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.

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.