[索拉非尼诱导的肝癌耐药细胞系的建立及基因表达分析]
[Establishment and gene expression analysis of drug-resistant cell lines in hepatocellular carcinoma induced by sorafenib].
作者信息
Ma B, Tian Z H, Qu L, Liu Y X, Zhang H, Ding H R
机构信息
Department of Lymphoma, Ministry of Education Key Laboratory of Carcinogenesis and Translational Research, Peking University Cancer Hospital and Institute, Beijing 100142, China.
Department of Laboratory, Ministry of Education Key Laboratory of Carcinogenesis and Translational Research, Peking University Cancer Hospital and Institute, Beijing 100142, China.
出版信息
Beijing Da Xue Xue Bao Yi Xue Ban. 2020 Apr 18;52(2):207-213. doi: 10.19723/j.issn.1671-167X.2020.02.003.
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.
目的
建立索拉非尼诱导的肝癌耐药细胞系，筛选索拉非尼诱导的肝癌耐药细胞系中的高表达基因，进而探索肝癌中与索拉非尼耐药相关的基因。
方法
获取人PLC和Huh7细胞系，然后通过体外间歇诱导法用索拉非尼诱导PLC和Huh7耐药细胞系。采用CCK8法检测索拉非尼的IC50值，以评估PLC和Huh7中肝癌细胞系的药物敏感性。利用高通量cDNA测序（RNA-Seq）筛选出索拉非尼诱导的PLC和Huh7耐药细胞系中所有上调基因，使用Ualcan数据库分析索拉非尼诱导的PLC和Huh7耐药细胞系中的上调基因与肝癌的四个临床生物学特征之间的相关性，包括正常样本与肿瘤样本之间的基因表达、肿瘤分期、肿瘤分级和患者总生存期，以寻找可能参与肝癌索拉非尼耐药机制的基因。
结果
通过高通量cDNA测序（RNA-Seq）在PLC和Huh7耐药细胞系中检测到的所有上调基因，通过以下条件进一步筛选：（1）在索拉非尼诱导的PLC和Huh7耐药细胞中共表达的基因；（2）倍数变化大于4倍且差异具有统计学意义（P<0.05），找到了PLC和Huh7耐药细胞系中上调的前12个基因，分别为TPSG1、CBX4、CLC、CLEC18C、LGI4、F2RL1、S100A6、HABP2、C15ORF48、ZG16、FOLH1和EPCAM。通过Ualcan数据库分析这12个基因与临床生物学特征之间的相关性，筛选出潜在的重要基因CBX4。
结论
成功建立了索拉非尼诱导的人肝癌PLC和Huh7耐药细胞系。通过高通量cDNA测序（RNA-Seq）和使用Ualcan数据库进行进一步分析，筛选出了肝癌中与索拉非尼耐药相关的基因CBX4，为进一步研究肝癌索拉非尼耐药机制提供了有力依据。
Zotero 插件