SIRT1 Contributes To The Chemo-Resistance Of Cervical Cancer Through PARP1 Signaling Pathway In Vivo

Objective:Silent information regular1(SIRT1),a NAD~+dependent class III histone deacetylases,belongs to the sirtuin family.SIRT1 could participate in cell proliferation,apoptosis,metabilism,stress response and so on.It has been proved that SIRT1 was abnormally expressed in a variety of tumors and related to chemo-resistance.This study aims to investigate the effects of silencening SIRT1 on the cell proliferation and chemo-resistance to cisplatin(DDP)in vivo,to futher explore the relationship between SIRT1 and chemo-resistance associated proteins.Methods:Three recombinant RNA lentiviral vectors carrying different SIRT1 targets were constructed and applied to infect Hela cells,western blot was used to selected the best interference vector for the subsequent experiments.Hela cells were divided into 6groups:blank Hela cell control group,polybrene group,negative letiviral vector control group,SIRT1-shRNA group,DDP group and SIRT1-shRNA combined DDP group.CCK-8 method was used to asses the proliferation rate of Hela cells.4 weeks BALB/c female nude mice were divided into 5 groups randomly:blank Hela cell group,negative letiviral vector control group,SIRT1-shRNA group,DDP group and SIRT1-shRNA combined DDP group,6×10~6 Hela cells were injected subcutaneously into the right axillary to establish xenotransplanted model of human cervical cancer in nude mice.After tumor formation,DDP(2mg/kg,200?l)were intraperitoneally injected twice one week for 3w.Tumor dimensions were measured every 3d and the volumes were calculated 3d after DDP treatments completed,all the mice were sacrificed and the tumor weight were evaluated.HE staining were used to observe the histopathological changes and immunohistochemical staining were performed to asses the expression of SIRT1.Western blot were applied to measure the expression of SIRT1 and chemo-resistance associated proteins.Results:By well tried optimizing experiments,the best infection condition was as follows:MOI=20,polybrene=6ug/ml complete medium solution,cell infection rate of SIRT1-shRNA-2 lentivirus was>90%,cell survival rate was>95%,which was applied for following experiments.CCK-8 assay indicated that,when compared with blank Hela cell control group,the cell inhibition rates of polybrene group and negative letiviral vector control group were 5.7%and 11.2%respectively(P>0.05),while in SIRT1-shRNA group and DDP groupthe inhibition rate were 65.2%and 21.6%respectively(P<0.05).In vivo experiments,tumor growth curves confirmed that there were no significant differences between blank Hela cell group and negative letiviral vector control group(P>0.05),however,SIRT1-shRNA combined DDP group showed significantly suppression compared to the control group(P<0.05).HE staining depicted that in SIRT1-shRNA group,DDP group and SIRT1-shRNA combined DDP group,there were remarkable morphological changes in the cells,such as cell shrinkage and necrosis.Immunohistochemistry staining demonsrated that SIRT1 was down-regulated in SIRT1-shRNA group and SIRT1 shRNA combined DDP group when compared with blank Hela cell group(P<0.05).Western blot results displayed that in SIRT1-shRNA,DDP and SIRT1-shRNA combined DDP groups,the expression of ABCC1 were down-regulated,while PARP1 and DNA-PKcs were significantly up-regulated when compared with blank Helacell group(P<0.05).Conclusions:Lentivirus mediated SIRT1-shRNA could inhibit the expression of SIRT1protein and suppress Hela cells proliferation in vitro.In vivo,SIRT1exerted an activity in chemo-resistance to cisplatin by upregulating the expression of resistance-related proteins PARP1,DNA-PKcs and downregulating the expression of ABCC1 protein.