干扰Sphk1基因表达对人乳腺癌MCF-7细胞增殖-第三军医大学学报.DOCVIP

Sphk1基因对人乳腺癌MCF-7细胞增殖、凋亡和迁移能力的影响 张彦 郑晓东 唐鹏 张毅 姜军（400038重庆，第三军医大学西南医 院乳腺中心） 基金项目：国家自然科学基金资助项目 作者简介：张彦（1980-10），男，湖南泸溪人，硕士生，医师，主要从事乳腺癌基础与临床研究。电话：（023通讯作者：姜军,电话：（023e-mail:jcbd@;张毅,电话：（023e-mail：zy53810@163.com [摘要] 　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＜0.05）。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。 [关键词]：乳腺癌；RNA干扰；siRNA；Sphk1； [Abstract] Objective To inhibit the express of sphingosine kinase-1 (Sphk1) gene of breast cancer MCF-7 cells through RNA interference（RNAi），and to explore the proliferation, apoptosis and migration change after gene silencing. Methods sphk1 siRNA was chemically synthesized and transfected into MCF-7 cells mediated by Lipofectamine2000. Transfection efficiency was examined by flow cytometry（FCM）. mRNA and protein levels of Sphk1 in MCF-7 cells before and after transfection were detected by real time quantitative reverse transcription polymerase Chain reaction and Western-boltting .Inhibitory effect of Sphk1 siRNA on MCF-7 cell proliferation was assessed by CCK-8 assay. Cell apoptosis was examined by flow cytometry（FCM）.Cell migration was examined by Transwell assay.Result The efficiency of siRNA transfection into human MCF-7 cells were 89%.The siRNA targeting sphk1 gene significantly reduced mRNA and the protein expressions of sphk1 in MCF-7 cells（Ｐ＜0.05）, with the most significant effect of siRNA-2（Ｐ＜0.05）。 MCF-7 cells transfected with sphk1 siRNA-2 showed a significant decrease of cell proliferation and migration, while, significant increase of proportion of apoptotic cells