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成纤维细胞生长因子13通过ROS/Caspase-3通路调控非小细胞肺癌A549细胞的凋亡

刘天宇 唐铖铖 冯光 雷静静 孙晨皓 王令 路宏朝

刘天宇, 唐铖铖, 冯光, 雷静静, 孙晨皓, 王令, 路宏朝. 成纤维细胞生长因子13通过ROS/Caspase-3通路调控非小细胞肺癌A549细胞的凋亡[J]. 中国肿瘤生物治疗杂志, 2021, 28(5): 451-459. doi: 10.3872/j.issn.1007-385x.2021.05.005
引用本文: 刘天宇, 唐铖铖, 冯光, 雷静静, 孙晨皓, 王令, 路宏朝. 成纤维细胞生长因子13通过ROS/Caspase-3通路调控非小细胞肺癌A549细胞的凋亡[J]. 中国肿瘤生物治疗杂志, 2021, 28(5): 451-459. doi: 10.3872/j.issn.1007-385x.2021.05.005
LIU Tianyu, TANG Chengcheng, FENG Guang, LEI Jingjing, SUN Chenhao, WANG Ling, LU Hongzhao. Fibroblast growth factor 13 regulates apoptosis of A549 cells through the ROS/Caspase-3 pathway[J]. Chin J Cancer Biother, 2021, 28(5): 451-459. doi: 10.3872/j.issn.1007-385x.2021.05.005
Citation: LIU Tianyu, TANG Chengcheng, FENG Guang, LEI Jingjing, SUN Chenhao, WANG Ling, LU Hongzhao. Fibroblast growth factor 13 regulates apoptosis of A549 cells through the ROS/Caspase-3 pathway[J]. Chin J Cancer Biother, 2021, 28(5): 451-459. doi: 10.3872/j.issn.1007-385x.2021.05.005

成纤维细胞生长因子13通过ROS/Caspase-3通路调控非小细胞肺癌A549细胞的凋亡

doi: 10.3872/j.issn.1007-385x.2021.05.005
基金项目: 陕西省教育厅科技计划资助项目(No. 20JK0570)
详细信息
    作者简介:

    刘天宇(1994–),男,硕士,主要从事细胞工程研究,E-mail:1206141742@qq.com

    通讯作者:

    路宏朝(LU Hongzhao,corresponding author),博士,教授,硕士生导师,主要从事细胞工程研究,E-mail:zl780823@126.com

  • 中图分类号: R734.2; R730.54

Fibroblast growth factor 13 regulates apoptosis of A549 cells through the ROS/Caspase-3 pathway

Funds: Project supported by the Science and Technology Plan Foundation from Department of Education of Shaanxi Province (No. 20JK0570)
  • 摘要:   目的:  探讨成纤维细胞生长因子13(fibroblast growth factor 13,FGF13)对非小细胞肺癌A549细胞活性氧(reactive oxygen species,ROS)的生成和凋亡的影响及其调控机制。  方法:  WB法检测FGF13在人正常肺上皮细胞BEAS-2B和肺癌A549、H460细胞中的本底表达量。采用FGF13过表达载体转染BEAS-2B和A549细胞;设计两组靶向FGF13的shRNA序列,构建慢病毒干扰载体,包装病毒后侵染A549细胞,采用qPCR和WB法检测干扰效果,DCFH-DA探针结合荧光酶标仪分析敲减FGF13对A549细胞内ROS水平的影响,MitoSOX与WB法检测对线粒体ROS水平及烟酰胺腺嘌呤二核苷酸磷酸氧化酶4(nicotinamide adenine dinucleotide phosphate oxidase 4,NOX4)蛋白表达量的影响,Annexin V-FITC-PI双染法检测对细胞凋亡和Caspase-3及Cleaved Caspase-3蛋白表达的影响。  结果:  与BEAS-2B细胞相比,FGF13蛋白在两种肺癌细胞中均高表达(均P<0.05)。成功构建FGF13过表达、低表达的A549细胞系。过表达FGF13后,BEAS-2B和A549细胞内ROS水平显著降低(P<0.05);敲减FGF13表达后,A549细胞内ROS水平显著升高(P<0.05);然而过表达及干扰FGF13对A549细胞内线粒体ROS水平无显著影响,但NOX4蛋白表达量显著下调(P<0.05)及显著上调(P<0.05)。FGF13干扰后A549细胞凋亡率显著升高(P<0.01),Caspase-3及Cleaved Caspase-3蛋白表达量显著上调(P<0.05)。  结论:  FGF13可能通过NOX家族途径调控ROS的生成,并通过ROS/Caspase-3通路调控A549细胞凋亡。
  • 图  1  FGF13表达水平及ROS水平检测

    Figure  1.  Determination of expression level of FGF13 and ROS

    *P<0.05 vs BEAS-2B cells group A, B: Protein expression level of FGF13 in BEAS-2B, A549 and H460 cells; C: Protein expression level of FGF13 in A549 cells transfected with empty vector or pcDNA3.1-FGF13; D: ROS levels in BEAS-2B and A549 cells transfected with empty vector or pcDNA3.1-FGF13

    图  2  重组质粒的鉴定

    Figure  2.  Identification of recombinant plasmid

    A: Electrophoretic profile of colony PCR product; M: Marker; 1-4: pLKO.1-FGF13-shRNA1 positive clones (264 bp); 5: Negative control; 6-9: pLKO.1-FGF13-shRNA2 positive clones(264 bp); 10: Negative control. B: Sequencing results of recombinant plasmid; shRNA-1: pLKO.1-FGF13-shRNA-1; shRNA-2: pLKO.1-FGF13-shRNA-2

    图  3  A549-FGF13-shRNA稳转细胞株的建立及对ROS的影响

    Figure  3.  Establishment of A549-FGF13-shRNA stably transfected cell line and its effect on ROS

    *P<0.05, **P<0.01 vs NC group A: Pictures of positive cells after 48 h of purinomycin screening (×100); Blank: A549 cells; NC: Negative control; shRNA-1:pLKO.1-FGF13-shRNA-1; shRNA-2: pLKO.1-FGF13-shRNA-2; B: qPCR for FGF13 mRNA expression in shRNA-transfected A549 cells; C, D: Protein expression level of FGF13 in shRNA-transfected A549 cells; E: ROS levels in A549-FGF13-shRNA stably transfected cell line

    图  4  FGF13对A549细胞内线粒体ROS水平及NOX4蛋白表达的影响

    Figure  4.  Effects of FGF13 on ROS levels and NOX4 protein expression in A549 cells

    *P<0.05 vs NC group A, B: Protein expression level of NOX4 in BEAS-2B, A549 and H460 cells; C: MitROS levels in A549 cells transfected with empty vector or pcDNA3.1-FGF13,NC or shRNA-1; D, E: Protein expression level of NOX4 in A549 transfected with empty vector or pcDNA3.1-FGF13; F,G: Protein expression level of NOX4 in shRNA-transfected A549 cells; shRNA-1: pLKO.1-FGF13-shRNA-1; NC: Negative control

    图  5  干扰FGF13对A549细胞凋亡的影响

    Figure  5.  The effect of interfering FGF13 on A549 cell apoptosis

    *P<0.05, **P<0.01 vs NC groupA, B: A549 cell apoptosis was determined by Annexin V-FITC-PI staining followed by fluorescence microscope (×100); Phase contrast: Survival cells; Annexin V-FITC: Apoptosis cell in the early; PI: Apoptosis cell in the late; Merge: Dead cells. C-E: Protein expression level of Caspase-3 and Cleaved Caspase-3 in shRNA-transfected A549 cells; NC: Negative control; shRNA-1: pLKO.1-FGF13-shRNA-1

    表  1  shRNA序列

    Table  1.   Sequence of shRNA

    shRNASequence (5’-3’)
    FGF13-shRNA-1-F CCGGGAACAAGCCTGCAGCTCATTTCTCGAGAAATGAGCTGCAGGCTTGTTCTTTTTG
    FGF13-shRNA-1-R AATTCAAAAAGAACAAGCCTGCAGCTCATTTCTCGAGAAATGAGCTGCAGGCTTGTTC
    FGF13-shRNA-2-F CCGGCAAGCTGTACTTGGCAATGAACTCGAGTTCATTGCCAAGTACAGCTTGTTTTTG
    FGF13-shRNA-2-R AATTCAAAAACAAGCTGTACTTGGCAATGAACTCGAGTTCATTGCCAAGTACAGCTTG
    NC-shRNA-F CCGGTACAACAGCCACAACGTCTATCTCGAGATAGACGTTGTGGCTGTTGTATTTTTG
    NC-shRNA-R AATTCAAAAATACAACAGCCACAACGTCTATCTCGAGATAGACGTTGTGGCTGTTGTA
    下载: 导出CSV
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  • 收稿日期:  2020-08-21
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