Zhang Yang, DU Qiang, Qiu Xiao-yan, Tian Xin-xia, Fang Wei-gang
Department of Pathology, Peking University Health Science Center, Beijing 100191, China.
Zhonghua Bing Li Xue Za Zhi. 2010 Apr;39(4):259-63.
To investigate the effect of over expression of human hyperplasia suppressor gene (HSG) on proliferation, invasion, apoptosis and cell cycle of human breast cancer cells and to determine the relationship between HSG and Ras-dependent signaling pathway.
Full length HSG coding sequences were cloned into plasmid pcDNA3.0. The recombinant plasmids were transfected into MDA-MB-231, a highly malignant breast cancer cell line. Vacant pcDNA3.0 was used as the control. MTT, Matrigel transwell assay and flow cytometric analysis were used to test for proliferation, invasion, cell cycle distribution and apoptosis of tumor cells after transient transfection of HSG.GST-pulldown and Western blotting assays were performed to investigate the activity of Ras protein.
HSG transfection inhibited proliferation of MDA-MB-231 cells, and significantly decreased the number of invading cells in Matrigel transwell assay compared with the vector/231 group (78.5 +/- 5.8 vs. 131.1 +/- 14.5) cells. FACS analyses demonstrated that compared with the vector/231 group, up-regulation of HSG promoted breast cancer cell apoptosis [(35.8 +/- 4.8)% vs. (25.6 +/- 3.5%)] and induced G(0)/G(1) phase arrest [(56.3 +/- 2.3)% vs. (50.4 +/- 1.9%)] after transfection for 18 hours. Furthermore, GST-pulldown assay showed that over-expression of HSG remarkably decreased the activity of Ras (about 65% lower than control).
HSG exibits multiple anticancer functions in breast cancer cells including inhibition of proliferation and in vitro invasion, G(0)/G(1) arrest and promotion of apoptosis. Besides, inhibition of Ras-dependent signaling pathway may be involved in these processes.
研究人增生抑制基因（HSG）过表达对人乳腺癌细胞增殖、侵袭、凋亡及细胞周期的影响，并确定HSG与Ras依赖性信号通路之间的关系。
将HSG全长编码序列克隆到质粒pcDNA3.0中。将重组质粒转染至高度恶性的乳腺癌细胞系MDA-MB-231中。空的pcDNA3.0用作对照。采用MTT法、基质胶侵袭实验和流式细胞术分析检测HSG瞬时转染后肿瘤细胞的增殖、侵袭、细胞周期分布和凋亡情况。进行谷胱甘肽-S-转移酶下拉实验（GST-pulldown）和蛋白质免疫印迹分析（Western blotting）以研究Ras蛋白的活性。
与载体/231组相比，HSG转染抑制了MDA-MB-231细胞的增殖，并显著减少了基质胶侵袭实验中侵袭细胞的数量（78.5±5.8对131.1±14.5）。流式细胞术分析表明，与载体/231组相比，转染18小时后，HSG的上调促进了乳腺癌细胞凋亡[（35.8±4.8）%对（25.6±3.5）%]，并诱导G(0)/G(1)期阻滞[（56.3±2.3）%对（50.4±1.9）%]。此外，GST-pulldown实验表明，HSG的过表达显著降低了Ras的活性（比对照低约65%）。
HSG在乳腺癌细胞中表现出多种抗癌功能，包括抑制增殖和体外侵袭、G(0)/G(1)期阻滞及促进凋亡。此外，抑制Ras依赖性信号通路可能参与了这些过程。
