NCEH1基因在肝癌组织与细胞系中的表达及生物学作用

曾裕文; 张芳雍; 谭国钳; 吴帆

doi:10.3969/j.issn.1001-5256.2021.08.023

NCEH1基因在肝癌组织与细胞系中的表达及生物学作用

DOI: 10.3969/j.issn.1001-5256.2021.08.023

曾裕文¹,
张芳雍¹,
谭国钳^{1, 2},
吴帆^{1, 2, ,}

1.
暨南大学附属广州红十字会医院肝胆外科, 广州 510220
2.
广州市红十字会医院肝胆外科, 广州 510220

基金项目:

国家自然科学基金 (81974442);

广东省自然科学基金 (2020A1515010799);

广东省医学科学技术研究基金 (A2018028);

广州市卫生计生科技项目 (2018A011022)

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: 曾裕文负责资料分析、拟定写作思路、收集数据、撰写论文、修改论文; 张芳雍、谭国钳参与指导撰写文章、收集数据、修改论文; 吴帆负责课题设计, 指导撰写文章并最后定稿。

详细信息

通信作者:
吴帆, wufan7911@ext.jnu.edu.cn

中图分类号: R735.7
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出版历程
- 收稿日期: 2021-03-09
- 录用日期: 2021-05-21
- 出版日期: 2021-08-20

Expression and biological role of the neutral cholesterol ester hydrolase 1 gene in liver cancer tissue and cell lines

Yuwen ZENG¹,
Fangyong ZHANG¹,
Guoqian TAN^{1, 2},
Fan WU^{1, 2
, ,}

1.
Department of Hepatobiliary Surgery, Guangzhou Red Cross Hospital Affiliated to Jinan University, Guangzhou 510220, China
2.
Department of Hepatobiliary Surgery, Guangzhou Red Cross Hospital, Guangzhou 510220, China

Research funding:

National Natural Science Foundation of China (81974442);

Province Natural Science Fund of Guangdong (2020A1515010799);

Guangdong Medical Science and Technology Research Fund (A2018028);

Guangzhou Health and Family Planning Science and Technology Project (2018A011022)

摘要

摘要: 目的了解中性胆固醇酯水解酶1(neutral cholesterol ester hydrolase 1, NCEH1)基因在肝癌组织及人肝癌细胞系中表达水平, 观察NCEH1基因敲减对SMMC-7721人肝癌细胞系增殖、凋亡、侵袭及转移能力的影响。方法选取2013年1月—2019年6月在暨南大学附属广州红十字会医院手术治疗的32例肝癌患者标本及对应的癌旁组织, 采用实时荧光定量PCR方法检测NCEH1基因的相对表达量。从ICGC数据库下载截至2020年9月份的肝癌样本基因表达数据, 应用R软件整理数据, 筛选出每个样本中NCEH1基因表达量, 分别采用配对Wilcoxon符号秩检验和Wilcoxon秩和检验分析肝癌与癌旁组织间的差异。采用实时荧光定量PCR方法检测NCEH1基因在SMMC-7721、Bel-7402、HepG2、Hep3B人肝癌细胞系和HL7702正常人肝细胞系中的表达水平。通过慢病毒介导的小干扰RNA(siRNA)技术构建NCEH1基因敲减的SMMC-7721人肝癌细胞系, 分为NCEH1敲减组(KD组)和阴性对照组(NC组), 以实时荧光定量PCR法检测NCEH1基因的敲减效率, 再以MTT检测实验、Annexin V-APC单染法流式细胞仪检测、划痕愈合实验、Transwell实验和Transwell侵袭小室实验检测2组SMMC-7721肝癌细胞的增殖、凋亡、转移和侵袭能力, 采用t检验对两组间数据进行统计学分析。结果 NCEH1基因在肝癌组织中的平均表达量高于癌旁组织(本院标本Z=2.263, P=0.024, ICGC数据库U=18 768, P＜0.001)。NCEH1基因在中等侵袭转移潜能的SMMC-7721细胞系中的表达量最高; 在低侵袭转移潜能的Bel-7402和HepG2细胞系中的表达水平次之, 在无侵袭转移潜能的Hep3B细胞系中的表达水平最低。KD组SMMC-7721细胞中NCEH1基因的表达水平明显低于NC组(t=11.578, P=0.000 3), NCEH1基因的敲减效率高达74.0%。较之NC组, KD组细胞生长速度明显减缓(t=32.10, P＜0.001);细胞凋亡率明显升高(t=27.303, P＜0.001);迁移率、转移和侵袭细胞数均明显降低(t值分别为9.51、38.123、22.331, P值均＜0.001)。结论 NCEH1基因在肝癌组织及细胞系中的表达明显升高, 且可促进肝癌细胞的生长增殖及侵袭转移并抑制凋亡, 提示其可能是一个潜在的肝癌治疗靶点。
- 癌, 肝细胞 /
- 中性胆固醇酯水解酶1 /
- RNA, 小分子干扰
Abstract: Objective To investigate the expression of the neutral cholesterol ester hydrolase 1 (NCEH1) gene in liver cancer tissue and human hepatoma cell lines and the effect of NCEH1 gene knockdown on the proliferation, apoptosis, invasion, and metastasis abilities of human hepatoma SMMC-7721 cells. Methods Liver cancer tissue samples and adjacent tissue samples were collected from 32 patients with liver cancer who underwent surgical treatment in Guangzhou Red Cross Hospital Affiliated to Jinan University from January 2013 to June 2019, and quantitative real-time PCR was used to measure the relative expression level of the NCEH1 gene. Gene expression data of liver cancer samples up to September 2020 were downloaded from the ICGC database, and R software was used to analyze the data and obtain the expression level of the NCEH1 gene in each sample. The paired Wilcoxon signed-rank test and the Wilcoxon rank-sum test were used to investigate the differences between liver cancer tissue and adjacent tissue. Quantitative real-time PCR was used to measure the expression level of the NCEH1 gene in human hepatoma SMMC-7721, Bel-7402, HepG2, and Hep3B cells and normal human HL7702 liver cells. The lentivirus-mediated small interfering RNA (siRNA) technique was used to establish a human hepatoma SMMC-7721 cell line with NCEH1 gene knockdown, and the cells were divided into NCEH1 knockdown group (KD group) and negative control group (NC group); quantitative real-time PCR was used to measure the knockdown efficiency of the NCEH1 gene, and then MTT assay, flow cytometry with Annexin V-APC single staining, wound healing assay, Transwell assay, and Transwell chamber invasion assay were used to measure the proliferation, apoptosis, metastasis, and invasion abilities of SMMC-7721 cells in both groups. The t-test was used for statistical analysis of data between the two groups. Results The mean expression level of the NCEH1 gene in liver cancer tissue was significantly higher than that in adjacent tissue (specimens from our hospital: Z=2.263, P=0.024; ICGC database: U=18 768, P < 0.001). SMMC-7721 cell line with moderate potential of invasion and metastasis had the highest expression level of the NCEH1 gene, followed by BEL-7402 and HepG2 cell lines with low potential of invasion and metastasis, and Hep3B cell line without the potential of invasion and metastasis had the lowest expression level. The KD group had a significantly lower expression level of the NCEH1 gene than the NC group (t=11.578, P=0.000 3), and the knockdown efficiency of the NCEH1 gene was as high as 74.0%. Compared with the NC group, the KD group had a significant reduction in cell growth rate, a significant increase in apoptosis rate, and significant reductions in migration rate and the number of metastatic and invasive cells (t=32.100, 27.303, 9.51, 38.123, and 22.331, all P < 0.001). Conclusion There is a significant increase in the expression of the NCEH1 gene in liver cancer tissue and cell lines, and the NCEH1 gene can promote the growth, proliferation, invasion, and metastasis of hepatoma cells and inhibit their apoptosis, suggesting that it may be a potential therapeutic target for liver cancer.
- Carcinoma, Hepatocellular /
- Neutral Cholesterol Ester Hydrolase 1 /
- RNA, Small Interfering

HTML全文

图 1 NCEH1基因的表达量

注: a, NCEH1基因在32例肝癌及癌旁组织中的相对表达量; b, ICGC数据库中NCEH1基因在肝癌中的表达量。

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图 2 慢病毒的感染率及敲减效率

注: a, KD组荧光视野(×200);b, NC组荧光视野(×200);C, KD组明视野(×200);d, NC组明视野(×200);e, KD组和NC组实时荧光定量PCR检测结果比较。

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图 3 MTT检测实验结果

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图 4 细胞凋亡结果

注: a, KD组流式细胞分析结果; b, NC组流式细胞分析结果; c, KD组和NC组肝癌细胞凋亡率比较。

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图 5 划痕愈合实验结果

注: a, KD组肝癌细胞划痕后0 h的荧光照片; b, NC组肝癌细胞划痕后0 h的荧光照片; c, KD组肝癌细胞划痕后56 h的荧光照片; c, NC组肝癌细胞划痕后56h的荧光照片; e, KD组和NC组肝癌细胞迁移率比较。

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图 6 Transwell实验结果

注: a, KD组肝癌细胞转移穿过聚碳脂膜的显微镜照片(×100);b, NC组肝癌细胞转移穿过聚碳脂膜的显微镜照片(×100);c, KD组和NC组肝癌细胞转移数比较。

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图 7 Transwell侵袭小室实验结果

注: a, KD组肝癌细胞侵袭小室实验显微照片(×100);b, NC组肝癌细胞侵袭小室实验显微照片(×100);c, KD组和NC组肝癌细胞中侵袭细胞数比较。

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