长链非编码RNA FOXD2相邻相反链RNA 1靶向调控微小RNA-760对前列腺癌细胞增殖和凋亡的影响

Targeted-regulating of miR-760 by lncRNA FOXD2 adjacent opposite strand RNA 1 and its impact on proliferation and apoptosis of prostate cancer cells

目的探讨FOXD2相邻相反链RNA 1(FOXD2-AS1)对前列腺癌细胞的增殖和凋亡的影响以及作用机制。方法实验于2019年1—11月进行,根据DU145细胞转染情况分为空载体质粒(pcDNA)组、FOXD2-AS1过表达质粒(pcDNA-FOXD2-AS1)组、小干扰RNA阴性对照(si-NC)组、FOXD2-AS1小干扰RNA(si-FOXD2-AS1)组、微小RNA(miR)-760组、miR-760阴性对照(miR-NC)组及si-FOXD2-AS1+抗miR-760阴性对照(anti-miR-NC)组、si-FOXD2-AS1+抗miR-760(anti-miR-760)组。实时荧光定量逆转录聚合酶链反应(qRT-PCR)检测miR-760和FOXD2-AS1表达水平;双荧光素酶报告基因实验检测荧光活性;蛋白质印迹法检测蛋白表达;MTT法检测细胞增殖;流式细胞术检测细胞凋亡。结果与正常前列腺上皮细胞RWPE-1相比,前列腺癌细胞DU145、LNCaP、22Rv1中miR-760表达水平显著降低[(0.34±0.03)、(0.56±0.05)、(0.42±0.04)比(1.01±0.08)](P<0.001),FOXD2-AS1表达水平显著升高[(2.28±0.23)、(2.13±0.21)、(2.45±0.24)比(1.00±0.09)](P<0.001)。与miR-NC组相比,miR-760组野生型FOXD2-AS1基因表达载体WT-FOXD2-AS1前列腺癌细胞DU145荧光素酶活性显著降低[(0.42±0.03)比(1.04±0.09)](P<0.001)。与pcDNA组相比,pcDNA-FOXD2-AS1组DU145细胞中miR-760的表达水平显著降低[(0.45±0.04)比(1.02±0.09)](P<0.001);与si-NC组相比,si-FOXD2-AS1组DU145细胞中miR-760表达水平显著升高[(2.34±0.23)比(1.01±0.08)](P<0.001)。通过抑制FOXD2-AS1表达和过表达miR-760均抑制B细胞淋巴瘤-2(Bcl-2)、细胞周期蛋白D1(cyclin D1)的表达,促进Bcl-2相关X蛋白(Bax)、周期素依赖激酶抑制剂p21(P21)蛋白的表达,抑制DU145细胞增殖,促进细胞凋亡。抑制miR-760过表达能逆转抑制FOXD2-AS1对前列腺癌细胞DU145增殖抑制和凋亡促进作用。结论lncRNA FOXD2-AS1可能通过靶向miR-760抑制前列腺癌细胞DU145的增殖、促进其凋亡,可为前列腺癌的预防和治疗提供新靶点。

Objective To investigate the impact of FOXD2 adjacent opposite strand RNA 1(FOXD2-AS1)on proliferation and apoptosis of prostate cancer cells and its mechanism.Methods Experiments were conducted from January to November of 2019.According to respective ways of DU145 transfection,there were pcDNA group,pcDNA-FOXD2-AS1 group,si-NC group,si-FOXD2-AS1 group,miR-NC group,miR-760 group,si-FOXD2-AS1+anti-miR-NC group,and si-FOXD2-AS1+anti-miR-760 group.The expression levels of miR-760 and FOXD2-AS1 were detected by real-time quantitative polymerase chain reaction(qRT-PCR),fluorescence activity by double luciferase reporter gene assay,protein expression by Western blotting,cell proliferation by thiazole blue(MTT)assay,and apoptosis by flow cytometry.Results Compared with normal prostate epithelial cells RWPE-1,miR-760 expressions in DU145,LNCaP and 22Rv1 were significantly decreased[(0.34±0.03),(0.56±0.05),(0.42±0.04)vs.(1.01±0.08)](P<0.001),while FOXD2-AS1 expressions were significantly increased[(2.28±0.23),(2.13±0.21),(2.45±0.24)vs.(1.00±0.09)](P<0.001).Compared with miR-NC group,the DU145 luciflucase activity of WT-FOXD2-AS1 prostate cancer cells in the MiR-760 group was significantly decreased[(0.42±0.03)vs.(1.04±0.09)](P<0.001).Compared with pcDNA group,the expression level of miR-760 in DU145 cells in PcDNAFOXD2-AS1 group was significantly decreased[(0.45±0.04)vs.(1.02±0.09)](P<0.001).Compared with si-NC group,miR-760 expression level in DU145 cells in si-FOXD2-AS1 group was significantly increased[(2.34±0.23)vs.(1.01±0.08)](P<0.001).Inhibition of FOXD2-AS1 expression and overexpression of miR-760 both inhibited the expressions of Bcl-2 and cyclin D1 proteins,promoted the expressions of Bax and P21 proteins,inhibited the proliferation of DU145 cells,and promoted cell apoptosis.Inhibition of miR-760 overexpression could reverse and inhibit the effect of FOXD2-AS1 on DU145 proliferation inhibition and apoptosis promotion.Conclu⁃sion LncRNA FOXD2-AS1 may inhibit the proliferation and promote apoptosis of prostate cancer DU145 cells by targeting miR-760,which may provide a new target for the prevention and treatment of prostate cancer.