The Research Of Effect And Mechanism Of SIRT1 Gene Silencing On The Growth Of Human Cervical Cancer Xenografts In Nude Mice

Objective:Silent information regulator 1(SIRT1),a NAD~+dependent class III histone deacetylase,was expressed abnormally in various tumor tissues.RNA interference targeting SIRT1 could inhibit the cell proliferation,migration,invasion and induce apoptosis of tumor cells.In this research,we applied lentiviral-vector mediated RNAi to inhibit SIRT1expression in vivo,to explore the relationship between SIRT1 protein and the proliferation of cervical cancer and also to investigate the correlation with microenvironment related proteins.Methods:Hela cells were divided into three groups:blank control group,lentiviral negative control group and lentiviral SIRT1 RNAi group;The later two groups were infected with negative control lentiviral and SIRT1 RNAi recombinant lentiviral vector respectively,to establish negative control cell lines and stable expressed SIRT1 RNAi cell lines.The total protein from the cells were extracted and the expression of SIRT1 was detected by Western blot.4-week BALB/c nude mice were divided into three groups randomly:blank control group,negative lentiviral control group and SIRT1 RNAi lentiviral group.6�10~6 Hela cells were injected into the right axillary subcutaneously to establish xenotransplanted model of human cervical cancer in nude mice.The tumor volume and nude mice weight were measured every 3d and the tumor growth curve was drawn.24d after the tumors emerge,the mice were sacrificed and the xenografts were separated and weighed.HE staining were used to observe the histopathological changes and immunohistochemistry were performed to evaluate SIRT1 expression.Western blot was conducted to evaluate the expression of SIRT1,Notch-1,Hes-1,HIF-1?and VEGF protein.Results:By well-tried optimizing experiments,the best condition for the SIRT1 RNAi recombinant lentivirus infection was as follows:MOI=20 and DMEM medium plus with8?g/ml Polybrene solution,the infection efficiency of Hela cells infected with the lentivirus was higher than 90%.Western blot indicated that,the expression of SIRT1 protein was down-regulated in SIRT1 RNAi lentiviral group when compared with the blank control group and lentiviral negative control group(p<0.05).In vivo experiments,the formation of cervical cancer xenografts in nude mice were 100%,the growth of the tumor in the control group mice and lentiviral negative control group mice were faster,while the growth of the tumor in the SIRT1 RNAi group was significantly inhibited.HE staining showed in the SIRT1 RNAi group,there were remarkable morphological changes in the cells,such as cell shrinkage and cell necrosis et al;Immunohistochemistry indicateded that the expression of SIRT1 in the SIRT1 RNAi group was dowregulated;Western blot results displayed that in the SIRT1 RNAi group the expression of HIF-1?and VEGF protein were down-regulated(p<0.05),while Notch-1 and Hes-1 protein were up-regulated when compared with the other two control groups(p<0.05).Conclusion:lentivirus mediated SIRT1 RNAi could inhibit the expression of SIRT1 protein and suppress cervical cancer xenograft in nude mice,which may related with tumor micro-environment and the regulation of Notch-Hes signaling pathway.