| Background:Mesangial proliferative glomerulonephritis, which is one of the highest incidence of kidney disease in China. The main pathological changes are diffuse proliferationof glomerular mesangial cells (GMC), as well as different levels of increased extracellular matrix (ECM). Meanwhile, MsPGN is the main reason of causing the chronic kidney disease (CKD), and can cause massive proteinuria, hypertension, kidney disease and other complications. If the disease continues to progress to deteriorate, it also can cause glomerulosclerosis and renal interstitial fibrosis, eventually become the major cause of leading to end-stage renal disease (ESRD). To inhibit the proliferation of mesangial cells is the main trerapeutic strategies for treating multiple mesangial proliferative glomerulonephritis. In recent years, a major breakthrough in the field of life science research is found out the microRNA which is the non-coding RNA in eukaryotic cells, these small RNA involved in the regulation of a wide range of biological proliferation/apoptosis, developmental/athological aging physiological processes, and is also closely related to human diseases. The present study showed, miR-34a may be involved in the regulation of multiple signaling pathways affecting cell proliferation.Objectives:Preparation of anti-Thyl antibody and then preparation of anti-Thyl mesangial proliferative glomerulonephritis rats model. Verify the level change of miR-34a at different point of pathological. Then, using siPDGFR-β to prove the effecting of miR-34a on the target-PDGFR-β,which is directly regulate the PDGFR-β and then trough the Ras/MAPK signal pathway to inhibit the proliferation of RMCs. And confirmed that the phenomenon of abnormal RMCs proliferation which is caused by exogenous stimulation can be antagonized by miR-34a in vitro.Methods:1. Using OX-7hybridoma cell lines to produce anti-Thyl antibody, and established the anti-Thy1mesangial proliferative nephritis rats model by using the Wistar rats through injected via the tail vein after antibody purification. Detect renal function and24-hour urinary protein excretion at different pathological time points (day0,3,5,7,10,14);2. Using immunohistochemical methods to detect the the expression level of Ki67and cyclin D1at different pathological point of time;3. Using the Western Blot to detect G0/G1phase of the cell cycle-related proteins cyclin D1, cyclin E, CDK2, CDK4, CDK6, p27kip1expression level at different pathological time points;4. Using TaqMan Realtime PCR to detect the expression of miR-34a at different pathological time points;5. transfected rat mesangial cells (RMC) by the siPDGFR-β or siRNA negative control, using the Cell Counting Kit-8method to detect the cell proliferation rate at different time points (24h,48h,72h);6. After transfected the siPDGFR-β or siRNA negative control in rat mesangial cells (RMC) for48h, then using flow cytometry to detect RMC cell cycle;7. After siPDGFR-P or siRNA negative control transfected rat mesangial cells (RMC) for48h, detecting the protein expression level of PDGFR-P, which is the direct target of miR-34a, various targets of Ras/MAPK signaling pathway and the cell cycle-related proteins; 8. Using dual luciferase reporter plasmid system detects the regulation of CDK2or cyclin E expression which two are the possible target of miR-34;9. Using of high concentrations of fetal bovine serum (20%FBS) to stimulate rat mesangial cells which are transfected with miR-34a mimics, testing the cell proliferation rate in each group and each time point;10. Using of high concentrations of fetal bovine serum (20%FBS) stimulated rat mesangial cells transfected with miR-34a mimics detected the cell cycle of RMC by flow cytometry;11. Using of high concentrations of fetal bovine serum (20%FBS) stimulated transfected miR-34a mimics rat mesangial cells, detected the different protein expression level of PDGFR-β, which is the direct target of miR-34a, and various targets of Ras/MAPK signaling pathway and the cell cycle-related proteins.Results:1. Successful to propare the anti-Thy1antibody and use the antibody to established the anti-Thy1nephritis rats model. Compared with the control group, the24h urinary protein excretion was significantly higher (p<0.05) at the5th and7th day;2. Ki67expression in various pathological time point of time. Compared with the control group, at the7th day, Ki67expression of pathological group increased significantly (p<0.05), and decreased with pathological outcomes. The expression of cyclin D1were the same trend with the Ki67;3. Western Blot detection the protein expression at different time point. The results showed that, compared with the control group, at the7th day, cyclin D1, cyclin E, CDK2, CDK4, CDK6expressed highest expression (p<0.05), while the p27kip1take minimum level (p<0.05).4. Using TaqMan Realtime PCR to detect the miR-34a expression levels change. The results showed that, compared with the control group miR-34a expression was negatively correlated with the pathological changes and at the7th day, the miR-34a expression was the lowest (p <0.05),;5. siPDGFR-P or siRNA negative control transfected rat mesangial cells (RMC), compared with the control group, siPDGFR-P proliferative activity decreased significantly from the beginning48h (p<0.05), more obvious to72h (p<0.01). Simultaneously detected the cell cycle by flow cytometry, siPDGFR-P cell cycle arrest was mainly extended the G0/G1phase (p<0.05);6. siPDGFR-P or siRNA negative control transfected rat mesangial cells (RMC), Western Blot detection the expression of PDGFR-β and p-PDGFR-β. Compared with the control group, siPDGFR-β group expression was significantly decreased (p<0.05);7. Using siPDGFR-β or siCON transfected RMCs, detected the protein chage of various targets of RAS/MAPK signaling pathway. The results showed that, compared with the control group, the total protein level of K-Ras decreased (p<0.05), total protein level of Rafl, MEK1and f ERK1/2were unchanged (p>0.05). p-Rafl, p-MEK1, p-ERK1/2protein levels were decreased (p<0.05);8. After usiung siPDGFR-P or siCON transfected RMC, we detected on the cell cycle protein cyclin D1and cyclin E, CDK protein CDK2, CDK4and CDK6, CKI protein p27kip1. The results showed that siPDGFR-β can be reduced cyclin D1, cyclin E, CDK2, CDK4, CDK6protein\evels(p<0.05), p27kip1expression was significantly up-regulated (p<0.05); 9. In order to verify whether the cyclin E or CDK2was the miR-34a target gene. The dual-luciferase reporter shown that, miR-34a mimics could decrease the both two luciferase activity (p<0.05);10. Using of high concentrations of fetal bovine serum (20%FBS) stimulated rat mesangial cells which were transfected with miR-34a mimics, proliferative capacity of20%FBS group or20%FBS+Con were upregulated than the10%FBS and20%FBS+miR34a group (p<0.05), but the10%FBS and20%FBS+miR34a group are no difference (p>0.05);11. Using of high concentrations of fetal bovine serum (20%FBS) stimulated rat mesangial cells which are transfected with miR-34a mimics, The results showed that, compared with10%FBS group, the G0/G1phase of20%FBS group are shorter(p<0.05), and compared to the20%FBS+Con group, G0/G1phase of20%FBS+miR34a group are significantly prolonged (p<0.05). Every time detection CV (%) were less than10%, indicating that results are credible;12. Using of high concentrations of fetal bovine serum (20%FBS) stimulated rat mesangial cells which are transfected with miR-34a mimics. The results showed that, the protein level of PDGFR-β and p-PDGFR-p of the20%FBS or20%FBS with miRNA Con are increased compared to the group with10%FBS or20%FBS with miR-34a,(p<0.05);13. Using of high concentrations of fetal bovine serum (20%FBS) stimulated rat mesangial cells which are transfected with miR-34a mimics. The results showed that after stimulation, the Ras/MAPK sigal pathway were more activity of20%FBS or20%FBS+Con group compared to10%FBS or20%FBS+miR-34a group(p<0.05)14. Using of high concentrations of fetal bovine serum (20%FBS) stimulated rat mesangial cells which are transfected with miR-34a mimics. The protein level of cell cycle related protein are increase expression of20%FBS or20%FBS+Con compared to the10%FBS or20%FBS+miR-34a group (p<0.05), at meanwhicl, p27kip1expression was significantly lower (p<0.05).Conclusions:1. The expression of miR-34a were negative with the change of at different time point of the anti-Thyl mesangial proliferative nephritis;2. siPDGFR-β may be act on PDGFR-β, and through the role of Ras/MAPK signaling pathway, extending G0/G1phase, and the result is decrease the activity of cell proliferation in. And this result strongly confirmed that the miR-34a made the same rold by act on the same target;3. miR-34a may antagonize the phenomenon of exogenous stimulation of cell proliferation. |
