Alu RNA及其突变体真核表达质粒构建以及对A549细胞活性氧簇产生的影响
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国家自然科学基金(81702733);肿瘤生物学国家重点实验室项目(CBSKL2015Z14,CBSKL201713);陕西省科技统筹创新工程(2016KTCL03-09)


Construction of eukaryotic expression plasmids for Alu RNA and its mutants and the effect on ROS production in A549 cells
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    目的:从氧化应激的A549细胞鉴定Alu RNA表达,构建Alu RNA及其2种功能缺失突变体的真核表达质粒,探讨过表达Alu RNA及其突变体对细胞氧化应激的影响。方法:设计Alu RNA特异性引物,H2O2应激A549细胞,RT-PCR扩增目的片段,通过定向克隆、点突变等方法构建pcDNA3.0-Alu(pAlu)重组质粒、右臂缺失突变体pcDNA3.0-Alu-left arm(pAlu-LA)、G25C突变体pcDNA3.0-Alu G25C(pAlu-M);转染A549细胞,DCFH-DA检测活性氧簇(reactive oxygen species,ROS)产生水平。结果:从应激的A549细胞成功扩增了Alu分子,重组的真核表达质粒pAlu、pAlu-LA、pAlu-M经酶切及DNA测序鉴定符合预期;pAlu、pAlu-LA转染显著促进A549细胞ROS产生;与pAlu相比,pAlu-M转染诱导产生的ROS显著降低。结论:成功克隆了氧化应激刺激下A549细胞产生的Alu RNA及其2种功能缺失突变体的真核表达质粒,体外实验显示Alu RNA通过翻译水平调控促进A549细胞ROS产生。

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    Objective:To identify Alu RNA expressed in oxidative-stressed A549 cells and to construct eukaryotic expression plasmids for Alu RNA and its two kinds of loss-of-function mutants so as to study the effect on oxidative stress in cells with overexpression of Alu RNA and its mutants. Methods:Alu RNA was amplified with RT-PCR from H2O2 treated A549 cells. pcDNA3.0-Alu(pAlu),pcDNA3.0-Alu-left arm(pAlu-LA)and pcDNA3.0-Alu G25C(pAlu-M)were constructed by directional cloning and point mutation. After recombinant transfection,reactive oxygen species(ROS) produced in transfected A549 cells was detected by DCFH-DA. Results:Alu fragment was successfully amplified from stressed A549 cells. Restriction analysis and DNA sequencing proved that recombinant eukaryotic expression plasmids of pAlu,pAlu-LA and pAlu-M were constructed as expected. pAlu or pAlu-LA transfection promoted ROS production significantly in A549 cells,while pAlu-M transfection induced ROS production was markedly decreased as compared with pAlu transfection. Conclusion:The eukaryotic expression plasmids for Alu RNA and its loss-of-function mutants were successfully constructed from oxidative-stressed A549 cells. Data in vitro suggested that Alu RNA promoted ROS production in A549 cells with translational regulation.

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雷恩宇,毛 壮,纪奇峰,束 毅,王 伟,颜 真. Alu RNA及其突变体真核表达质粒构建以及对A549细胞活性氧簇产生的影响[J].南京医科大学学报(自然科学版),2018,(5):577-581

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  • 收稿日期:2017-09-23
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  • 在线发布日期: 2018-05-16
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