文档介绍：靶向ATP6L的RNA干扰抑制乳腺癌细胞的体外侵袭能力
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作者:游海燕, 金洁, 唐宁, 邓云, 舒惠群, 沈秋瑾, 覃文新
【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。
【关键词】 ATP6L; RNA干扰; 侵袭; 肿瘤
[Abstract] 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 pared 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.
[Key words] ATP6L; RNA interference; invasion; cancer
人类对乳腺癌的发病及其发展过程有了进一步的认识,改变了过去认为乳腺癌只是局部疾病的观念。许多研究证实乳腺癌从起初发病时即表现为全身性疾病,而不仅仅是局部的肿瘤,且乳腺癌在早期即可发生血行播散。在美国,乳腺癌复发患者总的5年生存率为46%,远处转移率为51% [1,2]。乳腺癌肝转移的中位生存期仅为7~20个月[3]。复发和转移是导致乳腺癌患者死亡的主要原因[4],术后防范复发对于乳腺癌患者来说尤为关键。有研究发现,质子泵(vacuolar
H+ATPase, VATP