RNAi沉默IBP基因表达对乳腺癌细胞的抑制作用

Inhibitory effect of RNAi-silenced IBP gene expression in breast cancer cells

目的构建IRF-4结合蛋白(IBP)基因RNA干扰(RNAi)的真核表达载体,观察其对MDA-MB-231乳腺癌细胞内IBP基因表达及细胞增殖和侵袭力的影响。方法以IBP为靶基因,以pcDNATM6.2-GW/EmGFPmiR质粒为载体,设计构建4对重组体,并进行DNA测序鉴定。重组载体转染MDA-MB-231细胞后,选择转染效果最好的1对重组体建立稳定转染的细胞株,经RT-PCR和Westernblot检测重组表达质粒对IBPmRNA和蛋白表达的抑制效果;MTT法检测对各组细胞生长的影响;细胞体外侵袭实验测定对侵袭力的影响。结果重组体测序结果与目的序列完全一致;重组体转染MDA-MB-231细胞后IBP的mRNA和蛋白表达降低约70%;IBP表达下调后乳腺癌细胞增殖减缓(P<0.05);同时体外侵袭实验显示转染后乳腺癌发生迁移细胞数明显低于未转染组和空质粒对照组[分别为(25±7)、(67±6)、(68±5),P<0.05]。结论下调IBP能有效降低乳腺癌细胞的增殖和侵袭力。

Objective To construct the IRF-4 binding protein（IBP）eukaryotic expression vector for RNA interference and to observe its effect on proliferation and invasion of MDA-MB-231 breast cancer cells.Methods Four pairs of recombinant vectors were designed and established using IBP as the target gene and plasmid pcDNATM6.2-GW/EmGFPmiR as the vector,and the sequence of DNA was identified.After the recombinant plasmid was transfected into the cultured MDA-MB-231 cells,the most efficient expression vector was chosen to establish cell lines.Inhibitory effect of recombinant plasmid on expression of IBP mRNA and protein was detected by real-time PCR and Western blotting,respectively.The inhibitory effect of recombinant plasmid on cell growth was detected by MTT assay.The invasion of MDA-MB-231 cells was identified by cell invasion assay.Results The sequence of recombinant plasmid was consistent with that of the designed vector.The expression of IBP mRNA and protein was decreased about 70% in recombinant expression vector transfected into MDA-MB-231 cells.The proliferation of breast cancer cells was reduced with the decreased expression of IBP（P0.05）.The invasion assay demonstrated that the number of breast cancer cells was significantly lower in transfection group than in non-transfected and blank control groups（25±7 vs 67±6 and 68±5,P0.05）.Conclusion Down-regulation of IBP expression can significantly decrease the proliferation and invasion of breast cancer cells.