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目的研究Nanog表达下调对宫颈癌HeLa细胞恶性行为的影响。方法通过基因编辑工具转录激活样效应器核酸酶(TALENs)介导Nanog下调表达,基因测序分析Nanog的突变情况;挑取单个细胞培养以筛选出Nanog明显下调表达的单克隆HeLa细胞;RT-PCR检测mRNA表达水平;Western blot检测蛋白表达水平;HeLa细胞的集落形成能力、侵袭性及耐药性分别通过集落形成实验、Transwell侵袭实验及药物敏感性实验检测。结果 TALENs成功介导Nanog基因突变并导致其下调表达,Nanog突变的单克隆HeLa细胞Nanog mRNA和蛋白表达水平较野生型HeLa细胞均下调表达(P<0.05)。Nanog突变单克隆HeLa细胞相对于野生型HeLa细胞表现出明显减弱的侵袭、集落形成及化疗药物抵抗能力(P<0.05)。结论 Nanog突变减弱HeLa细胞的恶性行为,下调或沉默Nanog表达有望成为一种新型的治疗宫颈癌策略。
Objective To investigate the effect of down-regulation of Nanog expression on the malignant behavior of cervical cancer HeLa cells. Methods Transcriptional activation of effector nucleases (TALENs) mediated down-regulation of Nanog by gene editing tools and Nanog mutation were analyzed by gene sequencing. Single-cell culture was used to screen monoclonal HeLa cells with significantly down-regulated Nanog expression. RT-PCR The mRNA expression levels were detected by Western blot. The colony formation ability, invasiveness and drug resistance of HeLa cells were detected by colony formation assay, Transwell invasion assay and drug susceptibility assay respectively. Results TALENs successfully mediated the mutation of Nanog gene and resulted in down-regulated expression of Nanog gene. Nanog mutant HeLa cells showed lower expression of Nanog mRNA and protein than wild-type HeLa cells (P <0.05). Nanog mutant monoclonal HeLa cells showed significantly weaker invasion, colony formation and chemoresistance compared to wild-type HeLa cells (P <0.05). Conclusions Nanog mutation attenuates the malignant behavior of HeLa cells, and down-regulation or silencing of Nanog expression is expected to become a novel strategy for the treatment of cervical cancer.