Study On The Effect Of TRIM25 Regulating VSMC Function Involved In Aortic Dissection Formation

Part Ⅰ Expression and significance of P53、MDM2 and TRIM25 in AD mediaObjective:To investigate the expression of a-SMA,OPN,P53,MDM2 and TRIM25 protein in the middle layer of aorta in AD.Methods:The ascending aorta of 12 patients with type A AD was used as the experimental group,and the ascending aorta of 12 DCD donors served as the control group.The expression of a-SMA,OPN,P53,MDM2,TRIM25 and p-P53 were tested by western blot and immunohistochemical detection of in AD group and control group in the organization.P53,MDM2 and TRIM-25 mRNA were detected in AD group and control group in RT-PCR technology.Morphological changes of aorta were observed by HE staining and Masson staining.Results:By western blot and RT-PCR examination,we found that P53,MDM2 and TRIM-25 protein and mRNA content in AD was significantly increased in tissue.Immunohistochemical detection also showed that the increase of P53,MDM2 and TRIM25 content,but the level of p-P53 was decreased.The results of immunohistochemistry showed that a-SMA positive cells in the AD group were significantly lower than those in the control group,while the OPN positive cells increased in the AD group,which was confirmed by further western blot.HE staining and Masson staining showed that the elastic fibers and collagen fibers were significantly increased in AD group,and the number of VSMC decreased.Conclusion:The phenotype of VSMC in AD medial wall was changed to dedifferentiation.The expression of P53,MDM2 and TRIM25 increased,suggesting that TRIM25 may induce the VSMC phenotype to participate in AD formation by affecting the P53/MDM2 feedback loop.Part Ⅱ Effects of TRIM25 overexpression on VSMC function and P53/MDM2 feedback loopObjective:To investigate the changes of P53 and MDM2 contents in VSMC after overexpression of TRIM25 and their effects on the function of VSMC.Methods:TRIM25 lentivirus plasmid was constructed and infected the VSMC of aortic smooth muscle cells of mice.The VSMC were divided into TRIM25 over expression group,empty control group and normal control group.Western blot method was used to detect the content of α-SMA,OPN,P53,MDM2,TRIM-25 and PCNA protein in VSMC.The mRNA level of P53、MDM2 and TRIM-25 in each group were detected by RT-PCR technique.MTT method was used to detect the proliferation of VSMC in each group,the migration of VSMC was detected by Transwell assay,and the apoptosis was detected by flow cytometry.Comparison of a-SMA and OPN changes by immunofluorescence staining.Results:Compared with the empty control group and the normal control group,the expression of P53,MDM2,TRIM-25,PCNA and BAX protein in the over expression group of TRIM25 increased,the expression of p-P53 decreased,but there was no significant difference in the level of P53/MDM2 mRNA.Western blot showed that the expression of OPN in the over expression group of TRIM25 was increased,while the expression of-SMA was decreased,and the OPN was confirmed by immunofluorescence staining and the expression of-SMA was decreased.The results of MTT and Transwell showed that the proliferation and migration ability of VSMC in the TRIM25 group was increased,and the apoptosis was also detected by flow cytometry.Conclusion:VSMC phenotype is changed to dedifferentiated transformation by overexpression of TRIM25.Meanwhile proliferation and migration ability and apoptosis levels of VSMC were both enhanced through the intervention with TRIM25.Its mechanism may be that TRIM25 destroyed the VSMC P53/MDM2 negative feedback loop balance,which leaded to the expression of P53 and MDM2 protein increased and activity decreased.Part Ⅲ Effects of MDM2 blocker Nutlin-3a on VSMC function and P53/MDM2 feedback loop after TRIM25 overexpressionObjective:To investigate the effect of TRIM25 overexpression on the function of VSMC after intervention with MDM2 blocker P53/MDM2 in Nutlin3a feedback loop.Methods:Mouse aortic vascular smooth muscle cells were divided into four groups:TRIM25 group(TRIM25 overexpression plasmid lentiviral vector transfected VSMC),TRIM25 plasmid transfected +Nutlin3a interference group(Nutlin3a treated and TRIM25 lentiviral plasmid transfected),Nutlin3a interference group and normal control group.Western blot detect a-SMA,OPN,P53,MDM2,TRIM-25,p-P53,PCNA and BAX protein content in each group.The mRNA content of P53,MDM2 and TRIM-25 were detected by RT-PCR technology.MTT method was used to detect the proliferation of VSMC in each group,the migration of VSMC was detected by Transwell assay,and the apoptosis was detected by flow cytometry.Results:Contents of P53 and p-P53 in the TRIM25+Nutlin3a group and Nutlin3a group were significantly higher than those in the normal group,while the expression of MDM2 protein was decreased and the expression of P53 was higher than that of the TRIM25 transfection group,but the difference was not statistically significant.The mRNA levels of P53 and MDM2 in cells were higher than those in normal group.Western blot showed that the expression of a-SMA in TRIM25+Nutlin3a group and Nutlin3a group was higher than that in TRIM25 group and normal group,but the expression of OPN was significantly decreased.There was no significant difference between TRIM25+Nutlin3a group and Nutlin3a group.MTT and Transwell experiments showed that the proliferation and migration ability of TRIM25+Nutlin3a in group VSMC and group Nutlin3a were decreased.The apoptosis rate was increased in the intervention group and the highest in TRIM25+Nutlin3a group.Conclusion:After Nutlin3a blocked MDM2 VSMC phenotype was differentiated,proliferation and migration ability decreased,apoptosis increased significantly,even while overexpression of TRIM25 also can not be reversed.With the result of the second part,TRIM25 mainly regulated the balance of VSMC phenotype and function changes by affecting the P53/MDM2 negative feedback loop.