基因敲除去乙酰化酶SIRT1并靶向结合miR-138-5p诱导前列腺癌细胞PC-3氧化应激和凋亡

Gene knockout acetylase SIRT1 and targeting combined with miR-138-5p induced PC-3 oxidative stress and apoptosis in prostate cancer cells

目的沉默信息调节因子1(silent information regulator 1,SIRT1)是一种依赖烟酰胺腺嘌呤二核苷酸(NAD+)的组蛋白去乙酰化酶,可作为致癌基因通过抗凋亡活性在肿瘤发生中起作用,并涉及自噬,衰老,凋亡,增殖等多种细胞过程。因此,本研究通过基因敲除去乙酰化酶SIRT1,探究其对靶向结合miR-138b-5p对前列腺癌细胞PC-3氧化应激和凋亡的作用。方法设计合成SIRT1shRNA,将inhibitor和shRNA-SIRT1单独或联合转染PC-3细胞,RT-PCR检测miR-138b-5p和SIRT1的表达水平;克隆形成实验检测细胞的生长能力;Hoechst33258染色检测细胞凋亡;试剂盒检测细胞培养液中乳酸脱氢酶(LDH)和超氧化物歧化酶(SOD)活性以及细胞内丙二醛(MDA)含量;流式细胞术检测线粒体膜电位的变化;免疫印迹法检测Caspase-3,Caspase-9,Bax/Bcl-2的蛋白表达。结果miR-138-5p mimic转染PC-3细胞后,miR-138-5p的表达显著升高(P<0.05),SIRT1的表达显著降低(P<0.05)。inhibitor能明显减弱miR-138-5p对SIRT1的表达的抑制作用(P<0.05)。shRNA-SIRT1能显著降低SIRT1的表达水平(P<0.05)。敲除SIRT1明显抑制PC-3细胞克隆形成(P<0.05)。同时,敲除SIRT1线粒体膜电位显著升高,细胞凋亡率增加,促进细胞凋亡(P<0.05)。敲除SIRT1还能明显增加SOD活性,降低MDA含量及LDH活力(P<0.05)。此外,敲除SIRT1显著增加Caspase-3,Caspase-9及Bcl/Bax的蛋白表达水(P<0.05)。结论基因敲除SIRT1靶向结合miR-138-5p抑制前列腺癌PC-3细胞的生长,诱导PC-3细胞氧化应激和凋亡,以及促进凋亡相关蛋白的表达。

Objective Silent information regulator 1(SIRT1)is a histone deacetylase dependent on nicotinamide adenine dinucleotide(NAD+).As an oncogene,it can play a role in tumorigenesis through anti-apoptotic activity and is involved in various cell processes such as autophagy,aging,apoptosis and proliferation.Therefore,in this study,acetylase SIRT1 was removed by gene knockout to explore its effect on targeted binding of mir-138 b-5 p on PC-3 oxidative stress and apoptosis in prostate cancer cells.Methods SIRT1 shRNA was designed and synthesized,and inhibitor and shRNA-SIRT1 were transfected into PC-3 cells either alone or in combination.RT-PCR detected the expression levels of miR-138 b-5 p and SIRT1.The ability of cell growth was measured by clone formation experiment.Apoptosis was detected by Hoechst33258 staining.The activity of lactate dehydrogenase(LDH)and superoxide dismutase(SOD)as well as the content of malondialdehyde(MDA)in cell culture were detected by the kit.The changes of mitochondrial membrane potential were detected by flow cytometry.Western blotting was used to detect the protein expression of caspase-3,caspase-9,Bax/Bcl-2.Results After transfection of PC-3 cells with miR-138-5 p mimic,the expression of miR-138-5 p was significantly increased(P<0.05),and the expression of SIRT1 was significantly decreased(P<0.05).Inhibitor can significantly weaken the inhibitory effect of miR-138-5 p on SIRT1 expression(P<0.05).ShRNA-SIRT1 can significantly reduce the expression of SIRT1(P<0.05).SIRT1 knockout significantly inhibited PC-3 cell cloning(P<0.05).At the same time,the mitochondrial membrane potential of knocked out SIRT1 was significantly increased,and the apoptosis rate was increased,which promoted apoptosis(P<0.05).SIRT1 knockout can also significantly increase SOD activity,reduce MDA content and LDH activity(P<0.05).In addition,the protein expression of caspase-3,caspase-9 and Bcl/Bax was significantly increased by SIRT1 knockout(P<0.05).Conclusion Gene knockout SIRT1 and targeting miR-138-5 p inhibit the growth of prostate cancer PC-3 cells,induce oxidative stress and apoptosis of PC-3 cells,and promote the expression of apoptosis-related proteins.