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靶向Skp2基因siRNA对大肠癌细胞生物学特性的影响 被引量:1

Influence of Skp2-targeted siRNA on characteristics of human colorectal cancer cell line
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摘要 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.
出处 《世界华人消化杂志》 CAS 北大核心 2009年第8期775-779,共5页 World Chinese Journal of Digestology
关键词 SKP2基因 肠肿瘤 RNA干扰 基因表达 Skp2 gene Intestinal neoplasms RNAinterference Gene expression
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