| Background Peritoneal dialysis (PD) is an established alternative for the replacement therapy of end stage renal disease (ESRD). Studies show that with the improvement of peritoneal dialysis tube and technology, the incidence of peritonitis significantly decreased, but the long-term peritoneal dialysis patients because of peritoneal fibrosis, the effectiveness of PD decreasing, leading to ultrafiltration failure, which gradually become the main cause of patients withdrew from continuous ambulatory peritoneal dialysis (CAPD).TGF-βmediated pathogenesis of peritoneal fibrosis have been the concentration of PD study at home and abroad. Recently researchers found that EMT plays a critical role in TGF-β-induced peritoneal fibrosis Connective tissue growth factor (CTGF) as a factor of promoting fibrosis, involved in the pathophysiological process of peritoneal fibrosis, while also plays an important role in promoting the process of fibrosis which initiating the occurrence of EMT, and may become a new target for the treatment of peritoneal fibrosis.MicroRNA (miRNA) genes are present in animal and plant functional group, non-coding RNA, about 17-29 nucleotides. MiRNAs has been identified only 1% to 3% of the human genome sequence, but regulate in 1/3 of the gene expression, which play an important role in the growth and development, cell differentiation, proliferation, apoptosis, tumor and so on. The latest research report, the disorders of miRNAs expression associated with many diseases, and plays a key role in development process of EMT. We used high-channel miRNA chip to analyse miRNA expression profiles of peritoneal mesothelial cells from peritoneal effluent. We found that, compared with peritoneal dialysis start, the expression of many miRNA increased or decreased. CTGF is a downstream target of miRNA-302c. It suggests that the increased expression of CTGF may be related to abnormal expression of miRNA302c.Therefore, microRNA302c may plays a key role in the process of EMT in peritoneal mesothelial cells. Chapter I microRNA302c expression in a mice model of peritoneal EMT and the relationship with EMTObjective To investigate the expression of microRNA302c and the relationship with EMT in mesothelial cells in a mice model of peritoneal EMTMethods Male ICR mice were randomizely divided into normal control group, sham-operated groups and model groups. Sham groups were divided into NS 15d group and 30d group; model groups were divided into 4.25% PDS 15d group and 30d group. The mice in model groups received a daily infusion of 1.5ml 4.25% PDS. The mice in sham groups were intraperitoneally injected of saline.The animals were sacrificed at day 15 or 30 according to groups. Visceral peritoneum were harvested to extract tissue protein and mRNA. The expression of microRNA302c,zo-1,vimentin,CTGF were examined by Western blot and Realtime PCR.Results1. Compared with control, previous experiments have showed that accompanied with prolonged intraperitoneal injection of dialysis fluid, zo-1 and vimentin protein and mRNA expression in sham-operated group were not statistically different, but expression level of zo-1 of model group mice gradually declined, while vimentin expression up regulated gradually.2. In the peritoneal tissue of mice model, Realtime PCR showed that the expression of microRNA302c decreased accompanied with time of the injection of PDS extended.3. Western blot and Realtime PCR showed that zo-1 protein and mRNA level in peritoneaum of model mice gradually decreased; vimentin,CTGF protein and mRNA expression were both elevated.4. Linear correlation analysis showed that in model group, vimentin and CTGF mRNA expression were both negatively correlated with microRNA302c. but zo-1 positively correlated with microRNA302c; CTGF mRNA expression was negatively correlated with zo-1 mRNA, while positively correlated with vimentin mRNA.Conclusion During the EMT process, microRNA302c expression were significantly decreased in the peritoneum of mice peritoneal EMT model and both negatively correlated with EMT and CTGF, which suggests that microRN A302c might play an important role in EMT of mice peritoneal mesothelial cells. ChapterⅡExpression of microRNA302c in EMT induced by TGF-β1 and the relationship with EMTObjective To investigate the effect of microRNA302c in the process of TGF-β1 induced EMT in HPMC.Methods HPMCs were exposed to 5ng/ml TGF-β1. The expressions of microRNA302c, zo-1, vimentin were examined by Realtime PCR and Western blot. Furthermore HPMCs were transfected with microRNA302c precursor (pre-mir-302c) before exposing to TGF-β1. Then the expressions of microRNA302c, zo-1, vimentin were detected by Realtime PCR and Western blot.Results1. Stimulation of HPMCs with TGF-β1 resulted in a significant decrease of microRNA302c and zo-1, and increase of vimentin, all in time-dependent manner.2. The level of microRNA302c was decreased in TGF-β1 group, while that of pre-miR-302c+TGF-β1 group was dramatically increased.3. The downregulation of zo-1 mRNA and protein in HPMCs induced by TGF-β1 partly reversed by pre-miR-302c. The upregulation of vimentin mRNA and protein induced by TGF-β1 (5ng/ml) in HPMCs were attenuated by pre-miR-302c.Conclusion TGF-β1leads to EMT and represses microRNA302c level. Overexpression of microRNA302c can reverse the EMT of HPMC. Chapter III MicroRNA302c regulates TGF-β1 induced EMT via modulation of CTGF in HPMCObjective To investigate the mechanism by which microRNA302c modulates the process of TGF-β1 induced EMT in HPMC.Methods HPMCs were exposed to 5ng/ml TGF-β1. The expression of CTGF was examined by Realtime PCR and Western blot. Furthermore HPMCs were transfected with pre-mir-302c before exposing to TGF-β1. Then the expression of CTGF was detected by Realtime PCR and Western blot.Results1. Stimulation of HPMCs with TGF-β1 resulted in a significant increase of CTGF, in time-dependent manner.2. The level of CTGF was increased in TGF-β1 group, while the upregulation of CTGF mRNA and protein induced by TGF-β1 (5ng/ml) in HPMCs were attenuated by pre-miR-302c.Conclusion TGF-β1 leads to upregulation of CTGF. During the process of TGF-β1 induced EMT in HPMCs, microRNA302c modulates zo-1 and vimentin expression by posttranscriptional repression of CTGF. |
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