CXCR4-siRNA表达载体的构建及其对体外乳腺癌细胞侵袭能力的影响

Construction of a siRNA expression plasmid targeting CXCR4 and its effect on the invasion capability of the breast cancer cells in vitro

目的构建趋化因子受体(CXCR4)小分子干扰RNA(siRNA)表达载体,研究其对体外乳腺癌细胞侵袭能力的影响。方法选择CXCR4高表达的乳腺癌MDA-MB-231细胞系,设计合成人CXCR4基因不同靶点的能编码siRNA的2条双链DNA序列,克隆到真核表达载体pGE-1-U6/kna中构建siRNA表达载体,体外脂质体介导转染MDA-MB-231细胞,用Western blot分析CXCR4蛋白表达,Transwell小室检测细胞的侵袭能力。结果成功构建了CXCR4-siRNA表达载体,瞬时转染乳腺癌细胞后能显著降低CXCR4的蛋白表达,可有效抑制人类乳腺癌MDA-MB-231细胞的侵袭能力。结论CXCR4-siRNA表达载体通过降低CXCR4基因的蛋白表达能显著抑制人类乳腺癌细胞的侵袭能力,有望为乳腺癌转移的基因治疗开辟新途径。

Objective To construct and identify the siRNA eukaryotic expression vector targeting gene CXC chemokine receptor-4 and explore its role in invasion process of breast cancer cells in vitro.Methods Two siRNAs were designed and synthesized according to the coding sequence of CXCR4 gene and cloned into eukaryotic expression plasmid pGE-1-U6/kna.The constructed CXCR4-siRNA expression vector was transfected into MDA-MB-231 cells by liposome.Western blot was used to evaluate the suppression of CXCR4 expression in different groups.The invasion and migration of MDA-MB-231 cells were evaluated by cell invasion assay in vitro.Results Enzyme digestion and DNA sequencing confirmed that the CXCR4-siRNA expression vector was successfully constructed.After transfection,the CXCR4-siRNA obviously suppressed the expression of CXCR4 compared with control groups and the ability of cell migration was decreased markedly.Conclusion CXCR4-siRNA expression vector can effectively suppress CXCR4 expression in the breast cancer cells and decrease potential of cell invasion,which may provide a novel strategy for gene therapy of breast cancer metastasis.