Effect Of Lentivirus-mediated MiRNA-302c On EMT Of Peritoneal Mesothelial Cells And The Mechanism

Background Chronic kidney disease (CKD) is a public health problem all over the world, the morbidity is about11%, and the incidence increased year by year.5%of the CKD patients will eventually develop into the end-stage renal failure, and should have life-long renal replacement therapy. Peritoneal dialysis (PD) is an important modality of renal replacement therapy according with China catual condition. But long-term PD can lead to peritoneal fibrosis and ultrafiltration failure which ultimately limits the long term clinical application of PD and makes patients drop out of PD. Transforming growth factor-β (TGF-β) is a key factor in the mechanism of peritoneal fibrosis, recently some studies have found that epithelial mesenchymal transition (EMT) plays a important role in TGF-βinduced peritoneal fibrosis, it is the initiate and reversible process of fibrosis. Connective tissue growth factor (CTGF) can accelerate cell proliferation, migration and differentiation. Some studies have showed that CTGF acts both as an effector in the downstream cascade of fibrosis induced by TGF-β and as a pro-fibrotic factor, it is closely related to the occurrence and development of many organs fibrosis. It also has been proved that CTGF not only play a important role in PD related peritoneal fibrosis, but also facilitate EMT in the process of fibrosis. MicroRNAs are non-coding RNAs that post-transcriptionally regulate gene expression by inducing target mRNA degradation or inhibiting its translation. They participate in regulating growth and development, cell differentiation, proliferation, apoptosis and theevelopment of tumour, nowadays become the hot topic in the field of life sciences. Some studies have proved that microRNAs can participate in regulating the development of EMT, and they are important regulators3n the process of TGF-β induced EMT such as microRNA-200family and microRNA-205.With the development of gene therapy technology and gene delivery system, amounts of significant results have been achieved in rnderstanding the mechanism of PD related peritoneal fibrosis. Promising results from numerous studies in animal models of peritoneal dialysis have demonstrated that gene delivery method is an innovative and feasible strategy for mechanistic investigation and potential treatment of peritoneal fibrosis. Lentiviral vector (LV) has been widely used as an effective genomic vector, it has a relatively large capacity (close to10kb) and high transfection efficiency that can mediate stable gene transfer in dividing and non-dividing cells without potent immunogenic or inflammatory response. It is very meaningful to apply it in peritoneal fibrosis research.As mentioned above, we suppose that novel microRNA may participate in regulating EMT and peritoneal fibrosis in PD patients. Using the tool of lentiviral vectors, it has important theoretical and practical significance to research the role of microRNA in EMT and peritoneal fibrosis in the process of PD.Chapter I Effect of lentivirus infecting peritoneum in mice through intraperitoneal injectionObjectiveTo investigate the effect of lentivirus infecting peritoneum in mcting lentivirus carring GFP (LV-GFP) with different titer.and determine the best effective titer.Methods Male ICR mice were randomly divided into control group (n=5), LV-GFP1group (n=5), LV-GFP2group (n=5), LV-GFP3group (n=5). The mice reoeived an infusion of1.5ml0.9%N.S. or LV-GFP with different titer. The mice were sacrificed at day28. Parietal peritoneum (?) collected for-HE and Masson staining or frozen section to゜serve was morphology of peritoneum and the fluorescence of GFP. Visceral p(?)toneum were harvested to extract tissue protein and mRNA for examining the GFP expression by Real Time PCR and western blot.Results1. Observing through fluorescence microscope, the frozen sections of control group have no fluorescence of GFP, the frozen sections of LV-GFP1group, LV-GFP2group and LV-GFP3group have fluorescence of GFP. 2. The paraffin sections of mice peritoneal with HE、Masson staining showed that compared with the control group, LV-GFP1group, LV-GFP2group and LV-GFP3group have no apparent morphology changes.3. Real Time PCR and western blot showed that the control group has no GFP expression, LV-GFP1group, LV-GFP2group and LV-GFP3group have GFP expression.4. The expression of GFP in LV-GFP2group and LV-GFP3-group is stronger than LV-GFP1group, the expression of GFP between LV-GFP2group and LV-GFP3group is similar.Conclusion The lentivirus can successfully infect the peritoneum-of mouse by intraperitoneal injection, and2×108GTU is the best effcetive tiler. Chapter Ⅱ The effect of miRNA-302c in EMT of peritoneum in mice on peritoneal dialysis and the mechanismObjective To investigate the effect of miRNA-302c in EMT of peritoneum in mice on peritoneal dialysis and the mechanism by intraperitoneal injecting LV-mmu-miR-302c to infect mice peritoneum.Methods Male ICR mice were randomly divided into control group (n=5), PDF group (n=5), LV-mmu-miR-302c+PDF group (n=5), LV-pGIPZ+PDF group (n=5). The mice received an infusion of1.5ml0.9%N.S. or1.5ml4.25%PDF in the presence or in the absence of LV-mmu-miR-302c for28days. The mice were sacrificed at day28. Parietal peritoneum was collected for HE and Masson staining or frozen section to observe the morphology of peritoneum and the fluorescence of GFP. Visceral peritoneum were harvested to extract tissue protein and mRNA for examining the expression of miRNA-302c、E-cadherin、 α-SMA、collagen Ⅰ、CTGF by Real Time PCR and western blot.Results1. By TaqMan quantitative fluorescence probe Real Time PCR, we found that compared with control group, the expression of miRNA-302c was downregulated with the intraperitoneal injection of4.25%PDF, but (?) expression of miRNA-302c was rised in the presence of LV-mmu-miR-302c.2. The structure of mice peritoneum has changed with the intraperitoneal injection of4.25%PDF, which manifesting the loss of mesothelial cell monolayer, the thickness and fibrosis of submesothelial zone, but the presence of LV-mmu-miR-302c can ameliorate the structural change.3. Real Time PCR and western blot showed that intraperitoneal injection of4.25%PDF for28days can cause mice peritoneum EMT and fibrosis with a decrease in the expression of E-cadherin and increase in the expression of a-SMA and collagen I, but the presence of LV-mmu-miR-302c can ameliorate the change. Meanwhile, compared with the control group, the expression of CTGF was rising with the intraperitoneal injection of4.25%PDF, but the presence of LV-mmu-miR-302c can ameliorate the increase of CTGF.Conclusion Treat the mice with intraperitoneal injection of4.25%PDF for28days can cause mice peritoneum EMT; intraperitoneal injection of LV-mmu-miR-302c can successfully upregulated the expression of miR-302c;:miR-302c can ameliorate the EMT and fibrosis of peritoneum in mice on peritoneal dialysis through suppresssing the expression of CTGF. ChaptcrⅢ miRNA-302c modulate TGF-β1induce human peritoneal mesothelial cells EMT and the mechanismObjectiveTo investigate the mechanism of miRNA-302c modulating TGF-β1-induced human peritoneal mesothelial cells EMT by infecting human peritoneal mesothelial cells with LV-hsa-miR-302c.Methods We treated the human peritoneal mesothelial cells with5ng/ml TGF-β1for48hours in the presence or absence of LV-hsa-miR-302c. To estimate the transfection efficiency of LV-hsa-miR-302c by observing the fluorescence of GFP through fluorescence microscope. To detect the expression of miRNA-302c、 E-cadherin、α-SMA、collagen I、CTGF by Real Time PCR and western blot.Results1. By TaqMan quantitative fluorescence probe Real Time PCR, we found that compared with control group, the expression of miRNA-302c was downregulated with the stimulation of TGF-β1, but the expression of miRNA-302c was rised in the presenc of LV-hsa-miR-302c.2. The morphology of human mesothelial cells has changed from epithelial phenotype to mesenchymal phenotype with the stimulation of but the presence of LV-hsa-miR-302c can ameliorate the morphology change.3. Real Time RCR and western blot showed that treating the human peritoneal mesotholial cells with5ng/ml TGF-β1for48hours can cause EMT and fibrosis with a decrease in e expression of E-cadherin and increase in the expression of α-SMA and collagen I, but the presence of LV-hsa-miR-302c can ameliorate the change. Mesanwhile, compared with the control group, the expression of CTGF was rising with the stimulation of TGF-β1, but the presence of LV-hsa-miR-302c can ameliorate the increase of CTGF.Conclusion Treat the human peritoneal mesothelial cells with5ng/ml TGF-β1for48hours can cause HPMCs EMT and fibrosis; the infection of LV-hsa-miR-302c can upregulate the expression of miR-302c; miR-302c can ameliorate the TGF-β1-induced human peritoneal mesothelial cells EMT and fibrosis through suppressing the expression of CTGF.