Kruppel样因子6对肝癌HepG2细胞的作用及其缺失对斑马鱼肝脏发育的影响

摘要

目的 探讨Kruppel样因子6(KLF6)低表达对肝癌细胞凋亡及迁移能力的影响,并以斑马鱼作为动物模型,研究klf6基因沉默对肝脏发育的作用. 方法 构建敲除KLF6基因质粒,转染肝癌细胞(HepG2)及正常人肝细胞(L-02),通过免疫印迹(WB)、凋亡及细胞周期测定、划痕实验检测KLF6基因敲除后对HepG2细胞的影响;设计合成沉默KLF6基因的吗啉代寡核苷酸(Morpholino),显微注射入转基因斑马鱼Tg(lfabp:eGFP)胚胎中,通过免疫荧光法观察KLF6蛋白表达的变化以及对转基因斑马鱼胚胎肝脏发育表型的影响. 结果 体外实验表明,L-02细胞中的KLF6蛋白的表达量明显高于HepG2细胞;敲除KLF6基因后,HepG2细胞KLF6蛋白表达明显减少、凋亡减弱、细胞周期主要集中于S期、迁移能力增强;体内实验表明,klf6基因沉默后,斑马鱼胚胎肝脏中的KLF6蛋白表达量减少,肝脏发育明显迟缓. 结论 KLF6基因低表达促进肝癌细胞的增殖和迁移能力,减少凋亡;且影响斑马鱼肝脏的正常发育.探讨KLF6基因低表达及其功能,可为以后斑马鱼肝癌模型建立及药物筛选提供理论基础.%Objective To investigate the effects of Kruppel-like factor 6 (KLF6) on the apoptotic and migration ability of HepG2 cell, and the developmental role of KLF6 on zebrafish liver.Methods Constructed plasmid with shRNA-KLF6 was transfected in HepG2 and L-02.The impacts of loss of KLF6 on HepG2 was investigated by Western bolt, apoptosis analyses, cell cycle detection and scratch experiment;KLF6 morpholino oligonucleotides was microinjected into the Tg(lfabp:eGFP) transgenic zebrafish embryos.The morphant phenotype of the liver was imaged and the protein expression of KLF6 after knockdown of KLF6 was analyzed by Immunofluorescence staining.Results The expression of KLF6 in L-02 was significantly higher than in HepG2.After knockout of KLF6, KLF6 protein expression and apoptosis were significantly reduced.In addition, the cell cycle mainly stated in S phase and the migration ability of HepG2 was enhanced.After klf6 knockdown in transgenic zebrafish larvae, the development of zebrafish liver was delayed and KLF6 expression was obviously decreased in the liver.Conclusions The reduction of KLF6 expression increased the proliferation and migration ability, and reduced the apoptosis of HepG2.Loss of KLF6 affects the development of zebrafish liver, which may open a possibility to use zebrafish as a liver cancer model and for anti-liver cancer drug screening.