骨桥蛋白下调对乳腺癌细胞生物学行为及MMP-2表达的影响

Effect of downregulation of osteopontin on biological behaviors and MMP-2 expression of breast cancer cells

目的:研究下调骨桥蛋白(osteopontin,OPN)对乳腺癌MDA-MB-231细胞生物学行为及基质金属蛋白酶-2(matrix metalloproteinase-2,MMP-2)表达的影响。方法:采用重组的OPN mRNA干扰质粒转染MDA-MB-231细胞,建立稳定转染细胞株。RT-PCR和Western印迹法检测细胞中OPN和MMP-2的表达,MTT法、流式细胞仪检测和软琼脂生长实验观察OPN下调对细胞生长、细胞周期分布和瘤细胞恶性表型的影响,酶联免疫吸附法(ELISA)检测细胞培养液中MMP-2的分泌水平。结果:与MDA-MB-231细胞相比,建立的转染细胞株M-OPNS1和M-OPNS2细胞的OPN mRNA及其蛋白表达均显著下调,细胞生长受到抑制,在细胞周期上表现为S期阻滞;OPN下调的细胞在软琼脂上的生长能力明显下降,MMP-2表达也明显降低。结论:RNA干扰介导的OPN下调能抑制MDA-MB-231细胞增殖、下调细胞恶性程度和MMP-2的表达,这可能为将来乳腺癌治疗提供新的方法。

Objective： To study the effects of downregulation of osteopontin （OPN） on biological behaviors and matrix metallo-proteinase-2 （MMP-2） expression of breast cancer cells MDA-MB-231. Methods： siRNA expression vectors were designed to target OPN and named as pSilence-OPN S 1 and pSilence-OPN S 2. The two plasmids were transfected into MDA-MB-231 cells. The mRNA and protein expression of OPN and MMP-2 were measured by RT-PCR and Western blotting, respectively. The effect of downregulation of OPN on cell proliferation, cell cycle distribution, and malignant phenotype of MDA-MB-231 cells were determined by MTT assay, flow cytometry, and soft agar assay, respectively. The level of MMP-2 protein in the supernatant of cultured cells was measured with ELISA. Results ： The expression of OPN in the transfected cells （ M-OPN S 1 and M-OPN S 2） was significantly downregulated at both mRNA and protein levels compared with MDA-MB-231 cells. The proliferation of transfected cells was inhibited and the cells were arrested at S phase. Downregulation of OPN caused a significant reduction in colony formation rate and MMP-2 expression. Conclusions： The stable silencing of OPN gene expression with siRNA inhibited the proliferation of MDA-MB-231 cells, downregulats malignant degree and MMP-2 expression. It may provide a novel therapeutic approach for breast cancer therapy.