Department of Pathology, School of Basic Medicine Sciences, Chong Qing University of Medical Sciences, Chong Qing, 400016, China.
J Exp Clin Cancer Res. 2011 May 19;30(1):60. doi: 10.1186/1756-9966-30-60.
MTA1(metastasis associated-1) is a tumor metastasis associated candidate gene and overexpression in many human tumors, including breast cancer. In this study, we investigated depressive effect on MTA1 by MTA1-specific short hairpin RNA(shRNA) expression plasmids in human breast cancer cell lines MDA-MB-231 and MCF-7, and effect on protein levels of ER alpha, MMP-9, cyclinD1, and tumor cell invasion, proliferation.
ShRNA expression vectors targeting MTA1 was constructed and transfected into human breast cancer cell lines MDA-MB-231 and MCF-7. The transfection efficiency was evaluated by fluorescence microscopy, mRNA levels of MTA1 were detected by reverse transcription-polymerase chain reaction (RT-PCR), protein levels of ER alpha, MMP-9 and cyclinD1 were detected by Western blotting, respectively. Tumor cells invasive ability were evaluated by Boyden chamber assay, the cells proliferation were evaluated using cell growth curve and MTT analysis, the cell cycle analysis was performed using flow cytometry.
Down-regulation of MTA1 by RNAi approach led to re-expression of ER alpha in ER-negative breast cancer cell lines MDA-MB-231, and reduced protein levels of MMP-9 and CyclinD1, as well as decreased tumor cell invasion and proliferation, more cells were blocked in G0/G1 stage(P < 0.05). However, after inhibiting mRNA levels of MTA1, protein expression of ER alpha, MMP-9, cyclinD1 and the changes of cancer cells invasiveness, proliferation, cells cycle were no statistical difference in ER-positive human breast cancer cell lines MCF-7 (P > 0.05).
ShRNA targeted against MTA1 could specifically mediate the MTA1 gene silencing and consequentially recover the protein expression of ER alpha, resulting in increase sensitivity of antiestrogens, as well as suppress the protein levels of MMP-9 and cyclinD1 in ER-negative human breast cancer cell lines MDA-MB-231. Silencing effect of MTA1 could efficiently inhibit the invasion and proliferation in MDA-MB-231 cells. The shRNA interference targeted against MTA1 may have potential therapeutic utility in human breast cancer.
MTA1（转移相关-1）是一种肿瘤转移相关的候选基因，在许多人类肿瘤中过度表达，包括乳腺癌。在这项研究中，我们通过 MTA1 特异性短发夹 RNA（shRNA）表达质粒在人乳腺癌细胞系 MDA-MB-231 和 MCF-7 中研究了 MTA1 的抑制作用，以及对 ERα、MMP-9、cyclinD1 和肿瘤细胞侵袭、增殖的蛋白水平的影响。
构建靶向 MTA1 的 shRNA 表达载体并转染人乳腺癌细胞系 MDA-MB-231 和 MCF-7。通过荧光显微镜评估转染效率，通过逆转录-聚合酶链反应（RT-PCR）检测 MTA1 的 mRNA 水平，通过 Western 印迹分别检测 ERα、MMP-9 和 cyclinD1 的蛋白水平。通过 Boyden 室测定评估肿瘤细胞的侵袭能力，通过细胞生长曲线和 MTT 分析评估细胞增殖，通过流式细胞术进行细胞周期分析。
RNAi 方法下调 MTA1 导致 ER 阴性乳腺癌细胞系 MDA-MB-231 中 ERα 的重新表达，并降低 MMP-9 和 cyclinD1 的蛋白水平，以及降低肿瘤细胞的侵袭和增殖，更多的细胞被阻滞在 G0/G1 期（P<0.05）。然而，在抑制 MTA1 的 mRNA 水平后，ER 阳性人乳腺癌细胞系 MCF-7 中 ERα、MMP-9、cyclinD1 的蛋白表达以及癌细胞侵袭、增殖、细胞周期的变化无统计学差异（P>0.05）。
针对 MTA1 的 shRNA 可特异性介导 MTA1 基因沉默，并随后恢复 ERα 的蛋白表达，导致抗雌激素敏感性增加，以及抑制 ER 阴性人乳腺癌细胞系 MDA-MB-231 中 MMP-9 和 cyclinD1 的蛋白水平。MTA1 的沉默作用可有效抑制 MDA-MB-231 细胞的侵袭和增殖。针对 MTA1 的 shRNA 干扰可能在人类乳腺癌中具有潜在的治疗应用价值。
