Construction of recombinant lentivirus vector containing retinoic acid-induced gene G and its effect on human lung cancer A549 cell line

吴军录; 权文强; 姚懿雯

doi:10.3969/j.issn.1007-3969.2015.08.002

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Construction of recombinant lentivirus vector containing retinoic acid-induced gene G and its effect on human lung cancer A549 cell line

更新时间：2025-12-31

- Construction of recombinant lentivirus vector containing retinoic acid-induced gene G and its effect on human lung cancer A549 cell line
- China Oncology Vol. 25, Issue 8, Pages: 566-571(2015)
- 作者机构：
  
  同济大学附属同济医院检验科,上海,200065
- 作者简介：
- 基金信息：
- DOI：10.3969/j.issn.1007-3969.2015.08.002
  CLC：
- Published Online：14 December 2015，
  
  Published：14 December 2015
- 稿件说明：
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吴军录,权文强,姚懿雯,等. 维甲酸诱导基因G慢病毒表达载体的构建及对肺癌细胞A549的影响[J]. 中国癌症杂志, 2015, 25(8): 566-571.

吴军录, 权文强, 姚懿雯. Construction of recombinant lentivirus vector containing retinoic acid-induced gene G and its effect on human lung cancer A549 cell line[J]. China Oncology, 2015, 25(8): 566-571.
吴军录,权文强,姚懿雯,等. 维甲酸诱导基因G慢病毒表达载体的构建及对肺癌细胞A549的影响[J]. 中国癌症杂志, 2015, 25(8): 566-571. DOI： 10.3969/j.issn.1007-3969.2015.08.002.

吴军录, 权文强, 姚懿雯. Construction of recombinant lentivirus vector containing retinoic acid-induced gene G and its effect on human lung cancer A549 cell line[J]. China Oncology, 2015, 25(8): 566-571. DOI： 10.3969/j.issn.1007-3969.2015.08.002.

摘要

背景与目的：维甲酸诱导基因G(retinoic acid-induced gene G，RIG-G)是从急性早幼粒细胞性白血病细胞系NB4细胞中克隆出的肿瘤抑制基因。我们通过调控基因(Tet-on)系统构建受强力霉素(doxycycline，DOX)诱导RIG-G基因表达的A549细胞系，并观察其对A549细胞增殖的作用。方法：采用实时定量PCR(quantitative real-time PCR，qRT-PCR)技术扩增RIG-G基因片段，利用LR重组系统构建pLenti6/TO/V5-GIM-RIG-G慢病毒载体，对该慢病毒载体和Tet-on慢病毒载体包装和病毒滴度测定后，感染A549细胞；采用有限稀释法筛选稳定株；使用细胞免疫荧光和蛋白［质］印迹法(Western blot)鉴定RIG-G基因受DOX调控表达的效果；CCK-8试验检测细胞增殖能力。结果：成功构建pLenti6/TO/V5-GIM-RIG-G慢病毒载体，包装后测得其活性滴度为1.0×10

TU/mL；慢病毒经Tet-on包装后物理滴度为4×10

VP/mL；RIG-G蛋白成功地在慢病毒感染后的A549稳定株中合成和表达，并且受DOX的诱导调控。RIG-G蛋白表达成功后，A549细胞的增殖与对照组相比显著降低(1.168±0.107 vs 2.099±0.162，P0.05)。结论：本研究成功建立了RIG-G基因可调控表达的A549稳定株；RIG-G蛋白对A549的增殖有抑制作用。

Abstract

Background and purpose: Retinoic acid-induced gene G (RIG-G) is a tumor suppressor gene which is cloned by NB4 cell line from a acute promyelocytic leukemia cell. This study aimed to investigate the effect of RIG-G in lung cancer cells A549 by constructing a lentiviral vector expressing RIG-G under doxycycline (DOX) regulation. Methods: RIG-G gene amplification was performed by quantitative real-time PCR (qRT-PCR). pLenti6/ TO/V5-GIM-RIG-G lentiviral vector with GFP was built by LR recombination system.

The concentration of pLenti6/ TO/V5-GIM-RIG-G lentiviral vector and Tet-on lentiviral vector were measured by virus titer method. After infecting A549 cells

stably transfected lines were selected via limiting dilution analysis. RIG-G gene expression was examined by immunofluorescence staining and Western blot assay. Cellular proliferation was determined by CCK-8 assay. Results: The concentrations of pLenti6/TO/V5-GIM-RIG-G lentiviral vector and Tet-on lentiviral vector were 1.0×10

TU/mL and 4×10

VP/mL

respectively. RIG-G was expressed in lentivirus infected A549 cells after adding DOX

and the amount of cells with GFP could be observed by fluorescence microscopy. After the expression of RIG-G protein

the proliferation activity of A594 cell was significantly inhibited compared to the control group (1.168±0.107 vs 2.099±0.162

P0.05). Conclusion: The regulated expression of RIG-G gene was established in A549 lung cancer cell line. The RIGG protein has potential abilities to inhibit the proliferation of lung cancer cell A549.

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