靶向ATP6L的RNA干扰抑制乳腺癌细胞的体外侵袭能力  

Small interfering RNA targeting the subunit ATP6L of proton pump V-ATPase suppresses the invasion of breast cancer cells in vitro

作　　者：游海燕[1] 金洁[2] 唐宁[1] 邓云[1] 舒惠群[1] 沈秋瑾[1] 覃文新[1] 

机构地区：[1]上海交通大学肿瘤研究所癌基因及相关基因国家重点实验室,上海200032 [2]江苏大学基础医学与医学技术学院,江苏镇江212013

出　　处：《江苏大学学报（医学版）》2010年第2期126-130,135,共6页Journal of Jiangsu University：Medicine Edition

基　　金：国家自然科学基金资助项目(30973492);国家重点基础研究发展计划973课题(2009CB521803)

摘　　要：目的:通过靶向ATP6L的RNA干扰抑制质子泵的功能,检测MCF-7/ADR乳腺癌细胞的体外侵袭情况。方法:采用有效ATP6L干扰片段及干扰对照分别转染MCF-7/ADR细胞,通过荧光分光光度计检测BCECF与细胞分泌上清孵育后在不同激发光激发后发射的荧光量;用实时荧光定量PCR检测细胞中基质金属蛋白酶2(MMP-2)的表达情况;用明胶酶谱方法检测细胞分泌上清中的MMP-2活性;用细胞侵袭实验(Transwell)检测细胞体外侵袭能力。结果:在MCF-7/ADR细胞中,下调ATP6L基因的表达可以使细胞的泌H+能力减弱;细胞中MMP-2表达水平无变化;细胞分泌上清中的MMP-2活性减弱;细胞的体外侵袭能力下降。结论:靶向ATP6L的RNA干扰可抑制质子泵的功能,从而抑制MCF-7/ADR乳腺癌细胞的体外侵袭能力。Objective：To explore whether the invasion of MCF-7/ADR cells could be affected by in vitro inhibition of V-ATPase activity.Methods：The expression of ATP6L was knocked down by specific siRNA in breast cancer cell line,MCF-7/ADR.Then the capability of proton extrusion was analyzed by detecting the extracellular pH of the supernatant using pH-sensitive fluorescent dye BCECF on a luminescence spectrometer.The expression of MMP-2 and activity of gelatinase were determinated by real-time fluorogentic quantitative PCR and zymography.The ability of cell invasion in vitro was tested by transwell assay.Results：Proton extrusion was decreased in MCF-7/ADR cells treated with siRNA-ATP6L as compared with the control.There was no difference in the expression of MMP-2 between siRNA-ATP6L treated group and control group.The gelatinase activity in the supernatant of MCF-7/ADR cells treated with siRNA-ATP6L was decreased.The invasive ability of MCF-7/ADR cells was inhibited by ATP6L knockdown.Conclusion：After knockdown of ATP6L expression,the invasion of MCF-7/ADR cells was inhibited by the decrease of proton extrusion and the down-regulation of gelatinase activity.

关 键 词：ATP6L RNA干扰 侵袭 肿瘤 

分 类 号：R737.9[医药卫生—肿瘤]