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于爱清, 李成林, 杨 毅等.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.四川大学学报(医学版),2016,47(1):23-27
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
  
中文关键词:  TALENs Nanog HeLa细胞 宫颈癌
英文关键词:TALENs Nanog HeLa cells Cervical cancer
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中文摘要:
      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。
英文摘要:
      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 3 times compared to thoses of wild-type HeLa cells (P<0.05). Additionally, significant weakened abilities of colony-formation, invasion, and chemoresistance in monoclonal HeLa cells were observed when compared to those of wild-type 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.
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