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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 an eukaryotic expression vector for RNA interference (RNAi) of IRF-4 binding protein (IBP) gene and study its effect on IBP gene expression and cell proliferation and invasiveness in MDA-MB-231 breast cancer cells. Methods Using IBP as target gene and pcDNATM6.2-GW / EmGFPmiR plasmid as carrier, four pairs of recombinant plasmids were designed and constructed, and DNA sequencing was performed. After transfection of recombinant vector into MDA-MB-231 cells, one pair of recombinants with the best transfection efficiency were selected to establish stable transfected cell lines. The inhibitory effect of the recombinant plasmid on the expression of IBP mRNA and protein was detected by RT-PCR and Western blot. The effect of MTT assay on the cell growth of each group; the influence of cell invasion assay in vitro on the invasiveness. Results The sequencing results of the recombinants were completely consistent with the target sequences. The mRNA and protein expression of IBP decreased about 70% after transfected with MDA-MB-231 cells. The proliferation of breast cancer cells was decreased after IBP was down-regulated (P <0.05) Invasion experiments showed that the number of transplanted breast cancer cells after transfection was significantly lower than that of untransfected and empty plasmid control groups (25 ± 7, 67 ± 6, 68 ± 5, P <0.05, respectively). Conclusion Down-regulation of IBP can effectively reduce the proliferation and invasiveness of breast cancer cells.