沉默CC型趋化因子受体5对人乳腺癌细胞黏附及迁移的影响

Effect of inhibiting CC chemokine receptor 5 on human breast cancer cell adhesion and migration

构建CC型趋化因子受体5(CCR5)基因shRNA的真核表达载体,探讨沉默CCR5对乳腺癌细胞黏附与迁移能力的影响。在多种人乳腺癌细胞中检测CCR5的基因表达,选择CCR5表达较高的MDA-MB-231细胞做为基因沉默的研究对象;设计并合成2对靶向人CCR5基因的RNA沉默序列,连入基因沉默载体pSilencer 2.1-U6中,构建沉默质粒shCCR5-1和shCCR5-2,定量PCR和蛋白印迹法检测CCR5基因沉默的效率;以细胞黏附实验和Transwell趋化小室实验分别检测沉默CCR5对MDA-MB-231细胞黏附和迁移能力的影响。结果表明,沉默CCR5可以抑制MDA-MB-231细胞的黏附能力,同时显著降低MDA-MB-231细胞的迁移和趋化。

To construct shRNA eukaryotic expression vectors that targeting human CC chemokine receptor 5（CCR5） and to investigate the biological function of CCR5 in breast cancer cell adhesion and migration,the gene expression of CCR5 in MDA-MB-231,MDA-MB-468,T47D human breast cancer cell lines was measured by quantitative PCR,and MDA-MB-231 cells highly expressing CCR5 were used in the next experiments.Secondly,to construct shRNA eukaryotic expression vectors that express shRNA targeting human CCR5,small fragments of siRNA were designed and cloned into the Ambion shRNA vector pSilencer 2.1-U6.The recombinant plasmids（shCCR5-1 and shCCR5-2） were then transiently transfected in MDA-MB-231 cells and CCR5 gene and protein expression were measured by quantitative PCR and Sestern blot.Thirdly,the effect of knockdown of CCR5 on MDA-MB-231 cell adhesion and migration was determined by adhesion assay and transwell chamber test,respectively.It was found that silencing of CCR5 greatly inhibited MDA-MB-231 cell adhesion and migration.