论文部分内容阅读
目的:观察MRP-1/CD9对高转移人乳腺癌细胞系MDA-MB-231体外增殖和侵袭的抑制效应,并初步探讨其作用机制。方法:应用MRP-1/CD9特异性扩增引物,借助RT-PCR获得MRP-1/CD9全长cDNA片段,正向插入pcDNA3.1表达载体中,并将重组质粒导入MDA-MB-231细胞中,G418稳定筛选;应用CFSE-流式细胞仪检测,单层伤口愈合实验,软琼脂克隆形成实验等方法观察了MDA-MB-231细胞转染前后,细胞体外增殖及侵袭能力等指标的变化。Western blot检测AKT、p-AKT和SRC在转染前后的细胞中的表达变化。结果:成功构建全长MRP-1/CD9真核表达载体,将其转染入MDA-MB-231细胞后获得稳定表达MRP-1/CD9的MDA-MB-231克隆株(MDA-MB-231/CD9)。与空质粒转染后的MDA-MB-231细胞相比,MDA-MB-231/CD9细胞运动能力明显减弱,侵袭能力降低;p-AKT和SRC在MDA-MB-231/CD9细胞中表达明显降低。结论:MRP-1/CD9对细胞的运动和侵袭有抑制作用,这种作用与p-AKT和SRC的下调引起E-cadherin介导的粘附增强有关。
OBJECTIVE: To observe the inhibitory effect of MRP-1 / CD9 on proliferation and invasion of highly metastatic human breast cancer cell line MDA-MB-231 in vitro and to explore its possible mechanism. Methods: MRP-1 / CD9 specific primers were used to amplify the full-length cDNA of MRP-1 / CD9 by RT-PCR and inserted into pcDNA3.1 expression vector. The recombinant plasmid was introduced into MDA-MB-231 cells , And G418 was stably selected. The changes of proliferation and invasion ability of MDA-MB-231 cells before and after transfection were observed by CFSE-flow cytometry, monolayer wound healing test and soft agar colony formation assay . Western blot was used to detect the expression of AKT, p-AKT and SRC in the cells before and after transfection. Results: The full-length MRP-1 / CD9 eukaryotic expression vector was successfully constructed and transfected into MDA-MB-231 cells to obtain the MDA-MB-231 clone stably expressing MRP-1 / CD9 / CD9). Compared with the MDA-MB-231 cells transfected with empty plasmid, the ability of MDA-MB-231 / CD9 cells decreased significantly and the invasive ability decreased. The expressions of p-AKT and SRC in MDA-MB-231 / reduce. Conclusion: MRP-1 / CD9 can inhibit cell motility and invasion. This effect is related to the enhanced E-cadherin-mediated adhesion induced by downregulation of p-AKT and SRC.