Effect Of Silencing SIRT1 Expression On Cisplatin Resistance In Human Cervical Cancer Cells And Its Mechanism

Objective:Silent information regulator 1?SIRT1?is a nicotinamide adenine dinucleotide?NAD⁺?-dependent class III histone deacetylase.A large number of studies have shown that SIRT1 is highly expressed in a variety of tumors,exert an activity in the development of tumors,and is closely related to tumor prognosis and chemo-resistance.In this study,small interfering RNA targeting SIRT1 was used to silence the expression of SIRT1,and to study the effect of SIRT1 on the proliferation and cell cycle in cervical cancer SiHa cells and C33A cells.Subsequently,siRNA-SIRT1 combined with cisplatin were used to treat SiHa cells and C33A cells to asses the effect of SIRT1 on cisplatin resistance of cervical cancer cells and the related drug resistance genes,as to explore its mechanism.Methods:Chemically synthesized small interfering RNA?siRNA?targeting SIRT1and its negative control sequences were transfected into cervical cancer SiHa cells and C33A cells by Lipofectamine RNAi MAX liposome respectively.The experiments were divided into three groups:blank cell control group,negative siRNA transfection group and siRNA-SIRT1 transfection group.72 h post transfection,total RNA and total protein were extracted from each group.The expression of SIRT1 mRNA was measured by RT-PCR.The expression of SIRT1 protein was detected by Western blot.CCK-8 method was used to determine the cell proliferation and IC₅₀0 of SiHa cells and C33A cells.RT-PCR,qRT-PCR and Western blot were used to evaluate the expressionof ABCC1 in different cell groups.SiHa cells and C33A cell cycle were assesed by flow cytometry;Western blot was used to detect the expression levels of PARP-1,DNA-PKcsproteins in SiHa cells and C33A cells.Results:The CCK-8 results indicate that the IC₅₀0 values of cisplatin to SiHa cells were 11.16?M?24h?and 5.11?M?48h?,respectively.Compared with the blankSiHa cells,in the siRNA-SIRT1 combined with cisplatin group,the inhibition rate of SiHa cell proliferation was 60%?P<0.05?.The IC₅₀0 values of cisplatin to C33A cells were 3.01?M?24h?and 1.05?M?48h?,respectively.Compared with the blank C33A cell group,in the siRNA-SIRT1 combined with cisplatin,the proliferation inhibition rate of C33A cells was 45%?P<0.05?.RT-PCR and qRT-PCR demonstrated that the expression of SIRT1 mRNA and ABCC1 mRNA in the siRNA-SIRT1 transfected group was significantly lower than that in blank control group?P<0.05?.Western blot proved that compared with the blank cell control group,the expression of SIRT1protein and ABCC1 protein in siRNA-SIRT1 group was significantly down-regulated?P<0.05?,and the expression of PARP-1 and DNA-PKcs protein was significantly up-regulated?P<0.05?.Cell cycle results showed that compared with the blankSiHa cell control group,the cells in the siRNA-SIRT1 transfection group and the siRNA-SIRT1 transfection and cisplatin combination pointed to significant S phase arrest?P<0.05?.Compared with the blank C33A cell group,there were significant S and G2 phase arrests in the siRNA-SIRT1 transfection group?P<0.05?,and significant G1 arrest in the siRNA-SIRT1 transfection and cisplatin combination?P<0.05?.Conclusion:siRNA-SIRT1 not only inhibits the proliferation of SiHa cells and C33A cells,but also causes cell cycle arrest,activates the DNA damage repair pathway,SIRT1 contributes to cisplatin resistance in huaman cervical cancer cells by down-regulating the expression of the drug resistance gene ABCC1,and up-regulating PARP-1 and DNA-PKcs DNA damage repair proteins.SIRT1 is expected to be a potential target for the treatment of cervical cancer.