hAFP及hTERT双启动子调控的针对人IGF-II基因的siRNA特异性抑制人肝癌细胞生长

Human IGF-II siRNA driven by hAFP /hTERT dual promoters specifically suppresses growth of human hepatocellular carcinoma cells

目的:设计并筛选高效沉默胰岛素样生长因子II(IGF-II)基因的小干扰RNA(siRNA)序列,构建由重组人甲胎蛋白(hAFP)和人端粒酶逆转录酶(hTERT)双启动子调控的该siRNA表达载体,观察其对肝癌细胞生长的影响。方法:根据siRNA设计原则,参照IGF-II mRNA序列设计3对siRNA序列及1对阴性对照序列,转染人肝癌Huh7细胞,转染24 h后采用实时荧光定量PCR检测IGF-II mRNA表达量变化,筛选干扰效率最高的siRNA序列。采用PCR扩增出hAFP及hTERT启动子的核心序列,应用基因重组技术构建重组hAFP和hTERT双启动子调控的该siRNA表达载体。将上述siRNA表达载体转染Huh7细胞及L-02人正常肝细胞,观察IGF-II mRNA表达及细胞生长情况变化。结果:实时荧光定量PCR显示,siRNA3在25 nmol/L浓度时抑制效率最高,约90%。成功扩增hAFP及hTERT启动子核心序列,将其分别克隆入pGL3-Basic载体,构建成重组pGL3-hAFP-hTERT载体;将siRNA3克隆至pGL3-hAFP-hTERT载体,构建成重组pGL3-hAFP-hTERT-siRNA3表达载体。Huh7细胞IGF-II mRNA表达量显著降低,抑制效率达86%,细胞增殖受到明显抑制,G1期细胞比例显著增加;L-02细胞上述指标无明显改变。结论:成功构建双重RNA聚合酶II启动子(hAFP及hTERT双启动子)调控的针对IGF-II基因的siRNA表达载体,即pGL3-hAFP-hTERT-siRNA3;该siRNA表达载体可特异性抑制IGF-II mRNA表达及肝癌细胞生长。

AIM： To design and screen the most effective siRNA targeting human insulin-like growth factor II （IGF-H） gene, to construct an siRNA expression vector driven by hAFP （human alpha-fetoprotein）/hTERT （human te- lomerase reverse transcriptase） dual promoters, and to observe the effect of the siRNA on the growth of human hepatocellu- lar carcinoma ceils. METHODS： Three siRNAs targeting IGF-H gene and one negative control siRNA were designed and synthesized. They were transfected into human hepatocellular carcinoma Huh？ cells. The expression IGF-II mRNA in Huh7 cells was detected by real-time fluorescence quantitative PCR 24 h after transfection to screen the most effective siRNA. The core sequences of hAFP and hTERT promoters and the most effective siRNA were cloned into pGL3-basic vector to con- struct the siRNA expression vector driven by hAFP and hTERT promoters. The siRNA expression vector was transfected into Huh7 cells and human normal liver L-02 cells, and then the IGF-II mRNA expression in these cells and the cell growth were evaluated. RESULTS： The inhibitory effect of siRNA3 on IGF-II mRNA expression was the strongest at the concentration of 25 nmol/L, with an inhibitory rate of approximately 90%. The core sequence fragments of hAFP and hTERT promoters and siRNA3 were successfully cloned into pGL3-basic vector. The expression of IGF-II mRNA in the transfected Huh7 cells was reduced by approximately 86%, and proliferation inhibition and G^-phase arrest of the cells were also ob- served. However, there were no differences of the above parameters in L-02 cells before and after transfection. CONCLU- SION： The siRNA expression vector targeting human IGF-H gene driven by hAFP/hTERT dual promoters was successfully constructed. The siRNA3 expression vector may specifically suppress the expression of IGF-II mRNA and the growth of hepatocellular carcinoma cells. [