缺氧诱导因子1α对肝癌细胞HepG2干细胞特性及表阿霉素敏感性的影响

赵金金; 张海光; 崔非非; 汪磊; 莫清江; 焦路阳

doi:10.3969/j.issn.1001-5256.2021.02.021

缺氧诱导因子1α对肝癌细胞HepG2干细胞特性及表阿霉素敏感性的影响

DOI: 10.3969/j.issn.1001-5256.2021.02.021

a.
新乡医学院第一附属医院检验科, 河南新乡 453100
b.
新乡医学院第一附属医院妇产科, 河南新乡 453100

基金项目:

新乡医学院第一附属医院博士科研项目启动基金 (10243);

河南省医学科技攻关计划(联合共建)项目 (LHGJ20190453)

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

作者贡献声明：赵金金负责课题设计，资料分析，撰写论文；张海光负责实验操作，参与数据分析；崔非非、汪磊参与实验操作；莫清江参与收集数据，修改论文；焦路阳负责拟定写作思路，指导撰写论文并最后定稿。

详细信息

作者简介:
赵金金(1987—)，男，主管技师，博士，主要从事肿瘤耐药及肿瘤干细胞的研究

通信作者:
焦路阳，jiaoluyang2009@163.com

中图分类号: R735.7
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出版历程
- 收稿日期: 2020-08-12
- 录用日期: 2020-10-10
- 出版日期: 2021-02-20

Effect of hypoxia-inducible factor-1α on stemness and epirubicin sensitivity of HepG2 hepatoma cells

a.
Department of Clinical Laboratory, The First Affiliated Hospital of Xinxiang Medical University, Xinxiang, Henan 453100, China
b.
Department of Obstetrics and Gynecology, The First Affiliated Hospital of Xinxiang Medical University, Xinxiang, Henan 453100, China

摘要

摘要: 目的探讨缺氧诱导因子(HIF)1α对肝癌细胞HepG2干细胞特性及表阿霉素敏感性的影响。方法以肝癌细胞为研究对象，肝癌细胞HepG2脂质体转染过表达HIF-1α的质粒作为实验组，转染pcDNA3.1空质粒作为对照组，单独HepG2细胞为HepG2组。实时荧光定量PCR检测HIF-1α mRNA表达，Western Blot检测HIF-1α蛋白表达；流式细胞术检测细胞表面CD133表达。不同浓度表阿霉素(0、6.25、12.5、25、50 μmol/L)作用3组细胞24 h，MTT法检测细胞活性，流式细胞术检测表阿霉素(50 μmol/L)处理后细胞凋亡情况。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t检验。结果相较于HepG2组及对照组，实验组HIF-1α mRNA的表达水平明显升高，差异具有统计学意义(P值均<0.001)；Western Blot结果显示实验组HIF-1α蛋白高表达。HepG2组、对照组和实验组细胞的CD133比例分别为0.040%±0.003%、0.030%±0.010%、20.110%±0.600%，实验组的CD133阳性率显著高于HepG2组和对照组(P值均<0.001)。表阿霉素浓度为25、50 μmol/L时，HepG2组和对照组的细胞活性明显受到抑制，显著低于实验组(P值均<0.05)。50 μmol/L表阿霉素作用48 h后，实验组的细胞凋亡率(67.9%±2.5%)较HepG2组(93.6%±1.5%)和对照组(93.0%±1.2%)明显降低(P值均<0.001)。结论过表达HIF-1α的质粒成功转染至HepG2细胞，HIF-1α可提高肝癌细胞干细胞比例使其对表阿霉素耐药。
- 肝肿瘤, 实验性 /
- 缺氧诱导因子1, α亚基 /
- 表柔比星 /
- 干细胞 /
- 抗药性, 肿瘤
Abstract: Objective To investigate the effect of hypoxia-inducible factor-1α (HIF-1α) on the stemness and epirubicin sensitivity of hepatoma cells. Methods Hepatoma cells were selected for experiment. HepG2 hepatoma cells transfected with HIF-1α overexpression plasmid were selected as experimental group, and those transfected with pcDNA3.1 empty plasmid were selected as control group; HepG2 cells alone were selected as HepG2 group. Quantitative real-time PCR was used to measure the mRNA expression of HIF-1α; Western blot was used to measure the protein expression of HIF-1α; flow cytometry was used to measure the expression of CD133 on the surface of hepatoma cells. The three groups of cells were treated with epirubicin at different concentrations (0, 6.25, 12.5, 25, and 50 μmol/L) for 24 hours; MTT assay was used to measure cell viability, and flow cytometry was used to measure apoptosis after treatment with epirubicin (50 μmol/L). A one-way analysis of variance was used for comparison of continuous data between multiple groups, and the t-test was used for further comparison between two groups. Results Compared with the HepG2 group and the control group, the experimental group had a significant increase in the mRNA expression of HIF-1α (both P < 0.001), and Western blot showed high expression of HIF-1α in the experimental group. The percentage of CD133 cells was 0.040%±0.003% in the HepG2 group, 0.030%±0.010% in the control group, and 20.110%±0.600% in the experimental group, and the experimental group had a significantly higher positive rate of CD133⁺ than the HepG2 group and the control group (both P < 0.001). At an epirubicin concentration of 25 and 50 μmol/L, the HepG2 group and the control group had significantly inhibited cell viability and a significantly lower cell viability than the experimental group (both P < 0.05). After the treatment with 50 μmol/L epirubicin for 48 hours, the experimental group had a significantly lower cell apoptosis rate than the HepG2 group (67.9%±2.5% vs 93.6%±1.5%, P < 0.001) and the control group (67.9%±2.5% vs 93.0%±1.2%, P < 0.001). Conclusion HepG2 cells are successfully transfected with HIF-1α overexpression plasmid, and HIF-1α can increase the percentage of liver cancer stem cells and improve their resistance to epirubicin.
- Liver Neoplasms, Experimental /
- Hypoxia-Inducible Factor 1, alpha Subunit /
- Epirubicin /
- Stem Cells /
- Drug Resistance, Neoplasm

HTML全文

图 1 HIF-1α质粒在HepG2细胞的表达

注：a，real-time qPCR检测HIF-1α质粒在HepG2细胞的表达；b，Western Blot检测HIF-1α质粒在HepG2细胞的表达。

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图 2 流式细胞术检测HepG2细胞CD133表达

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图 3 流式细胞术检测细胞凋亡情况

下载: 全尺寸图片幻灯片

表 1 不同浓度表阿霉素作用24 h的细胞活性

组别	表阿霉素浓度(μmol/L)
组别	0	6.25	12.5	25	50
HepG2组(%)	100	96.33±1.21	85.47±2.28	67.10±3.56	42.27±0.67
对照组(%)	100	92.60±1.54	86.80±2.40	59.03±2.60	45.50±0.56
实验组(%)	100	92.50±0.35	85.53±0.81	84.10±1.18¹⁾²⁾	72.30±2.07¹⁾²⁾
F值		3.618	0.696	23.570	162.200
P值		0.093	0.535	0.001	<0.001
注：与对照组比较，1)P<0.05;与HepG2组比较，2)P<0.05。

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