Effect of MicroRNA302c on Epithelial-mesenchymal Transition in Peritoncal Mesothelial Cells
|School||Central South University|
|Keywords||Epithelial cells - mesenchymal cell transdifferentiation High glucose peritoneal dialysate microRNA302c TGF-β1 Human peritoneal mesothelial cells CTGF|
Background peritoneal dialysis (peritoneal dialysis, PD) is one of the most important alternative treatment of patients with end-stage renal disease (end stage renal disease, ESRD). Studies have shown that, as the improved dialysis tube and catheter technology improved, the incidence of peritonitis was significantly decreased, but the long-term peritoneal dialysis patients due to peritoneal fibrosis, there will be a reduction in performance of peritoneal dialysis, ultrafiltration failure and gradually and become continuous ambulatory peritoneal dialysis (continuous ambulatory peritoneal dialysis, CAPD) patients withdrew from the main reason. TGF-p-mediated the peritoneal fibrosis pathogenesis of research is the focus of the of PD field research at home and abroad. The recent researchers found that epithelial cells - transdifferentiation of mesenchymal cells (epithelial-mesenchymal transition, EMT) played a key role in occurrence of peritoneal fibrosis induced by TGF-p [2,3]. CTGF (connective tissue growth factor, CTGF) as a fibrogenic factor involved in the pathophysiological process of peritoneal fibrosis, also played an important role in the occurrence of fibrosis in the process of initiating EMT [4 microRNA (miRNA) is present in the plant and animal genome functional non-coding RNA, about 17 to 29 nucleotides. Identification of miRNAs has accounted for only 1% to 3% of the human genome sequence, but in 1/3 of the regulation of gene expression in the growth and development, cell differentiation, proliferation, apoptosis, tumor play an important role. The latest research reports the miRNAs expression disorders associated with many diseases and play a key role in the process of development of EMT. High channel miRNA microarray PD peritoneal effluent peritoneal mesothelial cells miRNA expression pattern analysis. Found beginning with peritoneal dialysis compared to many miRNAs table up-or down-regulated. The which microRNA302c based CTGF as a downstream target of. The prompted CTGF increased expression of PD peritoneal mesothelial cells may be related to abnormal expression microRNA302c. Based on the above analysis, we believe, microRNA302c involved in the regulation of peritoneal mesothelial cells EMT and fibrosis. Chapter microRNA302c expression in mouse peritoneal EMT model of peritoneal tissue and its relationship with EMT purpose using mouse peritoneal EMT model, explore the expression of microRNA302c peritoneal tissue EMT process and its relationship with EMT. Method of 12-week-old ICR male mice, 28-30 grams. Randomly divided into normal control group, sham-operated (sham) saline 15 days and 30 days group model 4.25% PDS 15 day group and 30 days group. sham group: intraperitoneal injection of 1.5ml saline, once a day. Model group: intraperitoneal injection of 1.5ml standard dialysate containing 4.25% glucose, once a day. The mice were killed in the first 15 days and 30 days, respectively, by the grouping. Visceral peritoneum of mice used to extract tissue protein and mRNA, and Western-blot, the Realtime PCR detected microRNA302, zo-l, vimentin, CTGF expression. Results Our previous experiments have confirmed: extended time with intraperitoneal injection of dialysate zo-1 and vimentin protein and mRNA expression of peritoneal tissue of the sham group was not statistically significant; model group zo-1 expression was decreased, vimentin expression gradually enhanced. Success prompted the peritoneal EMT modeling, saline can not be induced peritoneal EMT change. 2. Realtime PCR display the model mice peritoneal tissue microRNA302c expression decreased gradually with the extension of the injection the PDS time of the. Western Blot and Realtime PCR zo-1 protein and mRNA levels compared with normal control group, model group mice peritoneal tissue with injection PDS time extended gradually reduced, vimentin, CTGF protein and mRNA expression increased. 4. Linear correlation analysis showed that mouse peritoneal EMT process induced by high glucose peritoneal dialysate and normal control group and model group micro RNA302c expression of CTGF mRNA expression was negatively correlated; microRNA302c expression of vimentin mRNA expression was negatively correlated; microRNA302c expression the zo-1mRNA expression was positively correlated; CTGF mRNA and vimentin mRNA expression was positively correlated; CTGF mRNA and zo-l mRNA expression was negatively correlated. The conclusion in mouse peritoneal EMT model, with EMT progress, the peritoneal tissue microRNA302c expression significantly reduced and a negative correlation with the EMT, CTGF, suggesting that play an important role in the mouse peritoneal mesothelial cells EMT process microRNA302c. The second chapter microRNA302c TGF-β1-induced expression in peritoneal mesothelial cells EMT and EMT Objective To investigate the HPMC EMT in TGF-β1-induced microRNA302c the role. HPMC, with 5ng/ml TGF-β1 stimulation through Realtime PCR and Western blot detection cells microRNA302c and zo-1, vimentin expression; the transfection microRNA302c precursor (pre-miR-302c), be re-TGF-β1 stimulation Realtime PCR and Western blot of HPMC microRNA302c and zo-1, vimentin expression changes. Results 1.5ng/mlTGF-β1 stimulation, HPMC microRNA302c expression was time dependent decrease zo-1 mRNA and protein levels in a time-dependent reduction in positive for vimentin mRNA and protein expression levels in a time dependent increase. 2. Realtime PCR results showed that TGF-β1 group microRNA302c significantly reduced compared with the control group, TGF-β1 pre-miR-the 302c groups microRNA302c expression significantly increased compared with TGF-β1 group (p <0.05), while the control groups showed no significant difference (p> 0.05). 3. Realtime PCR and Western blot results, zo-1 mRNA and protein levels were significantly reduced compared with the control group, TGF-β1 group vimentin mRNA and protein levels were significantly raised above change in pre-miR-302c TGF-β1 group were suppressed, were statistically significant. Conclusion TGF-β1-induced the HPMC EMT; TGF-β1 drop induced microRNA302c expression; raised microRNA302c HPMC EMT can be reversed. Chapter microRNA302c by regulating CTGF involved in TGF-β1-induced EMT in human peritoneal mesothelial cells to investigate microRNA302c involved in the regulation of TGF-β1-induced the HPMC EMT possible mechanism. Method 5ng/ml TGF-β1 stimulation of HPMC, by Realtime PCR and Western blot to detect the expression of CTGF cells; the transfection microRNA302c precursor (pre-miR-302c), shall then TGF-β1 stimulation by Realtime PCR and Western blot analysis HPMC CTGF expression changes. 1.5ng/ml TGF-β1 stimulation the HPMC of CTGF mRNA and protein levels in a time dependent upregulation. 2. Realtime PCR and Western blot showed that compared with the control group, TGF-β1-CTGF mRNA and protein expression levels were significantly increased above changes in the group of pre-miR-302c TGF-β1 was inhibited statistically significant. Conclusion TGF-β1-induced the HPMC CTGF upregulation the; microRNA302c regulation of CTGF affect zo-1, vimentin expression, and thus participate in the TGF-β1-induced peritoneal mesothelial cells EMT.