文档介绍：FAK基因表达对人结直肠癌细胞增殖及运动的影响
(作者:___________单位: ___________邮编: ___________)
作者:潘宁, 章蔼然, 牟茂森, 侯颖春
【摘要】目的: 探讨黏着斑激酶(FAK)表达水平对结直肠癌细胞增殖及运动的影响。方法: 针对FAK基因不同靶点设计siRNA序列, 构建siRNA重组子, 转染Caco2细胞, 以RTPCR和免疫细胞化学方法检测FAK mRNA和蛋白表达变化及时间效应, 同时检测FAK基因敲低对Caco2细胞的凋亡、增殖及迁移的影响。结果: FAK siRNA导入Caco2细胞后, FAK mRNA和蛋白表达水平明显下调, 细胞增殖及迁移能力受抑, 呈时间依赖关系, FAK mRNA水平下调在转染后48 h达到最大。结论: FAK siRNA可有效抑制靶基因表达, FAK表达水平下调后Caco2细胞的增殖及运动明显受抑制。
【关键词】小干扰RNA; 结直肠癌; FAK; 细胞增殖; 细胞运动
[Abstract] AIM: To investigate the effect of siRNA targeting FAK gene on proliferation and motility of colorectal cancer. METHODS: binant plasmids that produced siRNAs targeting FAK were designed and cloned, then transfected into Caco
2 cells. The changes of FAK expression levels were examined by RTPCR and immunocytochemistry. The effects of FAK gene knockdown on apoptotic morphological changes, proliferation, and motility were investigated. RESULTS: binant plasmids targeting FAK were essfully constructed, FAK mRNA and protein level was silenced in Caco2 cells significantly. The inhibition of mRNA level was achieved maximal at 48 h post transfection with time dependent. The ability of proliferation and motility of Caco2 cells were significantly decreased. CONCLUSION: Plasmidmediated FAK siRNA could inhibit FAK gene expression. The proliferation, motility and apoptosis were inhibited effectively, which suggested that FAK expression is closely associated with the proliferation, motility and apoptosis, etc. The results may be used as the reference for gene therapy of colorectal cancer.
[Keywords]small interfering RNA; colorectal cancer; FAK; cell proliferation; cell motility
近年来以黏附相关因子为靶点的治疗成为基因治疗研究的新策略[1], 黏着斑激酶(focal adhesion kinase, FAK)是近年来发现的细胞黏附介导的信号通路的重要因子, 作为胞内信号蛋白酪氨酸激酶, 于1992年被独立鉴定为Src癌基因的底物。FAK的相对分子质量(Mr)为125 000, 定位于人染色体8q2
4, 与cmyc基因座接近, 此基因座在人癌细胞中往往被激活而强化[2]。FAK作为信号转导的关键分子, 可传递由胞外到胞内的多种信号, 其在结肠癌中表达升高而在正常组织细胞中表达呈弱阳性的特点开始引起研究人员的关注[3]。有研究显示, FAK基因在结直肠癌组织中表达升高[4], 并与癌细胞增殖、侵袭转移密切相关, 但作用机制还不明确。本实验将靶向FAK基因的siRNA表达