摘要
目的构建Peroxiredoxin2(PRDX2)基因RNA干扰(RNA interference,RNAi)慢病毒表达载体,探讨PRDX2基因干扰后对结直肠癌SW480细胞增殖的影响。方法设计、合成靶向PRDX2的RNA干扰的序列,构建pGC-EGFP-shPRDX2慢病毒载体并进行鉴定,同时应用qRT-PCR和Western blot方法观察转染的SW480结肠癌细胞PRDX2 mRNA和蛋白表达的抑制效果,并通过MTT、平板克隆形成实验检测细胞增殖变化。结果成功构建PRDX2基因慢病毒载体并经测序证实;pGCEGFP-shPRDX2可有效抑制结直肠癌SW480细胞PRDX2的表达,感染慢病毒的SW480细胞中PRDX2 mRNA和蛋白表达水平明显降低(P<0.05);SW480细胞经PRDX2 RNA干扰后其生长和增殖能力显著降低(P<0.05)。结论 PRDX2基因RNAi慢病毒表达载体在SW480细胞中表达稳定可靠,PRDX2基因干扰后有效抑制了结直肠癌SW480细胞的增殖和生长,为进一步探讨PRDX2在结直肠癌发生、发展及转移中的作用奠定基础。
Objective To construct a lentiviral expression vector of peroxiredoxin2（PRDX2） RNA interference （RNAi） and to investigate the effect of siRNA of PRDX2 genes on the proliferation of human colonrectal cancer SW480 cell .Methods RNAi target sequences were designed and synthesized towards the PRDX2 gene sequences .The lentiviral vector pGC-EGFP-shPRDX2 was constructed and identified .The vector was transformed into SW480 cells ,and the transfection efficiency was evaluated by fluorescence microscopy .The expression of PRDX2 was detected with Quantitative real-time PCR （qRT-PCR） and Western blot in the transfected cells .Cell growth and colony forming ability were detected with MTT and plate cloning technique .Results PRDX2 gene lentiviral vector was successfully established and was proved by gene sequencing .The expression of PRDX2 in mRNA and protein was significantly reduced（P〈0 .05） .The PRDX2 mRNA and protein expression in SW480 transfected with lentiviral were sig-nificantly reduced （P〈 0 .05） ,and the ability of growth and proliferation were significantly reduced（P〈 0 .05） .Conclusion PRDX2 gene lentiviral vector could be a stable and reliable tool .The proliferation and growth of SW480 cells transfected by pGC-EGFP-shPRDX2 could be effectively suppressed ,which could facilitate further investigation of the roles of PRDX2 gene in the development and progression of colorectal cancer .
出处
《重庆医学》
CAS
北大核心
2015年第1期14-17,20,共5页
Chongqing medicine
基金
国家自然科学基金资助项目(81172295)
