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北京大学学报（医学版） ›› 2020, Vol. 52 ›› Issue (2): 207-213. doi: 10.19723/j.issn.1671-167X.2020.02.003

• 论著 • 上一篇    下一篇

索拉菲尼诱导的肝癌耐药细胞株的建立及基因表达分析

马博1,田志华2,△(),曲莉1,刘月香1,张宏2,丁慧荣2   

  1. 1. 北京大学肿瘤医院暨北京市肿瘤防治研究所 恶性肿瘤发病机制及转化研究教育部重点实验室 淋巴瘤科
    2. 北京大学肿瘤医院暨北京市肿瘤防治研究所 恶性肿瘤发病机制及转化研究教育部重点实验室 中心实验室, 北京 100142
  • 收稿日期:2019-12-04 出版日期:2020-04-18 发布日期:2020-04-18
  • 通讯作者: 田志华 E-mail:tianzhihua@bjmu.edu.cn
  • 基金资助:
    国家自然科学基金(81872025);北京市自然科学基金(7182030);北京大学临床肿瘤学院科学研究基金(10-29)(10-29)

Establishment and gene expression analysis of drug-resistant cell lines in hepatocellular carcinoma induced by sorafenib

Bo MA1,Zhi-hua TIAN2,△(),Li QU1,Yue-xiang LIU1,Hong ZHANG2,Hui-rong DING2   

  1. 1. Department of Lymphoma
    2. Department of Laboratory, Ministry of Education Key Laboratory of Carcinogenesis and Translational Research,Peking University Cancer Hospital and Institute, Beijing 100142, China
  • Received:2019-12-04 Online:2020-04-18 Published:2020-04-18
  • Contact: Zhi-hua TIAN E-mail:tianzhihua@bjmu.edu.cn
  • Supported by:
    Supported by the National Natural Science Foundation of China(81872025);the Beijing Natural Science Foundation(7182030);Peking University School of Oncology Science Foundation(10-29)

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摘要：

目的 建立索拉菲尼诱导的肝细胞癌(hepatocellular carcinoma, HCC)耐药细胞株,筛选出耐药细胞株中的高表达基因,进一步探讨肝细胞癌中与索拉菲尼耐药的相关基因.方法: 选取人肝癌细胞株PLC及Huh7,采用药物间歇诱导法分别建立索拉菲尼诱导的耐药细胞株,运用高通量测序技术(RNA-Seq)测定两株耐药细胞株中高表达基因,运用生物信息学数据库分析高表达基因与肝癌临床生物学指标相关性,筛选出肝癌耐药机制中可能参与的基因.结果: 通过基因测序得到两株肝癌耐药细胞株中的高表达基因,在设定的限定条件下进行进一步筛选, 限定条件为两株耐药细胞中共同表达的基因且表达差异倍数大于4倍,差异具有统计学意义(P<0.05),发现两株耐药细胞共同表达前12位基因符合限定条件,进一步运用Ualcan数据库分析12个基因与肝癌组织表达水平,分期,分级及生存的关系,筛选出符合条件的基因CBX4.结论: 成功构建了索拉菲尼诱导的肝癌耐药细胞株,并筛选出与索拉菲尼耐药相关基因CBX4,为进一步耐药机制探讨提供依据.

关键词: 肝细胞癌, 索拉菲尼耐药, TCGA, CBX4

Abstract:

Objective: To establish the drug-resistant cell lines of hepatocellular carcinoma (HCC) induced by sorafenib, and to screen out the high expression genes in drug-resistant cell lines of HCC induced by sorafenib, then to explore the genes related to sorafenib resistance in hepatocellular carcinoma.Methods: The human PLC and Huh7 cell lines were obtained, then the PLC and Huh7 drug-resistant cell lines were induced with sorafenib by using intermittent induction in vitro. CCK8 assay was used to detect the IC50 value of sorafenib for evaluation of drug sensitivity of hepatocellular carcinoma cell lines in PLC and Huh7. All the up regulated genes in PLC and Huh7 drug-resistant cell lines induced by sorafenib were screened out using high-throughput cDNA sequencing (RNA-Seq), Ualcan database was used to analyze the correlations between the up regulated genes in PLC and Huh7 drug-resistant cell lines induced and four clinical biological characteristics of hepatocellular carcinoma, including the gene expressions between normal samples and tumor samples, tumor stage, tumor grade, and patient overall survival, to find the genes that might be involved in the mechanism of sorafenib resistance of hepatocellular carcinoma.Results: All the up regulated genes detected by the using high-throughput cDNA sequencing (RNA-Seq) in PLC and Huh7 drug-resistant cell lines were further screened out by following conditions:(1) genes co-expressed in PLC and Huh7 drug-resistant cells induced by sorafenib, (2) the fold change was more than 4 times and the difference was statistically significant (P <0.05), the top 12 up regulated genes in PLC and Huh7 drug-resistant cell lines were found, which were TPSG1, CBX4, CLC, CLEC18C, LGI4, F2RL1, S100A6, HABP2, C15ORF48, ZG16, FOLH1, and EPCAM. Compared with the correlations between the twelve genes and the clinical biological characteristics by Ualcan database, the potentially significant gene CBX4 was screened out.Conclusion: The human PLC and Huh7 drug-resistant cell lines of hepatocellular carcinoma induced by sorafenib were successfully established. CBX4, the gene related to sorafenib resistance in hepatocellular carcinoma, was screened out by the high-throughput cDNA sequencing (RNA-Seq) and further analysis using Ualcan database, which is providing a powerful basis for further research on the mechanism of sorafenib resistance of hepatocellular carcinoma.

Key words: Hepatocellular carcinoma, Sorafenib resistance, TCGA, CBX4

中图分类号: 

  • R735.7

图1

CCK8 法检测索拉菲尼对PLC和Huh7 细胞增殖抑制作用"

图2

基因测序PLC及Huh7耐药细胞株中的基因表达"

图3

肝细胞癌肿瘤组织与正常组织的基因表达差异"

图4

肝细胞癌肿瘤分期与基因表达差异"

图5

肝细胞癌肿瘤组织学分级与基因表达差异"

图6

肝细胞癌淋巴结转移与基因表达差异"

图7

肝细胞癌生存分析与基因表达差异"

表1

索拉菲尼耐药细胞株中的靶基因表达差异分析(TCGA数据库)"

Gene name Gene expression Tissues OS Metastasis Grade Scores
in Huh7 in PLC T/N>1 P<0.05 L/H>1 P<0.05 M1/M0>1 P<0.05 G4/N>1 P<0.05
TPSG1 11.82 4.00 1 1 0 0 0 0 0 0 2
CBX4 10.56 5.42 1 1 1 1 1 1 1 1 8
CLC 9.86 7.00 0 0 0 0 0 0 0 0 0
CLEC18C 8.74 4.04 1 1 0 0 1 0 1 0 4
LGI4 5.74 5.90 1 1 1 1 1 0 1 0 6
F2RL1 5.40 4.46 1 1 0 0 0 0 0 0 2
S100A6 5.02 5.00 1 1 1 1 1 1 1 0 7
HABP2 4.84 7.06 0 0 0 0 0 0 0 0 0
C15ORF48 4.38 4.46 1 1 1 1 0 0 1 1 6
ZG16 4.36 4.42 0 0 0 0 0 0 0 1 1
FOLH1 4.30 4.02 0 0 0 0 0 0 0 1 1
EPCAM 4.16 4.72 0 0 0 0 0 0 0 0 0
[1] Bray F, Ferlay J, Soerjomataram I , et al. Global cancer statistics 2018: GLOBOCAN estimates of incidence and mortality worldwide for 36 cancers in 185 countries[J]. CA Cancer J Clin, 2018,68(6):394-424.
[2] Kulik L, El-Serag HB . Epidemiology and management of hepatocellular carcinoma[J]. Gastroenterology, 2019,156(2):477-491.
[3] Llovet JM ,Zucman-rossi J,Pikarsky E, et al.Hepatocellular carcinoma[J]. Nat Rev Dis Primers, 2016,2(4):16018.
[4] Wilhelm S, Carter C, Lynch M , et al. Discovery and development of sorafenib: a multik inase inhibitor for treating cancer[J]. Nat Reviews Drug Dis, 2006,5(10):835-844.
[5] Keating GM, Santoro A . Sorafenib: a review of its use in advanced hepatocellular carcinoma[J]. Drugs, 2009,69(2):223-240.
[6] Cheng AL, Kang YK, Chen Z , et al. Efficacy and safety of sorafenib in patients in the Asia-Pacific region with advanced hepatocellular carcinoma: a phase Ⅲ randomised, double-blind, placebo-controlled trial[J]. Lancet Oncol, 2009,10(1):25-34.
[7] Llovet JM, Ricci S, Mazzaferro V , et al. Sorafenib in advanced hepatocellular carcinoma[J]. N Engl J Med, 2008,359(4):378-390.
[8] Zhang HM, Yang B, Guo B , et al. The role and practice of clinical bioinformatics in medical education[J]. Transpl Med J, 2018,3(4):175-178.
[9] Darshan SC, Bhuwan B, Sai AHB , et al. UALCAN: a portal for facilitating tumor subgroup gene expression and survival analyses[J]. Neoplasia, 2017,19:649-658.
[10] Peng L, Bian XW, Li DK , et al. Large-scale RNA-seq transcriptome analysis of 4043 Cancers and 548 Normal tissue controls across 12 TCGA cancer types[J]. Sci Rep, 2015,5:13413.
[11] Gebhardt ML, Mer AS , Andrade-Navarro MA. mBISON: finding miRNA target over-representation in gene lists from ChIP-sequencing data[J]. BMC Res Notes, 2015,16(8):157.
[12] Kagey MH, Melhuish TA, Wotton D . The polycomb protein Pc2 is a SUMO E3[J]. Cell, 2003,113:127-137.
[13] Li J, Xu Y, Long XD , et al. Cbx4 governs HIF-1alpha to potentiate angiogenesis of hepatocellular carcinoma by its SUMO E3 ligase activity[J]. Cancer Cell, 2014,25:118-131.
[14] Wang B, Tang J, Liao D , et al. Chromobox homolog 4 is corre-lated with prognosis and tumor cell growth in hepatocellular carcinoma[J]. Ann Surg Oncol, 2013,20(Suppl 3):S684-692.
[15] Yang JL, Cheng DD, Zhu B , et al. Chromobox homolog 4 is positively correlated to tumor growth,survival and activation of HIF-1α signaling in human osteosarcoma under normoxic condition[J]. J Cancer, 2016,7(4):427-435.
[16] Jiao HK, Xu Y, Li J , et al. Prognostic significance of Cbx4 expression and its beneficial effect for transarterial chemoembolization in hepatocellular carcinoma[J]. Cell Death Dis, 2015,6:e1689.
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