AIM; To investigate the effects of shRNA -mediated collagen type I alpha 1 (COL1A1) gene silencing on the proliferation and apoptosis of human breast cancer cell line MDA - MB - 231. METHODS: The specific re-combinant vector pSilencer2.1 - U6 - COL1 Al was transiently transfected into human breast cancer cell line MDA - MB -231 with lipofectamine. RT -PCR and Western blotting were performed to detect the expression levels of COL1A1. MTT assay was employed to evaluate the effect of COL1A1 gene silencing on the cell proliferation. Flow cytometry was used to determine the apoptosis and cell cycle of transfected cells . The morphological characteristics of apoptosis were observed by Hoechst 33258 staining. RESULTS: Compared with mock group and scrambled group , the mRNA and protein levels of COL1A1 were reduced by pshRNA -COL1A1 transfection (P<0.05). The proliferation of MDA -MB-231 cells treated in shRNA - COL1 Al was significantly inhibited in a time - dependent way. The percentages of G0/G, phase cells and early apoptotic rate were significantly higher in pshRNA - CO LI Al group than those in mock and scrambled group ( P < 0.05 ). The changes of apoptotic morphology such as cell shrinkage and nuclear condensation were also observed by staining with Hoechst 33258 under fluorescence microscope. CONCLUSION: Transfection of eukaryotic expression vector pshRNA -COL1 Al effectively inhibits the proliferation , induces apoptosis and arrests MDA - MB - 231 cells in G0/G, phase.%目的:探讨抑制Ⅰ型胶原α1链(COL1A1)基因表达对人乳腺癌MDA-MB-231细胞增殖及凋亡的影响.方法:用已构建的pSilencer2.1-U6-COL1A1重组质粒转染乳腺癌细胞MDA-MB-231,采用RT-PCR和Western blotting技术检测重组质粒对COL1A1基因表达的影响,MTT比色法测定细胞增殖的抑制情况,流式细胞术检测细胞周期和细胞凋亡的变化,Hoechst 33258荧光染色观察细胞凋亡的形态学变化.结果:RT-PCR及Western blotting结果证实重组质粒在mRNA和蛋白水平分别显著抑制COL1A1基因表达;pshRNA-COL1A1转染组细胞的增殖速度明显减慢且呈时间依赖关系;与对照组相比,pshRNA-COL1A1组细胞明显阻滞于G0/G1期,细胞凋亡率显著升高(P<0.05),出现细胞质浓缩、核凝聚等凋亡形态学变化.结论:pshRNA-COL1A1能有效抑制乳腺癌MDA-MB-231细胞增殖,并诱导其凋亡,为以COL1A1为靶点的乳腺癌基因治疗提供实验依据.
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