摘要
目的 构建人膜联蛋白A3（annexinA3）基因RNA干扰（miRNA）表达载体,体外研究其对人胆囊癌细胞SGC-996的增殖及凋亡作用.方法 构建4组针对annexinA3的pcDNATM 6.2-GW/EmGFPmiR/miRNA表达载体和1组阴性对照序列.脂质体介导瞬时转染人胆囊癌细胞株SGC-996.用RT-PCR和Western印迹检测转染前后annexinA3基因的表达.挑选干扰效率最强的一组表达载体,以MTT法和流式细胞仪检测经miRNA干扰annexinA3对人胆囊癌细胞SGC-996的体外作用.结果 经测序鉴定,annexinA3-miRNA载体构建成功,转染人胆囊癌细胞SGC-996后荧光定量PCR和westem印迹检测显示：annexinA3基因表达及蛋白表达明显降低（P＜0.05）;干扰后细胞体外增殖缓慢,增殖明显受抑（P＜0.05）.annexinA3基因干扰对细胞周期无明显影响,但能明显增加胆囊癌细胞的凋亡（P＜0.05）.结论 AnnexinA3-miRNA表达载体能有效抑制胆囊癌SGC-996细胞annexinA3的表达,抑制肿瘤细胞生长,引起胆囊癌细胞凋亡.AnnexinA3可能是胆囊癌治疗的一个分子靶点.
Objective To construct a recombinant interfering RNA（miRNA）plasmid vector targeting annexinA3 and observe its impact on the growth and apoptosis of human gallbladder cancer cell line SGC-996.Methods Four small fragments of miRNA and one negative control were sequenced and cloned into vector pcDNATM 6.2-GW/EmGFPmiR.The recombinant plasmids were then transfected into gallbladder cancer cell line SGC-996 by positive liposomal transfection.The transcription and translation level of annexinA3 expression were detected by RT-PCR and Western Blot.Meanwhile, the proliferation and apoptosis of transfected tumor cells were determined by MTT and flow cytometry.Results The recombinant plasmids containing annexinA3 miRNA were successfully constructed.AnnexinA3 expression was significantly knocked down after miRNA plasmid transfection.Transfection of annexinA3 gene miRNA could effectively inhibit the proliferation of gallbladder cancer cell and promote apoptosis.Conclusion AnnexinA3 gene silenced by miRNA can induce the apoptosis of human gallbladder cancer cell line SGC-996.The process might serve as a potential approach for cancer gene therapy.
出处
《中华肝胆外科杂志》
CAS
CSCD
北大核心
2010年第11期853-856,共4页
Chinese Journal of Hepatobiliary Surgery
作者简介
通信作者：吴浩荣，电子信箱：tmjtx2009@163．com
