Guo Shanyu, Xiao Dan, Liu Huihui, Zheng Xiao, Liu Lei, Liu Shougui
Department of General Surgery, Shanghai Ninth People's Hospital, Shanghai Jiaotong University School of Medicine, Shanghai 200011, P.R. China.
Oncol Lett. 2014 Oct;8(4):1557-1562. doi: 10.3892/ol.2014.2323. Epub 2014 Jul 7.
To investigate the effect and mechanism of the CXC chemokine receptor 4 (CXCR4) in the proliferation and migration of breast cancer, a short-hairpin RNA (shRNA) eukaryotic expression vector targeting CXCR4 was constructed, and the impact of such on the proliferation, adhesion and migration of human breast cancer MDA-MB-231 cells was observed. The fragments of CXCR4-shRNA were synthesized and cloned into a pGCsi-U6-Neo-green fluorescent protein vector. The recombinant plasmids were transfected into 293T cells and the most efficacious interfering vector was selected. MDA-MB-231 cells were transfected by liposome assay. The effects of silencing CXCR4 expression by shRNA on the growth, adhesion and migration of MDA-MB-231 cells were determined by Cell Counting Kit-8, cell-matrix adhesion and wound-healing assays. The shRNA eukaryotic expression vectors targeting CXCR4 (CXCR4-shRNA) were successfully constructed and transfected into 293T cells. Quantitative polymerase chain reaction and western blot analysis revealed that the maximum inhibitory rate of CXCR4 expression was 81.3%. CXCR4-shRNA transfection significantly inhibited the proliferation of MDA-MB-231 cells (P<0.05), as well as the adhesion between MDA-MB-231 cells and the extracellular matrix (P<0.05). Furthermore, wound-healing assays demonstrated that the migration distance of MDA-MB-231 cells in the CXCR4-shRNA transfection group was significantly smaller than that in the control plasmid and blank control groups (P<0.01). The CXCR4-shRNA interfering vector specifically inhibited CXCR4 expression, as well as the proliferation, adhesion and migration of MDA-MB-231 cells.
为研究CXC趋化因子受体4(CXCR4)在乳腺癌增殖和迁移中的作用及机制,构建了靶向CXCR4的短发夹RNA(shRNA)真核表达载体,并观察其对人乳腺癌MDA-MB-231细胞增殖、黏附和迁移的影响。合成CXCR4-shRNA片段并克隆至pGCsi-U6-Neo-绿色荧光蛋白载体。将重组质粒转染至293T细胞,筛选出最有效的干扰载体。采用脂质体法转染MDA-MB-231细胞。通过细胞计数试剂盒-8、细胞-基质黏附及伤口愈合实验,检测shRNA沉默CXCR4表达对MDA-MB-231细胞生长、黏附和迁移的影响。成功构建了靶向CXCR4的shRNA真核表达载体并转染至293T细胞。定量聚合酶链反应和蛋白质印迹分析显示,CXCR4表达的最大抑制率为81.3%。CXCR4-shRNA转染显著抑制MDA-MB-231细胞的增殖(P<0.05),以及MDA-MB-231细胞与细胞外基质之间的黏附(P<0.05)。此外,伤口愈合实验表明,CXCR4-shRNA转染组MDA-MB-231细胞的迁移距离显著小于对照质粒组和空白对照组(P<0.01)。CXCR4-shRNA干扰载体特异性抑制CXCR4表达以及MDA-MB-231细胞的增殖、黏附和迁移。