B7-H3沉默对乳腺癌MDA-MB-231细胞上皮间质转化及生物学功能的影响

目的:探讨B7-H3沉默对人乳腺癌MDA-MB-231细胞上皮间质转化及生物学行为的影响。方法:通过脂质体Lipofectamine TM 2000转染B7-H3 siRNA干涉载体pSilencer4.1-CMV neo/B7-H3到乳腺癌MDA-MB-231细胞,经G418筛选后获得B7-H3沉默细胞株MDA-MB-231-ShB7-H3,利用qRT-PCR及Western blot分别检测对照组(WT)、无关干涉组(ShNC)和B7-H3干涉组(ShB7-H3)细胞中B7-H3、EMT标志分子mRNA及蛋白表达水平;利用Transwell实验检测各组细胞迁移和侵袭能力;利用MTT细胞增殖实验评价各组细胞增殖情况。结果:通过qRT-PCR和Western blot证实B7-H3沉默表达的MDA-MB-231细胞株构建成功;与对照组细胞相比,B7-H3沉默表达抑制MDA-MB-231细胞EMT进程,上皮细胞标志分子E-cadherin和ZO-1表达上调,而间质细胞标志分子vimentin和αSMA表达下调;Transwell实验及MTT细胞增殖实验结果表明B7-H3沉默抑制MDA-MB-231细胞迁移、侵袭及增殖能力。结论:我们的研究结果强调了B7-H3在人乳腺癌细胞株MDA-MB-231中的重要作用,并提示B7-H3与乳腺癌细胞EMT、转移密切相关,并为以B7-H3为靶点的乳腺癌转移治疗提供初步的实验及理论基础。

Influence of B7-H3 silencing on epithelial-mesenchymal transition and biological function of breast cancer MDA-MB-231 cells

Objective:To investigate the effect of B7-H3 silencing on epithelial-mesenchymal transition(EMT)and biological behaviors of human breast cancer MDA-MB-231 cells.Methods:Lipofectamine TM 2000 were used to transfect B7-H3 siRNA vector pSilencer4.1-CMV neo/B7-H3 into breast cancer MDA-MB-231 cells,which was subsequently screened by G418 in order to acquire positive cell model whose B7-H3 level was decreased.qRT-PCR and Western blot were used to detect the mRNA and protein levels of B7-H3 and EMT marker molecules in control groups(WT),non-specific interference groups(ShNC)and B7-H3 interference groups(ShB7-H3).Transwell experiments were also used to detect cell migration and invasion.MTT assay was applied to evaluate cell proliferation.Results:The results of RT-PCR and Western blot demonstrated that the expression of B7-H3 was largely knocked down in MDA-MB-231 cells.Compared with the control groups,the EMT process of model cells with B7-H3 silencing were suppressed,accompanied by E-cadherin and ZO-1 were significantly up-regulated while vimentin andαSMA were down-regulated.Transwell experiments and MTT assays showed that the silent expression of B7-H3 inhibited cell migration,invasion and proliferation.Conclusion:Our present results emphasize the important role of B7-H3 in human breast cancer MDA-MB-231 cells,and suggest that B7-H3 is closely related with breast cancer EMT and metastasis,which lay good foundation concerning experiment and theory for exploring breast cancer metastasis therapy aiming at B7-H3.