特异性的shRNA抑制c－Jun蛋白表达对HepG2细胞增殖和迁移的影响  

Effects of c-Jun shRNA on the proliferation and migration of HepG2 cells

作　　者：罗文雅[1] 周凯[1] 胡磊[1] 李向阳[1] 孔凡运[1] 尤红娟[1] 汤仁仙[1] 

机构地区：[1]徐州医学院病原生物学与免疫学教研室感染与免疫实验室,江苏徐州221004

出　　处：《徐州医学院学报》2015年第10期648-653,共6页Acta Academiae Medicinae Xuzhou

基　　金：江苏省脑病生物信息重点实验室开放研究课题（Jsb11401）：江苏高校优势学科建设工程（2014）;徐州医学院科研课题（2012KJ05）;徐州市科技局研究课题（XZZD1330）

摘　　要：目的：构建c－Jun特异性的shRNA及研究其抑制c－Jun蛋白表达对人类肝癌细胞HepG2增殖和迁移的影响。方法根据c－Jun编码序列设计并构建shRNA质粒，利用LipofectamineTM 2000转染HepG2细胞，荧光显微镜观察转染效率。 Western blot检测其对c－Jun蛋白表达的影响。 CCK－8增殖实验、平板克隆形成实验观察HepG2细胞的增殖情况，transwell和划痕愈合实验方法观察细胞的迁移情况。结果构建了c－Jun小干扰质粒shRNA－1和shRNA－2。荧光显微镜观察到shRNA质粒在HepG2细胞转染效率约在80％。 Western blot显示shRNA－2小干扰质粒对c－Jun抑制效果要强于shRNA－1小干扰质粒。功能实验结果显示，与阴性对照组相比，shRNA－2能显著抑制HepG2细胞的增殖、克隆形成及其迁移能力（P均＜0．05）。结论成功构建了对c－Jun有效shRNA干扰质粒，下调c－Jun的表达能够明显抑制肝癌细胞的增殖和迁移，为进一步研究c－Jun在肝癌中的作用及相关机制提供了工作基础。Objective To construct specific short hairpin RNA （ shRNA） against c-Jun and explore its effects on the proliferation and migration of HepG2 cells.Methods According to the coding sequence of c-Jun, two shRNA plas-mids were designed and transfected into HepG2 cells by lipofectamineTM 2000.The transfection efficiency was observed by fluorescence microscopy.The effects of shRNA on the level of c-Jun were measured by Western blot.The proliferation of the cells was tested by CCK-8 assay and colony formation assay.The migration of the cells was determined by tran-swell assay and wound healing assay.Results Two shRNA plasmids were constructed （ named shRNA-1 and shRNA-2）, and their transfection efficiency was 80%by fluorescence microscopy.According to Western blot analysis, shRNA-2 produced stronger inhibitory effects against c-Jun than shRNA-2 （P〈0.05）.Compared with the negative control, shRNA-2 could remarkably block the proliferation, colony formation and migration of HepG2 cells （P〈0.05）.Con-clusion The shRNA plasmid against c-Jun is successfully constructed, which can markedly inhibit the proliferation and migration of HepG2 cells, providing experimental evidence for further research on the function and related mecha-nisms of c-Jun in liver cancer.

关 键 词：RNA小干扰 HEPG2细胞 增殖 迁移 

分 类 号：R73-351[医药卫生—肿瘤]