干扰B7-H4表达通过下调E2F家族相关转录因子抑制乳腺癌细胞的增殖

Interfering with B7-H4 expression can inhibit proliferation of breast cancer cells by down-regulating E2F family related transcription factors

目的:探讨干扰B7-H4表达对乳腺癌细胞增殖、凋亡、周期以及相关下游分子表达的影响。方法:利用脂质体转染技术分别将特异性靶向B7-H4的siRNA(siB7-H4)及其阴性对照(siNC)转染至对数生长期的乳腺癌T47D和MCF-7细胞,分别命名为T47D-siB7-H4、T47D-siNC、MCF-7-siB7-H4和MCF-7-siNC组。用qPCR法和WB法验证siRNA干扰效果及其对细胞周期分子cyclin D1表达的影响,CCK-8法和FCM分别检测干扰B7-H4表达对T47D和MCF-7细胞增殖、周期和凋亡的影响,qPCR法检测B7-H4干扰对E2F家族相关转录因子表达的影响。结果:成功构建干扰B7-H4表达的乳腺癌T47D和MCF-7细胞。与T47D-siNC和MCF-7-siNC组相比,T47D-siB7-H4和MCF-7-siB7-H4组细胞中B7-H4 mRNA和蛋白表达水平均显著降低、细胞增殖能力显著降低(均P<0.01),并伴有G1/S期细胞周期阻滞以及cyclin D1表达下调(均P<0.01),但细胞凋亡率差异无统计学意义(均P>0.05)。与T47D-siNC相比,干扰B7-H4后T47D细胞中E2F1、E2F2、E2F7和E2F8 mRNA水平有不同程度的降低(均P<0.01);与MCF-7-siNC相比,干扰B7-H4后MCF-7细胞中E2F1、E2F2、E2F3、E2F7和E2F8 mRNA水平均有不同程度的降低(P<0.05或P<0.01)。结论:干扰乳腺癌细胞B7-H4表达可下调cyclin D1和E2F家族相关转录因子的表达,导致细胞周期阻滞并抑制细胞增殖。

Objective:To investigate the effects of interfering with B7-H4 expression on proliferation,apoptosis,cell cycle and expression of downstream molecules in breast cancer cells.Methods:Breast cancer T47D and MCF-7 cells at logarithmic phase were transfected with siRNA specifically targeting B7-H4(siB7-H4)or its negative control(siNC)by using LipofectamineTM2000,namely T47D siNC,T47D-siB7-H4,MCF-7-siNC,and MCF-7-siB7-H4 group,respectively.The efficacy of siRNA interference and its effect on the expression of cyclin D1 were verified by quantitative PCR(qPCR)and Western blotting(WB).The effects of interfering with B7-H4 on cell proliferation,cell cycle and apoptosis of breast cancer cell lines T47D and MCF-7 were detected by CCK-8 assays and flow cytometry,respectively.The effects of interfering with B7-H4 on the expression of E2F family related transcription factors were examined by qPCR.Results:The T47D and MCF-7 cell lines with B7-H4 knockdown were successfully constructed.Compared with the cells in T47D-siNC and MCF-7-siNC groups,the mRNA and protein levels of B7-H4 were significantly decreased,and the proliferation was significantly inhibited in the cells of T47D-siB7-H4 or MCF-7-siB7-H4 groups,accompanied with G1/S cell cycle arrest as well as downregulation of cyclin D1(all P<0.01);however,there were no statistically significant differences in apoptotic rates(all P>0.05).Compared with the cells in T47D-siNC group,the mRNA levels of E2F1,E2F2,E2F7 and E2F8 in T47D cells decreased in varying degrees after interfering with B7-H4(all P<0.01);compared with cells in MCF-7-siNC group,the mRNA levels of E2F1,E2F2,E2F3,E2f7 and E2F8 in MCF-7 cells also decreased in varying degrees after interfering with B7-H4(P<0.05 or P<0.01).Conclusion:Interfering with B7-H4 in breast cancer cells can down-regulate the expression of cyclin D1 and E2F family related transcription factors,leading to cell cycle arrest and inhibition of cell proliferation.