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目的:探讨显性负抑制人转录激活因子5(ATF5)对卵巢癌细胞SKOV-3顺铂敏感性的影响。方法:采用RT-PCR和蛋白质印迹法检测不同卵巢癌细胞系ATF5的表达。采用脂质体介导法将真核表达质粒pLeGFP-C1-NT-Azip-ATF5和pLeGFP-C1分别转染SKOV-3细胞,同时设空白对照组;于转染后24h给予不同浓度的顺铂作用48h,通过MTT法测定细胞增殖抑制率;转染后24h给予4μmol/L顺铂作用0、12、24和48h,通过流式细胞术和蛋白质印迹法分别检测细胞凋亡和Bcl-2蛋白的表达。结果:ATF5在3种卵巢癌细胞系中均有不同程度的表达,其中SKOV-3细胞系表达最高;显性负抑制SKOV-3细胞内源性ATF5的功能联合顺铂作用后,与非特异性转染组和空白对照组相比,特异性转染组细胞增殖明显受抑制,顺铂的IC50由5.4μmol/L下降为3.6μmol/L;顺铂作用24h,特异性转染组细胞凋亡率显著升高,约为非特异性转染组3倍,抗凋亡基因Bcl-2蛋白表达明显下降,P<0.05。结论:显性负抑制ATF5的表达可明显增强卵巢癌细胞系SKOV-3对顺铂的化疗敏感性,其协同顺铂诱导细胞凋亡的机制可能与Bcl-2的下调相关。
Objective: To investigate the effect of dominant negative inhibitor of human transcriptional activator 5 (ATF5) on the sensitivity of ovarian cancer cell line SKOV-3 to cisplatin. Methods: RT-PCR and Western blotting were used to detect the expression of ATF5 in different ovarian cancer cell lines. The eukaryotic expression plasmids pLeGFP-C1-NT-Azip-ATF5 and pLeGFP-C1 were transfected into SKOV-3 cells respectively by liposome-mediated method. At the same time, a blank control group was established. The cells were treated with 4μmol / L cisplatin for 24h, 24h and 48h after transfection. The apoptosis and expression of Bcl-2 protein were detected by flow cytometry and Western blot respectively. expression. RESULTS: ATF5 was expressed in all three ovarian cancer cell lines in varying degrees, with the highest expression in SKOV-3 cell line. The negative staining of ATF5 negatively inhibited the function of endogenous ATF5 in SKOV-3 cells in combination with cisplatin, Transfection group and blank control group compared with the transfected group was significantly inhibited cell proliferation, cisplatin IC50 decreased from 5.4μmol / L to 3.6μmol / L; cisplatin 24h, the specific transfection group apoptosis (P <0.05). The expression of Bcl-2 protein in anti-apoptotic gene was significantly decreased, about 3 times higher than non-specific transfection group. Conclusion: The negative expression of ATF5 can significantly enhance the chemosensitivity of ovarian cancer cell line SKOV-3 to cisplatin. The mechanism of cisplatin-induced apoptosis may be related to the down-regulation of Bcl-2.