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Journal of Peking University (Health Sciences) ›› 2020, Vol. 52 ›› Issue (2): 207-213. doi: 10.19723/j.issn.1671-167X.2020.02.003

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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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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

CLC Number: 

  • R735.7

Figure 1

The inhibitory effect of sorafenib on the proliferation of PLC and Huh7 cells by CCK8 assay PLC and Huh7 drug-resistant cell lines were established after 12 weeks using intermittent induction with sorafenib. Cell proliferation assay was performed to calculate cell mortality based on sorafenib treatment. sr, sorafenib resistant."

Figure 2

Gene expression in PLC and Huh7 drug-resistant cell lines by High-throughput cDNA sequencing The up regulated genes in PLC and Huh7 drug-resistant cell lines were detected by RNA-Seq, the top twelve genes which fold change were more than 4 times."

Figure 3

Gene expression differences based on sample types in hepatocellular carcinoma Seven genes are screened out from all the twelve genes which gene expression in tumor sample are higher than in normal tissues (P<0.05)."

Figure 4

Gene expression differences based on tumor stage in hepatocellular carcinoma Two genes, CBX4 and S100A6, are screened out from the seven genes which gene expression in stage Ⅳ are higher than in normal tissues (P<0.05)."

Figure 5

Gene expression differences based on tumor grade in hepatocellular carcinoma Two genes, CBX4 and C15ORF48, are screened out from the seven genes which gene expression in grade IV are higher than in normal tissues (P <0.05)."

Figure 6

Gene expression differences based on nodal metastasis in hepatocellular carcinoma Gene expression of all the seven genes with nodal metastasis are higher than in normal tissues (P<0.05)."

Figure 7

Gene expression differences based on patient survival in hepatocellular carcinoma The overall survival of patients were prolonged with lower gene expression of these four genes, which are CBX4, LGI4, S100A6 and C15ORF48(P <0.05)."

Table 1

Screening the target genes in sorafenib resistant cell lines using TCGA dataset analysis"

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
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