α-辅肌动蛋白4对卵巢癌细胞增殖、凋亡及化疗敏感性的影响及其机制

Effects of Alpha-Actinin-4 on the Proliferation,Apoptosis and Chemosensitivity of Ovarian Cancer Cells and the Molecular Mechanisms

目的:探讨体外沉默α-辅肌动蛋白4(alpha-actinin-4,ACTN4)基因的表达对卵巢癌细胞增殖、凋亡及化疗敏感性的影响及其可能的机制。方法:化学合成靶向ACTN4基因的ACTN4-siRNA,体外转染至人卵巢癌细胞株SKOV3和OVCA429中,采用real-time PCR检测ACTN4 mRNA的表达,采用MTT法检测卵巢癌细胞的增殖能力及存活率,采用流式细胞术检测卵巢癌细胞的凋亡情况,采用Western blotting检测凋亡相关蛋白的表达。结果:ACTN4-siRNA转染SKOV3/OVCA429细胞后,ACTN4的mRNA及蛋白的表达均明显下降;细胞增殖能力明显受到抑制。在不同浓度紫杉醇作用下,与转染CtrlsiRNA的对照组相比,ACTN4-siRNA转染组SKOV3/OVCA429的细胞存活率均显著降低,即转染ACTN4-siRNA后的SKOV3/OVCA429细胞对紫杉醇的敏感性明显增加(P<0.01)。SKOV3细胞瞬时转染ACTN4-siRNA48 h后,细胞凋亡率[(7.22±0.31)%]明显高于转染Ctrl-siRNA的对照组[(2.07±0.08)%];OVCA429细胞的情况与之一致,转染ACTN4-siRNA后细胞凋亡率为[(23.37±0.18)%],对照组为[(19.49±0.19)%],差异均有统计学意义(P<0.05)。转染ACTN4-siRNA后,SKOV3/OVCA429中p-Akt和p-FAK蛋白表达明显减少,抗凋亡蛋白Bcl-2表达明显减少,而促凋亡蛋白Bid和Bim表达则明显增加。结论:ACTN4可以降低卵巢癌细胞的化疗敏感性,这种作用可能通过抑制细胞凋亡实现。

Objective： To investigate the effects of down-regulation of Alpha-actinin-4（ACTN4） expression on proliferation, apoptosis, chemosensitivity of ovarian cancer cells and the related molecular mechanisms. Methods：Chemically synthesized siRNA targeting ACTN4 gene was transfected into SKOV3 and OVCA429 cells. The expression of ACTN4 mRNA was detected by real-time PCR. MTT method was used to detect the proliferation ability and survival rates of ceils. Flow cytometry（FCM） assay and western blotting were performed to detect the apoptotic rates and the expression levels of relative apoptotic proteins, respectively. Results： The mRNA and protein expression levels of ACTN4 in SKOV3/OVCA429 cells were reduced after transfection with ACTN4-siRNA. SKOV3/OVCA429 cells transfected with ACTN4-siRNA had lower proliferation ability and cell survival rates, compared with SKOV3/OVCA429 cells transfected with Ctrl-siRNA after treatment with different concentrations of paclitaxel, that is, the sensitivity of ceils transfected with ACTN4-siRNA to paclitaxel was significantly increased （P〈0.01）. After transfection with ACTN4-siRNA for 48h, the apoptotic rate of SKOV3[（7.22±0.31）%] was higher than that of SKOV3 cells transfected with Ctrl-siRNA[（2.07 ± 0.08）%] ; the apoptotic rate of OVCA429 cells transfected with ACTN4 siRNA [（23.37 ± 0.18） %] was also higher than that of OVCA429 cells transfected with Ctrl-siRNA[（19.49 ±0.19） %]. The differences were statistically significant （P〈0.05）. After transfection with ACTN4-siRNA, in both cell lines, the levels of p- AKT, p-FAK, pro-surivival protein Bcl-2 were down-regulated, while pro-apoptosis proteins Bid and Bim were up-regulated. Conclusions： ACTN4 can weaken the chemosensitivity of ovarian cancer cells by inhibiting the apoptosis of ovarian cancer cells.