circ-0026462靶向miR-361-3p调控前列腺癌细胞对恩杂鲁胺耐药性的机制研究

circ-0026462 regulated the effect and mechanism of enzalutamide resistance in prostate cancer cells by targeting miR-361-3p

目的探讨circ-0026462靶向miR-361-3p调控前列腺癌细胞对恩杂鲁胺耐药性的机制研究。方法体外培养人前列腺癌细胞DU-145,建立前列腺癌恩杂鲁胺耐药性细胞DU-145/Enz,使用恩杂鲁胺处理DU-145、DU-145/Enz细胞,将其分为si-NC组、si-circ-0026462组、si-circ-0026462+anti-miR-NC组及si-circ-0026462+anti-miR-361-3p组。采用MTT法检测细胞的增殖能力;采用流式细胞术检测细胞凋亡率;采用qRT-PCR检测circ-0026462、miR-361-3p的表达量;采用双荧光素酶报告检测circ-0026462与miR-361-3p的靶向关系;采用Western blot法检测雄激素受体变异体(AR-V7)、细胞周期蛋白D1(Cyclin D1)、细胞周期蛋白依赖性抑制剂(P21)、B淋巴细胞瘤-2(Bcl-2)、Bcl-2相关蛋白(Bax)蛋白的表达量。结果与DU-145/Enz细胞比较,DU-145细胞的OD值及Cyclin D1、Bcl-2蛋白水平降低(均P<0.001),细胞凋亡率及P21、Bax蛋白水平升高(均P<0.001);与DU-145细胞比较,DU-145/Enz细胞中circ-0026462的表达水平升高(P<0.001),miR-361-3p的表达水平降低(P<0.001);转染si-circ-0026462可明显降低OD值、AR-V7、Cyclin D1及Bcl-2的表达水平(均P<0.001),提高细胞凋亡率及P21、Bax的表达水平(P<0.001);双荧光素酶报告实验证实circ-0026462可靶向结合miR-361-3p;抑制miR-361-3p表达可明显逆转沉默circ-0026462对DU-145/Enz细胞增殖、凋亡及耐药性的作用。结论沉默circ-0026462可通过上调miR-361-3p的表达而抑制细胞增殖及促进细胞凋亡,从而降低前列腺癌细胞对恩杂鲁胺的耐药性。

Objective To explore whether circ-0026462 regulated enzalutamide resistance in prostate cancer cells by targeting miR-361-3p.Methods Human prostate cancer cells DU-145 were cultured in vitro to establish prostate cancer enzalutamide-resistant cells DU-145/Enz,and enzalutamide was used to treat DU-145 and DU-145/Enz cells.si-NC,si-circ-0026462,si-circ-0026462 and anti-miR-NC,si-circ-0026462 and anti-miR-361-3p were transfected into DU-145/Enz cells respectively.MTT was used to detect cell proliferation ability.Flow cytometry was used to detect the apoptosis rate.qRT-PCR experiment was used to detect the expression level of circ-0026462 and miR-361-3p.The dual luciferase reporter experiment was used to detect the targeting relationship between circ-0026462 and miR-361-3p.Western blot method was used to detect the expression of AR-V7,Cyclin D1,P21,Bcl-2 and Bax protein.Results Compared with DU-145/Enz,the OD value and the protein levels of Cyclin D1 and Bcl-2 in DU-145 cells were decreased(all P<0.05),and the apoptosis rate and the protein levels of P21,Bax were increased(all P<0.05).Compared with DU-145,the expression level of circ-0026462 in DU-145/Enz cells was increased(P<0.05),and the expression level of miR-361-3p was decreased(P<0.05).Transfection of si-circ-0026462 could significantly reduce the OD value and the expression levels of AR-V7,Cyclin D1 and Bcl-2(all P<0.05),and increase the apoptosis rate and the expression levels of P21 and Bax(P<0.05).The dual luciferase report experiment confirmed that circ-0026462 could target miR-361-3p.Inhibition of miR-361-3p expression could significantly reverse the effects of silencing circ-0026462 on the proliferation,apoptosis and drug resistance of DU-145/Enz cells.Conclusions Silencing circ-0026462 could inhibit cell proliferation and promote cell apoptosis by up-regulating the expression of miR-361-3p,thereby reducing the resistance of prostate cancer cells to enzalutamide.