稳定干扰AGR2乳腺癌细胞系的建立及其功能  

Function of AGR2 by construction of cell line with stable interference AGR2

作　　者：邓鸣涛[1] 李飞飞[1] 吕忠显[1] 

机构地区：[1]厦门大学生物医学研究院,福建厦门361005

出　　处：《南昌大学学报（理科版）》2011年第4期389-392,共4页Journal of Nanchang University(Natural Science)

摘　　要：将AGR2基因的shRNA表达序列连接到pSUPER质粒中,获得pSUPER-shAGR2质粒,经测序鉴定后,将pSUPER-shAGR2和pSUPER质粒通过Lipofectamine2000转染MCF7细胞,经流式细胞仪和600μg.mL-1G418筛选获得稳定干扰AGR2的MCF7细胞系和对照细胞系,Western blot检测其RNA干扰效果,MTT检测细胞生长情况;用无血清酚红培养基饥饿12 h后,25 ng.mL-1EGF处理15 min,Western blot检测ERK活化和PARP水平。测序结果证实AGR2shRNA核苷酸链序列插入正确,RNA干扰能有效降低MCF7细胞中AGR2的蛋白表达水平;稳定干扰AGR2的MCF7细胞系,细胞生长缓慢,ERK活化水平显著降低,而凋亡核心蛋白PARP也明显上调。稳定干扰AGR2的MCF7细胞系成功建立,发现RNA干扰AGR2抑制其生长和促进其凋亡,为AGR2作为乳腺癌预后与治疗研究靶点提供更充分的理论依据。AGR2（Anterior Gradient-2） shRNA was inserted to the pSUPER plasmid to obtain a construct pSUPER-shAGR2.The positive clones were selected and further confirmed by DNA sequencing.MCF7 cells were transfected with pSUPER-shAGR2 or pSUPER plasmid using Lipofectamine2000.Then the cell lines with stable AGR2 interference and the control cell lines were selected by flow cytometry and G418（600μg/ml）.The interference efficiency was assessed with Western blotting.MTT assay was also performed to evaluate the proliferation of cells after selection.Cells were cultured with both serum-free and phenol red-free medium for 12 hours,before being treated with EGF（25 ng·mL-1） for 15 minute.The level of ERK phosphorylation and PARP were detected by Western blotting.DNA sequencing results showed interference sequence was correct.RNA interference could effectively reduce AGR2 expression level.Stableinterference of AGR2 cell line was grown slowly and the phosphorylation levels of ERK significantly decreased.The core apoptotic protein PARP increased in AGR2 knockdown cells.Therefore,breast cancer cell line with stable AGR2 interference was established successfully.AGR2 plays a very important role in MCF7 cell grow and apoptosis.Taken together,this finding may provide new theoretical basis for AGR2 acting as a target for prognosis and treatment of breast cancer.

关 键 词：AGR2 乳腺癌 凋亡 

分 类 号：R593[医药卫生—内科学]