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目的:构建CXC趋化因子受体4(CXC chemokine receptor4,CXCR4)RNA干扰真核表达载体,研究其对人乳腺癌细胞MDA-MB-231增殖、黏附及迁移能力的抑制作用。方法:构建针对CXCR4的带发夹结构的小RNA干扰序列,并连接到pGCsi-U6-Neo-GFP载体中,转染293T细胞,筛选出干扰效率最高的表达载体。脂质体法转染MDA-MB-231细胞。利用CCK8法、细胞-基质黏附实验和划痕修复实验检测shRNA干扰CXCR4表达对MDA-MB-231细胞增殖、黏附和迁移能力的影响。结果:成功构建CXCR4-shRNA重组质粒,并转染293T细胞,利用RT-PCR及Western blotting检测发现CXCR4沉默效率最高可达81.3%。CXCR4-shRNA转染能显著抑制MDA-MB-231细胞的增殖(P<0.05)以及细胞与细胞外基质的黏附(P<0.05)。CXCR4-shRNA转染组MDA-MB-231细胞的迁移距离明显低于对照质粒组和空白对照组(P<0.01)。结论:CXCR4-shRNA干扰载体能特异性抑制CXCR4的表达,从而抑制乳腺癌MDA-MB-231细胞的增殖、黏附及迁移。
OBJECTIVE: To construct the eukaryotic expression vector of CXC chemokine receptor 4 (CXCR4) and study its inhibitory effect on the proliferation, adhesion and migration of human breast cancer cell line MDA-MB-231. Methods: The small interfering RNA (shRNA) sequence of hairpin RNA targeting CXCR4 was constructed and ligated into pGCsi-U6-Neo-GFP vector. The recombinant plasmid was transfected into 293T cells and the most efficient expression vector was selected. Lipofectamine was transfected into MDA-MB-231 cells. The effects of CXCR4 expression on the proliferation, adhesion and migration of MDA-MB-231 cells were detected by CCK8 assay, cell-matrix adhesion assay and scratch repair assay. Results: CXCR4-shRNA recombinant plasmid was successfully constructed and transfected into 293T cells. The highest efficiency of CXCR4 silencing was 81.3% by RT-PCR and Western blotting. CXCR4-shRNA transfection could significantly inhibit the proliferation of MDA-MB-231 cells (P <0.05) and the adhesion of cells to extracellular matrix (P <0.05). The migration distance of MDA-MB-231 cells in CXCR4-shRNA transfected group was significantly lower than that in control plasmid group and blank control group (P <0.01). Conclusion: The CXCR4-shRNA interference vector can specifically inhibit the expression of CXCR4, thereby inhibiting the proliferation, adhesion and migration of breast cancer MDA-MB-231 cells.