应用RNA干扰技术下调ANXA3表达的MDA-MB-231乳腺癌细胞株的建立

Establishment of breast cancer cell line- MDA-MB-231 through down-regulating the expressions of annexin A3 gene by RNA interference technique

目的：建立针对膜联蛋白A3（Annexin A3， ANXA3）的shRNA稳定转染的乳腺癌细胞株MDA-MB-231，为后续进一步研究奠定基础。方法荧光定量RT-PCR及western blot方法检测两种乳腺癌细胞株（ MDA-MB-231及MCF-7）中ANXA3 mRNA及蛋白表达水平。构建沉默ANXA3基因的shRNA质粒3个（ ANXA3-sh1-3）及阴性对照质粒，脂质体法转染人乳腺癌细胞MDA-MB-231；选出ANXA3沉默效果最好的干扰质粒做后续实验。嘌呤霉素筛选出稳定转染细胞；Western blot 方法检测转染后细胞中ANXA3蛋白表达水平。结果 MDA-MB-231细胞中 ANXA3 mRNA及蛋白表达水平显著高于MCF-7中的表达（ P ＜0 k．05）；成功构建3个针对ANXA3基因的shRNA质粒；转染ANXA3-sh1～3及阴性对照质粒后MDA-MB-231细胞中ANXA3 mRNA表达水平分别为（0．0196±0．0002）、（0．0085±0．0002）、（0．0220±0．0035）、（0．0661±0．0057），未转染的MDA-MB-231细胞中ANXA3 mRNA表达水平为（0．0692±0．0050），脂质体组MDA-MB-231细胞中ANXA3 mRNA表达水平为（0．0652±0．0118），ANXA3-sh2对MDA-MB-231细胞中ANXA3 mRNA沉默效率最高，达87．72％，因此后续实验选择ANXA3-sh2；嘌呤霉素筛选出稳定转染细胞分别命名为MDA-MB-231-Sh及MDA-MB-231-NC细胞。 Western blot检测结果显示，MDA-MB-231-Sh细胞中ANXA3蛋白显著低于MDA-MB-231及MDA-MB-231-NC细胞中的表达（ P ＜0．01）。结论 MDA-MB-231细胞较MCF-7细胞高表达ANXA3，成功的建立了稳定下调ANXA3表达的乳腺癌细胞株MDA-MB-231，为后续进一步研究ANXA3的表达及特性打下基础。

Objective To establish a breast cancer cell line-MDA-MB-231 transfected with shRNA （ small hairpin RNA） targeting at annexin A3, in order to provide a basis for further study.Methods The expression levels of ANXA3 mRNA and protein in MDA-MB-231 cells and MCF-7 cells were detected by fluorescent quantitation RT-PCR and Western Blot,respectively.The three shRNAs （shRNA1, 2 and 3） plasmids of silencing ANXA3 gene and negative control plasmid were established.MDA-MB-231 human breast cancer cells were transfered with liposome method.The interference plasmid that could silence ANXA3 gene most effectively was selected for subsequent experiment.The stable transfection cells were selected out by using puromycin, and the expression levels of annexin A7 were detected by Western Blot.Results The expression levels of ANXA3 mRNA and protein in MDA-MB-231 cells were significantly higher than those in MCF-7 cells （ P 〈0.05） . The three shRNAs plasmids targeting at annexin A3 gene were successfully established.The expression levels of ANXA3 mRNA in MDA-MB-231 cells after transfected with ANXA3-sh1 3 and negative control plasmid were 0.0196 ±0.0002, 0.0085 ±0.0002, 0.0220 ±0.0035, 0.0661 ±0.0057, respectively, however, which in MDA-MB-231 cells without transfection and in liposomes group were 0.0692 ±0.0050, 0.0652 ±0.0118, respectively.The silencing efficiency of ANXA3-sh2 on ANXA3 mRNA in MDA-MB-231 cells was the highest （87.72%）, thus, ANXA3-sh2 was selected for subsequent experiment.The stable transfection cells selected by puromycin were named as MDA-MB-231-Sh cells and MDA-MB-231-NC cells. Western Blot showed that the expression levels of ANXA3 protein in MDA-MB-231-Sh cells were significantly lower than those in MDA-MB-231 cells and in MDA-MB-231-NC cells （ P 〈0.01 ） .Conclusion The expression levels of annexin A3 protein in MDA-MB-231 cells are higher than those in MCF-7 cells.The successful establishment of MDA-MB-231 cells of down-regulating the expressions of annexin A3 stably lays the foundation for furthere researchabout the expression and characteristics of ANXA 3.