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

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

兰州大学第二医院普通外科, 兰州 730030

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)

利益冲突声明: 本研究不存在研究者、伦理委员会成员、受试者监护人以及与公开研究成果有关的利益冲突。

作者贡献声明: 韩伟负责课题设计, 基础实验, 资料分析, 撰写论文; 魏丰贤参与基础实验, 修改论文; 张有成负责拟定课题设计和写作思路, 指导撰写文章并最后定稿。

详细信息

通信作者:
张有成, zhangycmd@126.com

中图分类号: R735.7
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出版历程
- 收稿日期: 2020-12-30
- 录用日期: 2021-02-03
- 出版日期: 2021-08-20

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

Department of General Surgery, Lanzhou University Second Hospital, Lanzhou 730030, China

Research funding:

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

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。
- 癌, 肝细胞 /
- 甲胎蛋白类 /
- 上皮-间质转化
Abstract: Objective To investigate the effect of alpha-fetoprotein (AFP) on the migration and invasion abilities of hepatocellular carcinoma (HCC) HepG2 cells. Methods HCC HepG2 cells were transfected with synthesized small interference RNA (siRNA) for targeted silencing of AFP, and then HepG2 cells were divided into blank control group, negative control group, and AFP siRNA group. After 48 hours of intervention, quantitative real-time PCR and ELISA were used to measure the efficiency of silencing after transfection; Transwell assay was used to analyze the invasion and migration abilities of HepG2 cells after silencing of the AFP gene; Western blot was used to investigate the effect of AFP silencing on the expression of epithelial-mesenchymal transition (EMT)-related proteins (N-cadherin, vimentin, and E-cadherin), AKT, and p-AKT. A one-way analysis of variance was used for comparison of continuous data between groups, and the LSD-t-test was used for further comparison between two groups. Results After transfection and silencing, compared with the blank control group, the AFP siRNA group had a significant reduction in the relative mRNA expression of AFP (P < 0.01), with an inhibition rate of 86.440%, and the AFP siRNA group also had a significant reduction in the protein expression level of AFP in cell supernatant (P < 0.01). Compared with the blank control group, the AFP siRNA group had significant reductions in the numbers of migrating and invading cells (both P < 0.01). After the expression of the AFP gene was silenced in HepG2 cells, compared with the blank control group, the AFP siRNA group had a significant increase in the expression level of the EMT-related protein E-cadherin (P < 0.01) and significant reductions in the expression levels of the EMT-related proteins N-cadherin and vimentin (both P < 0.05), as well as a significant reduction in the protein expression level of p-AKT involved in the PI3K/AKT signaling pathway (P < 0.01). Conclusion Silencing AFP can inhibit the migration of HepG2 cells, possibly by blocking the PI3K/AKT signaling pathway and inhibiting EMT based on the mechanism of action of HepG2 cell line.
- Carcinoma, Hepatocellular /
- alpha-Fetoproteins /
- Epithelial-Mesenchymal Transition

HTML全文

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。

下载: 全尺寸图片幻灯片

图 2 沉默AFP对肝癌HepG2细胞纵向迁移和侵袭能力的影响

下载: 全尺寸图片幻灯片

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

下载: 全尺寸图片幻灯片

图 4 AKT和p-AKT在蛋白水平的表达

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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值	P值
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值	92.17	49.74	32.45	46.97	37.00
P值	＜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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