腺苷脱氨酶ADAR1调控肺腺癌细胞放射敏感性的研究

陈才; 杨文娣; 陈柯宏; 张雅倩; 曾洪; 彭媛; 张晓月; 杨镇洲

doi:10.16016/j.2097-0927.202309052

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腺苷脱氨酶ADAR1调控肺腺癌细胞放射敏感性的研究

基础医学 | 更新时间：2026-01-09

- 腺苷脱氨酶ADAR1调控肺腺癌细胞放射敏感性的研究
- Adenosine deaminase acting on RNA-1 regulates the radiosensitivity of lung adenocarcinoma cells
- 陆军军医大学学报 2024年46卷第12期页码：1378-1386
- 作者机构：
  
  400000 重庆，重庆医科大学附属第二医院肿瘤中心
- 作者简介：
  
  [杨镇洲，E-mail：yangzz@cqmu.edu.cnYANG Zhenzhou, E-mail: yangzz@cqmu.edu.cn
- 基金信息：
  
  国家自然科学基金(82273572);重庆市自然科学基金(CSTC2021jscx-gksb-N0022)
- DOI：10.16016/j.2097-0927.202309052
  中图分类号： R394-33;R730.55;R734.2
- 收稿：2023-09-11，
  
  修回：2023-11-03，
  
  纸质出版：2024-06-30
- 稿件说明：
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陈才, 杨文娣, 陈柯宏, 等. 腺苷脱氨酶ADAR1调控肺腺癌细胞放射敏感性的研究[J]. 陆军军医大学学报, 2024,46(12):1378-1386.

Cai CHEN, Wendi YANG, Kehong CHEN, et al. Adenosine deaminase acting on RNA-1 regulates the radiosensitivity of lung adenocarcinoma cells[J]. Journal of Army Medical University, 2024, 46(12): 1378-1386.
陈才, 杨文娣, 陈柯宏, 等. 腺苷脱氨酶ADAR1调控肺腺癌细胞放射敏感性的研究[J]. 陆军军医大学学报, 2024,46(12):1378-1386. DOI： 10.16016/j.2097-0927.202309052.

Cai CHEN, Wendi YANG, Kehong CHEN, et al. Adenosine deaminase acting on RNA-1 regulates the radiosensitivity of lung adenocarcinoma cells[J]. Journal of Army Medical University, 2024, 46(12): 1378-1386. DOI： 10.16016/j.2097-0927.202309052.

摘要

目的

研究下调ADAR1基因表达对肺腺癌细胞放射敏感性的影响。

方法

慢病毒转染下调肺腺癌细胞A549的ADAR1基因

分别设阴性对照组(shNC)和ADAR1敲降组(shADAR1)

同时以单次剂量0 Gy和6 Gy X射线照射为干预条件。采用蛋白免疫印迹和RT-qPCR分别检测A549细胞转染后ADAR1的蛋白和mRNA表达水平。CCK-8实验、伤口愈合实验及Transwell迁移实验检测细胞增殖、迁移能力。克隆形成实验检测下调ADAR1对A549细胞放射敏感性的影响。流式细胞术、蛋白免疫印迹实验检测细胞凋亡及相关蛋白Bax和Bcl-2的表达水平。细胞免疫荧光、蛋白免疫印迹实验检测γ-H2AX的表达水平。彗星实验检测细胞DNA损伤程度。4~6周、体质量16~18 g雌性裸鼠12只

用随机数表法分为(

=3): shNC组、shADAR1组、阴性对照联合电离辐射(Ionizing radiation

IR)组(shNC+IR)和ADAR1敲低联合IR组(shADAR1+IR)

通过皮下成瘤实验检测不同组细胞在体内生长情况。

结果

蛋白免疫印迹和RT-qPCR实验显示A549 shADAR1细胞ADAR1的蛋白及mRNA表达量均明显降低(

0.05)。CCK-8实验、伤口愈合实验及Transwell迁移实验结果显示下调ADAR1抑制A549细胞的增殖、迁移能力

IR后这种抑制趋势更加明显(

0.01)。细胞克隆形成实验结果显示随着辐射剂量增加

两组的克隆形成率均有下降

但shADAR1组形成的克隆数低于shNC组。流式细胞术及蛋白免疫印迹实验结果显示下调ADAR1增加A549细胞凋亡率、Bax蛋白表达(

0.01)

减少Bcl-2蛋白表达(

0.05)

IR后A549 shADAR1细胞凋亡率、Bax蛋白水平进一步升高(

0.01)

Bcl-2蛋白水平进一步降低(

0.01)。A549 shADAR1细胞经IR后γ-H2AX foci数目及蛋白表达量明显升高(

0.05)

彗星实验结果显示A549 shADAR1细胞经IR后DNA损伤更加明显(

0.01)。裸鼠皮下成瘤实验结果显示A549 shADAR1细胞皮下成瘤经IR后其生长受到明显抑制(

0.01)。

结论

下调ADAR1可显著抑制IR后A549细胞的增殖、迁移

促进细胞凋亡及DNA损伤

从而增加肺腺癌细胞的放射敏感性。

Abstract

Objective

To investigate the effect of down-regulating adenosine deaminase acting on RNA-1(ADAR1) on the radiosensitivity of lung adenocarcinoma cells.

Methods

Lentiviral transfection was used to establish an ADAR1 knockdown cell line based on A549 cells.Then the cells were divided into negative control (shNC) and ADAR1 knockdown (shADAR1) groups

which were followed by a single-dose irradiation of 0 Gy and 6 Gy X-rays.Western blotting and RT-PCR were utilized to detect the expression of ADAR1 at protein and mRNA levels

respectively.CCK-8 assay

wound healing assay and Transwell migration assay were applied to measure cell proliferation and migration abilities.Meanwhile

clone formation assay was performed to detect the effect of down-regulating ADAR1 on the radiosensitivity of A549 cells.Flow cytometry and Western blotting were conducted to detect the expression levels of apoptosis and apoptosis-related proteins Bax and Bcl-2.Immunofluorescence assay and Western blotting were used to detect the expression level of γ-H2AX.Comet assay was performed to detect the level of cellular DNA damage.Twelve female nude mice (4~6 weeks old

weighing 16~18 g) were divided into shNC group

shADAR1 group

shNC+ionizing radiation (IR) group and shADAR1+IR group

with 3 mice in each group.The growth of tumor of different groups was observed with subcutaneous tumorigenesis assay.

Results

Western blotting and RT-qPCR showed that the protein and mRNA expression of ADAR1 were significantly reduced in A549 shADAR1 cells (

0.05).CCK-8 assay

wound healing assay and Transwell migration assay indicated that down-regulation of ADAR1 inhibited the proliferation and migration abilities of A549 cells

and this inhibition trend became more obvious (

0.01) after IR.Cell clone formation assay showed that the clone formation rate of both groups was decreased

with the increase of radiation dose.But the number of formed clones was lower in the shADAR1 group than the shNC group.Flow cytometry and Western blotting displayed that down-regulation of ADAR1 increased the apoptotic rate and Bax expression in A549 cells (

0.01) and decreased Bcl-2 expression (

0.05)

and the apoptotic rate and Bax protein level were further increased in A549 shADAR1 cells after IR (

0.01)

and the Bcl-2 protein level was further decreased (

0.01).The number of γ-H2AX foci and protein level in A549 shADAR1 cells were significantly

increased after IR (

0.05)

and the results of comet assay showed that the DNA damage was more obvious in A549 shADAR1 cells after IR (

0.01).Subcutaneous tumorigenesis assay in nude mice showed that the growth of subcutaneous tumour of A549 shADAR1 cells was significantly inhibited after IR (

0.01).

Conclusion

Down-regulation of ADAR1 significantly inhibits the proliferation and migration of A549 cells after IR and promotes apoptosis and DNA damage

and thereby increases the radiosensitivity of lung adenocarcinoma cells.

关键词

Keywords

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