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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

曾裕文, 张芳雍, 谭国钳, 等. NCEH1基因在肝癌组织与细胞系中的表达及生物学作用[J]. 临床肝胆病杂志, 2021, 37(8): 1867-1872. DOI: 10.3969/j.issn.1001-5256.2021.08.023
引用本文: 曾裕文, 张芳雍, 谭国钳, 等. NCEH1基因在肝癌组织与细胞系中的表达及生物学作用[J]. 临床肝胆病杂志, 2021, 37(8): 1867-1872. DOI: 10.3969/j.issn.1001-5256.2021.08.023
ZENG YW, ZHANG FY, TAN GQ, et al. Expression and biological role of the neutral cholesterol ester hydrolase 1 gene in liver cancer tissue and cell lines [J]. J Clin Hepatol, 2021, 37(8): 1867-1872. DOI: 10.3969/j.issn.1001-5256.2021.08.023
Citation: ZENG YW, ZHANG FY, TAN GQ, et al. Expression and biological role of the neutral cholesterol ester hydrolase 1 gene in liver cancer tissue and cell lines [J]. J Clin Hepatol, 2021, 37(8): 1867-1872. 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
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

详细信息
    通讯作者:

    吴帆, wufan7911@ext.jnu.edu.cn

  • 中图分类号: R735.7

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

Funds: 

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:   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。

     

  • 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。

    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。

    图  3  MTT检测实验结果

    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。

    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。

    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。

    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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  • 收稿日期:  2021-03-09
  • 修回日期:  2021-05-21
  • 刊出日期:  2021-08-16
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