摘要
目的利用小干扰RNA(small interfering RNA,siRNA)抑制肝癌细胞系中FoxO1基因的表达,观察其受抑制后对细胞增殖能力的影响。方法设计针对FoxO1基因的siRNA,构建于腺病毒穿梭载体pAdTrack-CMV中,与腺病毒骨架质粒pAdeasy-1在BJ5183细菌中进行同源重组,转染包装细胞293细胞,获得含Ad-siRNA-FoxO1的重组腺病毒。体外感染人肝癌细胞系HepG2,Western印迹检测Ad-siRNA-FoxO1对FoxO1蛋白的抑制效率。与PEPCK.luc共转染HepG2细胞,检测FoxO1表达水平对PEPCK启动子活性的影响,并观察FoxO1表达水平的改变对肝癌细胞系增殖的影响。结果成功构建3个针对FoxO1的siR-NA重组腺病毒载体,重组腺病毒均能显著抑制HepG2细胞中FoxO1蛋白的表达,并抑制PEPCK启动子的活性和显著促进细胞增殖。结论肝癌细胞系中FoxO1参与了细胞增殖的调节。
Objective To observe the effect on the proliferation of human liver cancer HepG2 cell line by inhibiting the expression of FoxO1 gene. Methods Small interfering RNA （siRNA） for FoxO1 gene was designed using Ambion software and synthesized and inserted into pAd-Track-CMV vector. The recombinant plasmid pAd-Track-CMV-siRNA-FoxOl was constructed by homologous recombination with adenoviral backbone plasmid pAdeasy-1 in bacteria E. coli BJ5183. The resultant recombinant adenoviral vector Ad-siRNA-FoxO1 was transfected into 293 cells for packaging of re- combinant adenovirus. Ad-siRNA-FoxO1 infection of hepatocarcinoma HepG2 cells showed specific down-regulation of FoxO1 protein as detected by Western blotting. The PEPCK promoter luciferase activity in the cells transfect with Ad-siRNA-FoxO1 was determined, and cell proliferation was analyzed by MTT assay and 3H-TdR incorporation experiment. Results Adenoviral vector expressing small interfering RNA against FoxO1 gene, Ad-siRNA-FoxO1, was successfully constructed and observed to specifically inhibit FoxOl expression. Ad-siRNA-FoxO1 inhibited PEPCK promoter activity and enhanced cell proliferation as compared to si-negative control. Conclusion The Ad-siRNA- FoxOl effectively down-regulates PEPCK promoter activity and enhances cell proliferation in HepG2 cell line, having paved a way for further study on FoxO1 function.
出处
《医学分子生物学杂志》
CAS
CSCD
2008年第6期488-492,共5页
Journal of Medical Molecular Biology
基金
国家自然科学基金(No.30670998)
全军医学科研"十一五"计划国际合作项目(06H025)~~
