RNAi沉默结肠癌细胞LOVO中livin基因的表达

摘要

Objective: To observe the effects of RNAi - mediated livin gene silencing on colon carcinoma cell lines LOVO. Methods: Two target gene fragments were cloned into pSilencerTM 4.1 - CMV neo vector. Then transfected recombinant vectors into colon carcinoma cells LOVO. The interference effects were detected by RT - PCR and Western blot. The proliferation of LOVO cells were detected by MTT method. Colony formation of LOVO cells transfected with test plasmid were assayed by soft agar method. Results: Two recombinant eukaryotic expression vectors: pSi-Iencer4. 1 - LI and pSilencer4. 1 - L2 were constructed successfully. The results of RT - PCR and Western blot indicated that pSilencer4. 1 - LI vectors could knock down the transcription and expression of livin gene. After LOVO cells were transfected with pSilencer4. 1 - L1 vectors, the proliferation of LOVO cells become slowly and the cell number decreased about 30% , colony formation of LOVO cells were decreased about 70% compare with control groups. Conclusion: The study provided some material for study of the function of livin gene and indicate the livin may be a new target of gene therapy on colon carcinoma.%目的:运用RNA干扰(RNA interference,RNAi)技术阻断结肠癌细胞系LOVO中livin基因的表达,并研究livin基因沉默后对LOVO细胞增殖和克隆形成产生的影响.方法:用真核转录载体pSilencerTM4.1-CMV neo构建针对livin基因的重组RNAi真核转录载体pSilencer4.1-L1和pSilencer4.1-L2,脂质体法转染结肠癌细胞系LOVO,通过RT-PCR、免疫印迹实验检测livin的表达变化,并用克隆形成实验、MTT法检测转染后LOVO细胞在细胞增殖、克隆形成等方面的变化.结果:重组载体pSilencer4.1-L1有效地阻断了LOVO细胞中livin基因在mRNA和蛋白水平上的表达(P<0.01).pSilencer4.1-L1转染LOVO细胞后,与对照组相比细胞生长速度明显变慢,其细胞数在72h时与对照组相比减少约30%;克隆形成率仅为15%,与对照组相比下降了约70%.结论:成功构建了可有效沉默livin基因的RNAi干涉载体,初步证明livin基因在结肠癌细胞的分化增殖等方面所起的重要作用,为进一步阐明livin基因与结肠癌的关系以及以livin基因为靶点的结肠癌基因治疗研究奠定了基础.