Sphk1基因对人乳腺癌MCF-7细胞增殖、凋亡和迁移能力的影响

Sphk1 interference suppresses proliferation,apoptosis and migration in human MCF-7 breast cancer cells

目的通过抑制鞘氨醇激酶-1(sphingosine kinase-1,Sphk1)基因表达,观察其对人乳腺癌MCF-7细胞增殖、凋亡和迁移能力的影响。方法使用脂质体2000将化学合成的靶向Sphk1基因的小干扰RNA(small interfering RNA,siRNA)转入人乳腺癌MCF-7细胞。使用流式细胞术(FCM)检测转染效率,荧光定量RT-PCR(qRT-PCR)和Western blot法检测Sphk1的mRNA和蛋白表达水平。利用CCK-8法检测细胞的增殖能力,流式细胞术检测细胞凋亡,Transwell小室法检测细胞迁移能力。结果 Sphk1 siRNA转染效率达89%。转染siRNA后,MCF-7细胞的Sphk1 mRNA和蛋白表达水平均明显下降,以siRNA-2效果最为显著(P<0.01)。转染Sphk1 siRNA-2后,MCF-7细胞的增殖和迁移能力明显下降,凋亡细胞比例增加,以上差异均具有统计学意义(P<0.05)。结论干扰Sphk1基因表达可有效抑制MCF-7细胞的增殖、迁移能力,同时诱导细胞发生凋亡,Sphk1基因可能成为一个潜在的乳腺癌治疗靶标。

Objective To investigate the effect of down-regulating sphingosine kinase-1 （Sphk1） on the proliferation, apoptosis and migration in human breast cancer MCF-7 cell line. Methods The siRNA against Sphk1 was chemically synthesized and then transfected into MCF-7 cells using Lipofectamine 2000. Transfection efficiency was examined by flow cytometry （ FCM ） and the expression levels of Sphk1 mRNA and protein were determined by real-time fluorescent quantitative PCR and Western blotting. Cell proliferation and cell apoptosis was assessed by CCK-8 assay and FCM respectively. Cell migration was measured by transwell assay. Results The efficiency of siRNA transfection into human MCF-7 cells were 89%. The siRNA against sphkl gene significantly reduced the mRNA and protein expression of Sphk1 in MCF-7 cells （ P 〈 0.05 ）. The siRNA-2 showed most significant effect（ P 〈0.01 ）. The MCF-7 ceils transfected with Sphk1 siRNA-2 showed a significant decrease of cell proliferation and migration compared to the control cells. Moreover, significant increase of proportion of apoptotic cells were detected in the transfected cells （P 〈 0. 05 ）. Conclusion Sphk1 knockdown in breast cancer cells effectively inhibits the proliferation and migration and induces the apoptosis in MCF-7 ceils. Sphk1 may become a potential target for gene therapy of breast cancer.