慢病毒介导的siRNA干扰乳腺癌MCF-7细胞MIF对VEGF-C表达的影响

Down-regulation of macrophage inhibitory factor expression in MCF-7 cells with lentivirusmediated small interfering RNA vectors and its effect on expression of VEGF-C

目的探讨慢病毒介导的siRNA对乳腺癌MCF-7细胞巨噬细胞移动抑制因子(macrophage migration inhibitory factor,MIF)的下调作用,分析MIF与血管内皮生长因子-C(vascular endothelial growth factor-C,VEGF-C)在乳腺癌细胞中表达的相关性。方法构建慢病毒/siRNA载体,转染乳腺癌MCF-7细胞。倒置荧光显微镜观察siRNA转染效率,实时荧光定量PCR检测MIF与VEGF-C的mRNA表达,Western blot法检测乳腺癌细胞中MIF蛋白表达,酶联免疫吸附试验检测细胞培养上清中VEGF-C分泌情况。结果成功构建稳定低表达MIF的乳腺癌细胞株。慢病毒介导的siRNA对人乳腺癌细胞MCF-7的转染效率超过80%,其能有效阻断MIF mRNA和蛋白的表达,同时VEGF-C的表达水平相应降低。结论构建的慢病毒载体能有效阻断乳腺癌细胞中MIF的表达。MIF可以调控乳腺癌细胞VEGF-C的表达。

Purpose To explore the role of small interfering RNA（ siRNA） mediated by slow virus in reduction of macrophage migration inhibitory factor（ MIF） expression of MCF-7 cells,and to study the relationship between MIF and VEGF-C in breast cancer cells.Methods Lentivirus-mediated small interfering RNA vectors were constructed and transfected into breast cancer cells MCF-7. Inverted fluorescence microscope was used to observe the efficiency of transfection. The expression of MIF and VEGF-C mRNA was detected by real-time fluorescent quantitative polymerase chain reaction,and the expression of MIF protein in breast cancer cells was evaluated by Western blot. Using enzyme linked immunosorbent experiment,we examined the expression of VEGF-C in the supernatants of different cells. Results The stable low expression of MIF in breast cancer cells was correctly established. The transduction rate of Lentivirus-mediated siRNA to MCF-7 was more than 80%. The lentivirus-mediated siRNA was able to effectively block the expression of MIF mRNA and protein. At the same time,the expression level of VEGF-C was reduced correspondingly. Conclusion The lentivirus vector that we constructed could effectively block the expression of MIF in breast cancer cells. MIF can regulate the expression of VEGFC in breast cancer cells.