靶向Skp2基因siRNA对大肠癌细胞生物学特性的影响

Influence of Skp2-targeted siRNA on characteristics of human colorectal cancer cell line

目的:设计和构建Skp2特异性的siRNA真核表达载体,观察Skp2siRNA重组体对人大肠癌HCT116细胞株生物学特性的影响.方法:人工合成Skp2基因干扰序列并定向插入到RNAi真核表达载体pRNAT-U6.1/Neo,通过测序鉴定.用脂质体介导的基因转染方法转入HCT116细胞,经G418筛选出稳定转染的细胞系,半定量RT-PCR方法检测靶基因的表达.流式细胞技术分析细胞周期分布,MTT比色法检测细胞的生长抑制率.结果:测序表明Skp2干扰序列完全正确.RT-PCR结果显示2条Skp2siRNA真核表达质粒均能有效抑制核干细胞因子mRNA的转录表达(P<0.05).MTT比色法检测显示,与阴性对照组及未转染组细胞比较,干扰组细胞增殖率明显下降(29.21%±1.40%,30.10%±1.50%vs49.05%±4.50%,53.03%±4.92%,均P<0.05).流式细胞术检测结果显示转染后G0/G1期细胞数占生长周期细胞数的比例增多,即表现为G1期阻滞,S期细胞的比例降低.结论:成功构建了Skp2干扰真核表达载体,siRNA重组体能有效抑制人大肠癌细胞Skp2mRNA表达,细胞增殖能力减弱,为大肠癌基因治疗的可行性提供了有力的证据.

AIM： To design and construct the RNAi eukaryotic vector of Skp2 gene and to study its interfering effect on characteristics of human colorectal carcinoma cell line HCT116. METHODS： DNA oligonucleotide for small in- terfering RNA expression targeting Skp2 gene was synthesized and inserted into pRNAT-U6.1/ Neo plasmid after annealing. The inserted sequences were verified by DNA sequencing, and transfected into HCT116 cells with liposome-mediated transfection. The positive transfected cell clones were screened with G418 and the expression of Skp2 mRNA was detected by semi- quantitive RT-PCR. Distribution of cell cycle was assessed by flow cytometry. The cell growth suppression was analyzed by MTT assay. RESULTS： The sequence of specific siRNA was correct by sequence analysis. Compared with the negative control cells, RT-PCR showed the expression of Skp2 mRNA was down-regulated in the transfected cells （P 〈 0.05）. The subcloned recombinant plasmid expressing shRNA effec- tively inhibited HCT116 cell growth and prolif- eration while empty plasmid had no such specific effect. Decreased cellular proliferation activity was observed by MTT （29.21%±1.40%, 30.10%±1.50% vs 49.05% ± 4.50%, 53.03% ± 4.92%, all P 〈 0.05）. Flow cytometry revealed significant G1 ar- rest in cell cycle. CONCLUSION： The siRNA eukaryotic expression vector of Skp2 gene was constructed successfully which effectively down-regulate the expression of Skp2 in HCT116 cell line, and inhibit the cellular proliferation. It provides a powerful evidence for colorectal carcinoma gene therapy.