Preliminary Study On The Role Of SIRT1 In The Development And Chemotherapy Resistance Of Cervical Cancer

Objective:Silent information regulator 1?SIRT1?is a member of the class III histone deacetylase family.It was well known that SIRT1 is abnormally expressed in a variety of cancers and is closely related to tumor resistance.The purpose of this study was to investigate the effect of small interfering RNA targeting SIRT1 on the proliferation,migration,invasion and apoptosis of cervical cancer Hela cells in vitro,as to explore its role in the development of cervical cancer.On this basis,siRNA-SIRT1 was combined with cisplatin in Hela cells to measure the effect of SIRT1 on the related drug resistance genes.Methods:Chemically synthesized small interfering RNA?siRNA?targeting SIRT1 and its negative control sequence were transfected into cervical cancer Hela cells by Lipofectamine RNAi MAX liposome.The experiments were divided into three groups:blank control group,negative siRNA control group and siRNA-SIRT1 group.72 h post transfection,the total RNA and protein were extracted from each group of cells.The expression of SIRT1 mRNA was analyzed by RT-PCR.The expression of SIRT1 protein was detected by Western blot.HeLa cells were treated with the combination of siRNA-SIRT1 and cisplatin,the proliferation and IC₅₀0 of cisplatin on the Hela cells were detected by CCK-8 method.The mRNA expressions level of ABCC1 and ABCG2 in Hela cells were detected by RT-PCR and qRT-PCR.The expression of ABCC1 protein was analyzed by Western blot.The cell migration and invasion of Hela cells were assessed by Transwell chambers assay.The apoptosis of Hela cells were determined by Flow cytometry.Results:The RT-PCR results showed that the mRNA expression of SIRT1 was significantly downregulated in the siRNA-SIRT1 group when compared with the negative control si RNA group and blank control group?P<0.05?.The Western blot results displayed that the protein expression of SIRT1 was significantly downregulated in the siRNA-SIRT1 group when compared with the negative control siRNA group and the blank control group?P<0.05?.CCK-8 results indicated that the IC₅₀0 values of Hela cells to cisplatin were 5.34?M?24h?and 3.70?M?48h?respectively.When siRNA-SIRT1combined with cisplatin,the IC₅₀0 of cisplatin on Hela cells was 1.58?M?24h?,which was significantly decreased when compared with the effect of cisplatin alone.The inhibitory rate of cell proliferation in siRNA-SIRT1 group was 28.6%,the inhibition rate of cell proliferation in negative control group was 6.3%,and the inhibition rate of cell proliferation in blank control group was 4.7%.The proliferation inhibition rate of siRNA-SIRT1 group was significantly higher than that of other control groups?P<0.05?.Both of RT-PCR and qPCR results showed that the expression of ABCC1 mRNA was significantly downregulated in the siRNA-SIRT1 group compared with the negative control siRNA group and the blank control group?P<0.05?,while ABCG2 expression was not changed significantly.Western blot results indicated that the expression of ABCC1protein in siRNA-SIRT1 group was significantly lower than that in the negative control siRNA group and blank control group?P<0.05?.The expression of ABCC1 protein was significantly downregulated in the siRNA-SIRT1+cisplatin group compared with the negative control siRNA+cisplatin group and the blank control+cisplatin group?P<0.05?.The results of cell migration and invasion experiments displayed that the cell migration and cell invasion ability in the siRNA-SIRT1 group were significantly higher than that in control groups?P<0.05?.The flow cytometry showed that the apoptosis rate of the blank control group was 2.9%,the apoptosis rate of the negative siRNA control group was 3.7%,and the apoptosis rate of the siRNA-SIRT1 group was 11.7%.The apoptosis rate of the siRNA-SIRT1 group was significantly increased when compared to the negative control siRNA group and the blank cell control group?P<0.05?.Conclusion:siRNA-SIRT1 not only inhibited the the proliferation,migration and invasion of Hela cells and induced apoptosis,but also downregulated the expression of chemoresistance gene ABCC1.The combination of siRNA-SIRT1 and cisplatin could increase the sensitivity of Hela cells to cisplatin by decreasing the expression of the drug-resistance gene ABCC1.SIRT1 is expected to overcome the chemotherapy resistance problem of cervical cancer by influencing the expression of ABCC1.