high glucose, which suggests that miRNA-29c may regulate the EMT of peritoneal mesothelial cells mediated by Spry-1.