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小干扰RNA沉默甲胎蛋白基因对HepG2细胞迁移及侵袭的影响

韩伟 魏丰贤 张有成

韩伟, 魏丰贤, 张有成. 小干扰RNA沉默甲胎蛋白基因对HepG2细胞迁移及侵袭的影响[J]. 临床肝胆病杂志, 2021, 37(8): 1873-1877. DOI: 10.3969/j.issn.1001-5256.2021.08.024
引用本文: 韩伟, 魏丰贤, 张有成. 小干扰RNA沉默甲胎蛋白基因对HepG2细胞迁移及侵袭的影响[J]. 临床肝胆病杂志, 2021, 37(8): 1873-1877. DOI: 10.3969/j.issn.1001-5256.2021.08.024
HAN W, WEI FX, ZHANG YC. Effect of small interfering RNA silencing of the AFP gene on the migration and invasion of hepatocellular carcinoma HepG2 cells[J]. J Clin Hepatol, 2021, 37(8): 1873-1877. DOI: 10.3969/j.issn.1001-5256.2021.08.024
Citation: HAN W, WEI FX, ZHANG YC. Effect of small interfering RNA silencing of the AFP gene on the migration and invasion of hepatocellular carcinoma HepG2 cells[J]. J Clin Hepatol, 2021, 37(8): 1873-1877. DOI: 10.3969/j.issn.1001-5256.2021.08.024

小干扰RNA沉默甲胎蛋白基因对HepG2细胞迁移及侵袭的影响

DOI: 10.3969/j.issn.1001-5256.2021.08.024
基金项目: 

甘肃省青年科技基金计划资助项目 21JR1RA149

甘肃省青年科技基金计划资助项目 21JR1RA161

兰州市科技计划指导项目 2019-ZD-50

兰州市科技计划指导项目 2019-ZD-71

详细信息
    通讯作者:

    张有成, zhangycmd@126.com

  • 中图分类号: R735.7

Effect of small interfering RNA silencing of the AFP gene on the migration and invasion of hepatocellular carcinoma HepG2 cells

Funds: 

Gansu Province Science and Technology Foundation 21JR1RA149

Gansu Province Science and Technology Foundation 21JR1RA161

Lanzhou Science and Technology Plan Guidance Project 2019-ZD-50

Lanzhou Science and Technology Plan Guidance Project 2019-ZD-71

  • 摘要:   目的  研究甲胎蛋白(AFP)对肝癌HepG2细胞迁移和侵袭能力的影响及其机制。  方法  应用合成的靶向沉默AFP小干扰RNA(siRNA)转染肝癌HepG2细胞, 分为空白对照、阴性对照组及AFP siRNA组, 各组细胞干预48 h后, 采用实时定量PCR(qRT-PCR)和ELISA法检测细胞转染后的沉默效率, Transwell小室实验检测沉默AFP基因后HepG2细胞的侵袭、迁移能力, 用Western blotting法观察沉默AFP基因的表达对上皮-间质转化(EMT)相关蛋白(N-cadherin、Vimentin和E-cadherin)、AKT和p-AKT蛋白表达的影响。计量资料多组间比较采用单因素方差分析, 进一步两两比较采用LSD-t检验。  结果  转染沉默后, 与空白对照组相比, AFP siRNA组中AFP mRNA的相对表达量明显降低(P<0.01), 抑制率达86.440%, 同时AFP siRNA组细胞上清液中AFP蛋白表达水平同样明显降低(P<0.01)。与空白对照组相比, AFP siRNA组的迁移细胞数和侵袭细胞数显著下降, 差异具有统计学意义(P值均<0.01)。沉默HepG2细胞中AFP基因的表达后, 与空白对照组比较, AFP siRNA组EMT相关蛋白E-cadherin蛋白的表达水平升高(P<0.01), 而N-cadherin及Vimentin表达水平均明显降低(P值均<0.05), 且PI3K/AKT信号通路相关蛋白p-AKT的表达水平明显降低(P<0.01)。  结论  沉默AFP可抑制肝癌细胞转移, 基于HepG2细胞株的机制研究可能与阻断PI3K/Akt通路抑制EMT相关。

     

  • 图  1  HepG2细胞红色荧光标记寡核苷酸检测转染效果图(×200)

    注: a, 普通光镜视野下所见; b, 荧光显微镜同一视野下所见。

    图  2  沉默AFP对肝癌HepG2细胞纵向迁移和侵袭能力的影响

    图  3  Western blotting法检测各组细胞中AFP及EMT相关蛋白表达水平

    图  4  AKT和p-AKT在蛋白水平的表达

    表  1  沉默AFP在mRNA、蛋白水平表达以及肝癌HepG2细胞穿膜细胞率的变化

    方法 空白对照组 阴性对照组 AFP siRNA组 F P
    AFP mRNA 1 0.927±0.041 0.136±0.0531) 466.70 <0.01
    AFP(ng/ml) 235.500±19.340 217.400±11.750 33.930±11.6901) 172.60 <0.01
    迁移实验(%) 100.000±7.420 93.660±6.140 58.570±10.4301) 36.92 <0.01
    侵袭实验(%) 100.000±11.320 89.430±8.800 53.420±10.8101) 27.75 <0.01
      注: 与空白对照组比较, 1)P<0.05。
    下载: 导出CSV

    表  2  各组细胞AFP、N-cadherin、Vimentin、E-cadherin、p-AKT蛋白相对表达量统计分析

    组别 AFP E-cadherin Vimentin N-cadherin p-AKT
    空白对照组 0.106±0.009 0.223±0.022 0.519±0.028 0.408±0.045 0.200±0.019
    阴性对照组 0.103±0.011 0.222±0.014 0.430±0.063 0.355±0.062 0.194±0.010
    AFP SiRNA组 0.015±0.0041) 0.375±0.0261) 0.151±0.0472) 0.102±0.0391) 0.116±0.008
    F 92.17 49.74 32.45 46.97 37.00
    P <0.01 <0.01 <0.05 <0.01 <0.01
      注: 与空白对照组比较, 1)P<0.01, 2)P<0.05。
    下载: 导出CSV
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  • 收稿日期:  2020-12-30
  • 修回日期:  2021-02-03
  • 刊出日期:  2021-08-16
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