TALENs介导的Nanog基因表达下调对宫颈癌HeLa细胞恶性行为的影响

The Effect of TALENs-mediated Downregulation Expression of Nanog on Malignant Behavior of Cervical Cancer HeLa Cells

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 study the effect of downregulation expression of Nanog on malignant behavior of cervical cancer HeLa cells.Methods Gene editing tool TALENs was employed to induce downregulation expression of Nanog,and Nanog mutation was evaluated by sequencing.RT-PCR and Western blot was used to detect the mRNA and protein expression level,respectively.Colony-formation assay, Transwell invasion assay,and chemotherapy sensibility assay was carried out to assess the capacity of colony-formation,invasion,and chemoresistance,respectively.Results TALENs successfully induced Nanog mutation and downregulated Nanog expression.Nanog mRNA and protein expression of Nanog-mutated monoclonal HeLa cells downregulated 3times compared to thoses of wild-type HeLa cells（P〈0.05）.Additionally,significant weakened abilities of colonyformation,invasion,and chemoresistance in monoclonal HeLa cells were observed when compared to those of wildtype HeLa cells（P 0.05）.Conclusion Nanog mutation attenuates the malignant behavior of HeLa cells.Importantly,downregulation or silencing of Nanog is promising to be a novel strategy for the treatment of cervical carcinoma.