MRP-1/CD9与高转移人乳腺癌MDA-MB-231细胞恶性行为相关性的体外研究  

Correlation og motility relaued protein-1(MRP-1)/CD9 with malignant behavior of human highly metastatic breast cancer cell line MDA-MB-231 in vitro

作　　者：陈颖[1] 吴明富[1] 李琼[1] 游岚瑛[1] 魏军成 周剑锋[2] 徐刚[3] 卢运萍[1] 马丁[1] 

机构地区：[1]华中科技大学同济医学院附属同济医院妇产科,武汉430030 [2]华中科技大学同济医学院附属同济医院血液科,武汉430030 [3]华中科技大学同济医学院附属同济医院肾内科,武汉430030

出　　处：《肿瘤》2006年第9期793-797,共5页Tumor

基　　金：国家自然科学基金资助项目(编号:C03020705);国家重点基础研究发展规划2002年度项目(编号:2002CB513107)

摘　　要：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： Motility Related Protein-1（MRP 1）/CD9 belongs to the transmembrane 4 superfamily （TM4SF） with four highly conserved hydrophobic domains that are assumed to span the lipid bilayer of the cell membrane, which is involved in the regulation of motility of tumor cells as well as invasion and metastasis. This study was to observe inhibitory effect of MRP-1/ CD9 on proliferation and invasive potential of human highly metastatic breast cancer cell line MDA-MB-231 in vitro and explore the underlying mechanism. Methods： With specific amplified primer of MRP-1/CD9, MRP-1/CD9 full length cDNA fragment was obtained by reverse transcriptase polymerase chain reaction （RT-PCR）. RT PCR products were cloned into plasmid pcDNA3.1 in sense direction. The recombinant MRP 1/CD9 plasmid was transfected into MDA-MB-231 cells. The positive clones were screened by G418. The proliferation, migration, and invasion of MDA-MB-231 cells before and after transfection were assessed by CFSE-flow cytometry, monolayer wound healing assay, and soft agar colony formation test. The expression of AKT, p-AKT, and SRC protein in untransfected cells, mock transfected cells and CD9 erexpressing MDA MB-231 cells were determined by western blot analysis. Results：We successfully constructed full length MRP-1/CD9 eukaryotic expression vector and transfected it into MDA-MB 231 cells. The MRP 1/CD9 stable expressing clone （MDA-MB-231/CD9） was obtained. Compared with untransfected cells and mock-transfected cells, the proliferative ability, motility, and invasive potential of MDA-MB-231/ CD9 cells was obviously weakened. The protein levels of p AKT and SRC were decreased in the MdA MB 231/CD9 cells. Conclusion： MRP-1/CD9 plays a vital role in the suppression of cell motility and invasion, which may be relate to E cadherin mediated adherence enhancement induced by down regulation of p-AKT and SRC.

关 键 词：乳腺肿瘤 细胞系 肿瘤 肿瘤转移 基因表达 MRP-1/CD9 

分 类 号：R737.9[医药卫生—肿瘤]