| Background Peritoneal dialysis (PD) has become an alternative to hemodialysis for treatment of end stage of renal disease (ESRD). Unfortunately, peritoneal fibrosis is an almost invariable consequence of PD. Recent evidence suggests that the epithelial to mesenchymal transition (EMT) of human peritoneal mesothelial cells (HPMCs) is an early event during PD and is associated with high peritoneal transport and it is a key process leading to peritoneal fibrosis and function deterioration. TGF-β1 serves as a source of EMT and induces the EMT process in various epithelial cells and tissues fibrosis in vitro and in vivo.The microRNAs (miRNA, miR) are a class of short non-coding RNAs that are evolutionarily conserved, and function as negative regulators of gene expression. It has been well established that miRNAs play key roles in modulating gene expression during embryonic development and cell differentiation on post-transcriptional level. It is estimated that they regulate more than one-third of cellular messenger RN A by binding to the 3'untranslated region (UTR) of their target genes or by inducing mRNA degradation. Recent datas suggeted that miRNA play a role during EMT in diabetes nephropathy and mammary adenocarcinoma by modulating transcription factor and TGF-β1 signal pathway. We had detected the expression of microRNA194 in HPMCs isolated from effluents in dialysis fluid by Realtime PCR, which sugguets that miRNA194 was upregulated in long-term peritoneal dialysis patients. The upregulation of miRNA194 was negative correlated with E-cadherin but positive correlated with a-SMA, which suggested that miRNA might play a role in EMT of peritoneal mesothelial cells. miRNA distribute with tissue specificity, the miRNA profile of HPMCs undergoing EMT and the role of miRNAs in TGF-β1 induced EMT in HPMCs were remained unclear.As mentioned above, we suppose that TGF-β1 induced variation of miRNA profile as well as EMT in HPMCs. The EMT induced by TGF=β1 could be inhibited by modulating the level of definite miRNA. To this end, the experimental research is carried out as follows.Chapter one Profiling MicroRNA Expression in Human Peritoneal Mesothelial Cells Undergoing EMT Induced by TGF-β1Objective To profile the miRNA expression in HPMCs undergoing EMT induced by TGF-β1.To screening the EMT related miRNA in HPMCs. Methods HPMCs were divided into groups, control group (only FBS-free DMEM/F12), TGF-β1, group (treated with TGF-β1,5ng/ml for 12h,24h and 48h, respectively). Morphology of HPMCs was estimated by inverted microscope. The expressions of ZO-1 and vimentin in HPMCs were determined by immunofluorescence and realtime PCR, respectively. The level of E-cadherin in HPMCs was determined by realtime PCR and western blot, respectively. The profile of miRNA was determined by miRNA microarray and the target miRNA was verificated by realtime PCR. The target mRNAs of microRNA were estimated by program based on bioinformatics.Results The fibroblast like cells was observed in TGF-β1 treated groups. The expression of ZO-1 and E-cadherin were time dependently downregulated by TGF-β1 while the expression of vimentin was time dependently upregulated by TGF-β1,. Marked miRNA profile variation was obtained in HPMCs treated with or without TGF-β. hsa-miRPlus-E1114 and hsa-miRPlus-E1245 were upregulated but hsa-miR-589, hsa-miR-505*, hsa-miRPlus-F1147, hsa-miRPlus-E1033, hsa-miR-337-5p, hsa-miR-513a-5p, hsa-miRPlus-E1075, hsa-miR-891a, hsa-miR-1260, hsa-miR-1280, hsa-let-7e and hsa-miR-1255a were downregulated. The downregulation of miRNA589 was verificated by realtime PCR. Ets-1, the regulator of SIP1, was the target mRNA of miRNA589 and miRNA 1260. Conclusion TGF-β1, induced marked variation of miRNA profile as well as EMT in HPMCs. Downregulation of miRNA589 and miRNA1260 might play a role in TGF-β1, induced EMT in HPMCs.Chapter two The molecular mechanism of miRNA589 regulate Epithelial-Mesenchymal Transition in Human Peritoneal Mesothelial CellsObjective To investigate the effect of miRNA589 on EMT, and to understand the possible molecular mechanism of miRNA589 mediated EMT in HPMCs.Methods HPMCs were divided into three groups:control group (only FBS-free DMEM/F12), TGF-β1 group (treated with TGF-β1 5ng/ml for 24h) and pre-miR-589+TGF-β1 group (pre-treated with pre-miR-589, and treated with TGF-β1 5ng/ml for 24h). The level of miRNA589 was determined by realtime PCR. The expressions of ZO-1, vimentin, E-cadherin and Ets-1 in HPMCs were determined by realtime PCR and Western blot, respectively. The expression of SIP 1 mRNA in HPMC's was determined by realtme PCR.Results The level of miRNA589 was decreased in TGF-β1 group, while that of pre-miR-589+TGF-β1, group was dramatically increased. The downregulation of E-cadherin and ZO-1 mRNA and protein in HPMCs induced by TGF-β1 partly reversed by pre-miR-589. The upregulation of vimentin mRNA and protein induced by TGF-β1 (5ng/ml) in HPMCs were attenuated by pre-miR-589. The upregulation of Ets-1 mRNA was detected by realtime PCR in both TGF-β1 group and TGF-β1 +pre-miR-589 group. The upregulation of Ets-1 protein as well as SIPl mRNA induced by TGF-β1 (5ng/ml) in HPMCs were attenuated by pre-miR-589.Conclusion Downregulation of miRNA589 may mediate TGF-β1 induced EMT in HPMCs. Upregulation of miRNA589 may inhibit TGF-β1 induced EMT in HPMCs via degradation Ets-1 on post-transcriptional level. |
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