| Background Peritoneal dialysis (PD) is an established alternative for the replacement therapy of end stage renal disease (ESRD). Unfortunately, the limitation of long-term PD is peritoneal fibrosis (PF), resulting in ultrafiltration failure (UFF). Epithelial-mesenchymal transition (EMT) is the initiate and reversible stage of PF and extracellular matrix (ECM) accumulation is the key histological change of PF. It is established that transforming growth factor-P (TGF-P) plays a key role in these processes, and is regulated by multi-factors.microRNAs are 21～25 nucleotide small non-coding RNAs that participate in gene regulation, and exist in species ranging from plants to humans genome. They regulate gene expression by base-pairing to 3' untranslated region of target mRNAs. microRNAs have been implicated in various biological processes such as developing, differentiation, proliferation, apoptosis, tumorigenesis, et al. They are also related to pathological processes such as EMT. It is reported that EMT-related protein like Smad interacting protein 1 (SIP1) or connective tissue growth factor (CTGF) can be regulated by some microRNAs.Therefore, novel microRNAs may play a key role in the process of EMT and ECM accumulation in petitoneal dialysis. To this end, experimental research is carried out as follow. Objective To investigate the expression of microRNAs and the EMT status in HPMC from effluents, and to observe the relationship of microRNA-129-5p and EMT in peritoneal dialysis.Methods 22 patients undergoning continuous ambulatory peritoneal dialysis (CAPD) were enrolled into this study,10 patients undergoing PD start while 12 patients undergoing PD over 6 months. The isolated cells from effluents in dialysis fluid were cultured and identified by morphology and immunohistochemistry. Scaning the samples through micro array to determine the different microRNAs expression between PD-start group and PD-over-6-month group. Taqman assay was used to determine the expression of microRNA-129-5p. The expressions of E-cadherin, claudin1, vimentin and FN were tested by realtime PCR and western blot.Results Cells isolated from effluents of dialysis fluid were identified as HPMC. The morphology varied according to how long patients undergoing PD. Micro array showed that 33 miRNAs up-regulated and 58 miRNAs down-regulated in cells from patients undergoing PD over 6 months, compared to patients undergoing PD start. The down-regulation of microRNA-129-5p was clarified. Poor expression of E-cadherin and claudin1 and excessive expression of vimentin and fibronectin (FN) both in mRNA and protein level was remarkable in PD-over-6-month group, compared to PD-start group.Conclusion Multiple EMT-related microRNAs can be found in HPMC from effluents. Down-regulation of miRNA-129-5p along with EMT and ECM accumulation during long-term PD indicated that miRNA-129-5p was negatively related to peritoneal fibrosis. Methods HMrSV5 cells were exposed to 5ng/ml TGF-β1. The expressions of E-cadherin, claudin1, vimentin and FN were examined by realtime PCR and western blot, meanwhile realtime PCR for miRNA-129-5p. Furthermore HMrSV5 cells were transfected with miRNA-129-5p precursor (pre-mir-129-5p) or pre-miR negative control before exposing to TGF-β1. Then the expressions of E-cadherin, claudin1, vimentin and FN were detected by immunofluorescence, realtime PCR and western blot.Results Stimulation of HMrSV5 cells with TGF-β1 resulted in a significant decrease of E-cadherin and claudinl, and increase of vimentin and FN, all in time-dependent manner. TGF-β1 also repressed miRNA-129-5p. Compared to TGF-β1 group, overexpression of miRNA-129-5p in HMrSV5 cells upregulated the mRNA and protein expression of E-cadherin and claudinl, and downregulated the mRNA and protein expression of vimentin and FN. However, pre-miR negative control had no significant effect on the expression of E-cadherin, claudin1, vimentin and FN. Conclusion TGF-β1 leads to EMT and ECM accumulation and represses miRNA-129-5p level. Overexpression of miRNA-129-5p can reverse the EMT of HMrSV5 cells and inhibit the ECM accumulation.Ⅸ Objective To investigate the mechanism by which miRNA-129-5p modulates the process of TGF-β1 induced EMT and ECM accumulation in HMrSV5 cell lines.Methods HMrSV5 cells were exposed to 5ng/ml TGF-β1. The expression of SIP1 was examined by realtime PCR and western blot. Furthermore HMrSV5 cells were transfected with pre-mir-129-5p or pre-miR negative control before exposing to TGF-β1. Then the expression of SIP1 was detected by immunofluorescence, realtime PCR and western blot.Results Stimulation of HMrSV5 cells with TGF-β1 resulted in a significant increase of SIP1 in time-dependent manner. Compared to TGF-β1 group, overexpression of miRNA-129-5p in HMrSV5 cells downregulated the protein expression of SIP1, but had no effect on its mRNA expression. However, pre-miR negative control had no significant effect on the expression of SIP 1.Conclusion TGF-β1 leads to upregulation of SIP1. During the process of TGF-β1 induced EMT in HMrSV5 cells, miRNA-129-5p modulates E-cadherin and claudinl expression by posttranscriptional repression of SIP1. |
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