小RNA干扰PDK1抑制乳腺肿瘤细胞侵袭

Small Interfering RNA targeting PDK1 Inhibits Breast Cancer Cell Line Invasion and Metastasis

目的:在肿瘤侵袭转移的细胞和生物学机制中,包含有多种基因和信号传导通路的改变。PDK1是PI3K分子通路上的一个重要分子,为了研究PDK1对乳腺肿瘤细胞侵袭能力的影响,我们以乳腺癌MDA-MB-231细胞为研究模型,用小RNA干扰法特异下调PDK1的表达,研究其侵袭能力的变化,为乳腺癌临床治疗探索新的分子靶点。方法:通过构建PDK1特异的小RNA干扰质粒,利用脂质体介导的方法转染乳腺癌细胞MDA-MB-231,无限稀释法筛选单克隆细胞系,并通过Western Blot的方法检测各组细胞中PDK1蛋白的表达水平,细胞计数的方法观察各组细胞之间的增殖情况,并利用Matrigel Transwell的实验方法研究细胞在体外的侵袭能力差别。结果:MDA-MB-231细胞经小RNA干扰后PDK1蛋白表达水平明显下降,与对照组相比,细胞的生长增殖能力没有显著差异(P>0.05),细胞的侵袭能力显著下降,并且差异有统计学意义(P<0.05)。结论:MDA-MB-231是一种侵袭能力高的乳腺癌细胞系,本研究利用小RNA干扰技术,特异性下调了乳腺癌MDA-MB-231细胞中的PDK1蛋白表达,结果表明,乳腺癌细胞的生长能力与对照组的细胞相比没有明显差别,但是,PDK1蛋白表达水平下降后乳腺癌细胞的侵袭能力明显下降,提示我们PDK1可能与乳腺癌MDA-MB-231细胞的侵袭能力肾密相关。鉴于PDK1处于PDK1-Akt-PKC信号通路的上游,以PDK1为靶标的分子干预可能对乳腺癌细胞的侵袭转移起到有效的作用。

Objective： Understanding of the cellular and molecular basis of tumor metastasis inevitably brings up the challenging fact that metastasis is a complex multistep biological process most likely controlled by distinct genes and signaling pathways at each step. PDK1 is a key molecule that couples PI3K to cell proliferation and survival signals in response to growth factor receptor activation. PDK1 is oncogenic when expressed in mammary epithelial cells. In order to study the role of PDK1 in breast cancer cell invasion, we decreased PDK1 protein expression in MDA-MB-231 cells using small interfering RNA. Methods： We built a siRNA plasmid with the ability to specifically reduce PDK1 protein expression. Then we transfected the MDA-MB-231 breast cancer cells with the new plasmid in a lipid-mediated manner. We obtained the clone and assessed the PDK1 protein expression by Western blot. We assayed proliferation by cell counting and invasion using Transwell matrigel invasion chambers. Results： PDK1 protein expression was downregulated by siRNA expressed by the vector. Compared with the control group, cells transfected with the sJRNA vector had significantly decreased potency, with a statistical significance between the two groups （P〈0.05）. Conclusion： MDA-MB-231 is a highly invasive breast cancer cell line. PDK1, an upstream signal transducer in the PI3K-Akt-PKC pathway, may play an important role in MDA-MB-231 invasion and may serve as an ideal target for molecular intervention against breast cancer invasion and metastasis.