肝癌细胞仑伐替尼耐药的基因筛选及其通路研究

Research on genetic screening for lenvatinib resistance in hepatocellular carcinoma cells and its pathways

目的基于CRISPR/Cas9技术筛选肝癌仑伐替尼耐药相关基因并探究其潜在关系。方法应用CRISPR/Cas9 sgRNA全基因组文库技术结合Affymetrix表达谱芯片筛选肝癌对仑伐替尼耐药的相关基因。通过TCGA数据库分析这些基因在肝癌中的表达及预后的关系,最终确定采用THOC2为肝癌仑伐替尼耐药基因并用于后续研究。采用免疫组织化学(immunohistochemical,IHC)染色、Western blotting、RT-PCR检测THOC2在肝癌细胞株(MHCC-97H、MHCC-LM3、SMMC-7721)及仑伐替尼耐药/敏感肝癌组织中的表达情况。通过慢病毒感染构建高表达THOC2的SMMC-7721-THOC2肝癌细胞,采用免疫共沉淀(Co-IP)等技术探讨THOC2介导肝癌仑伐替尼耐药的可能机制。结果发现7个与肝癌仑伐替尼抵抗密切相关的耐药基因,包括THOC2、CTNNB1(β-catenin)、MET(c-Met/HGFR)、HTATIP2、C10orf11、PRDX3、MAST4。TCGA分析提示THOC2在肝癌与癌旁中表达存在统计学差异,且与肝癌临床分期及预后密切相关。IHC染色、Western blotting、RT-PCR检测发现THOC2主要分布于胞核中,其在仑伐替尼耐药组患者中的表达水平高于药物敏感组患者;THOC2在高转移潜能的肝癌细胞株(MHCC-97H、MHCC-LM3)中的表达较在SMMC-7721中更显著;Western blotting、Co-IP进一步表明上调THOC2后Wnt/β-catenin通路下游靶点BAX、cyclinD1和c-myc蛋白水平显著增高,且THOC2与β-catenin存在相互作用关系。结论THOC2基因高表达可能通过Wnt/β-catenin通路参与调控肝癌对仑伐替尼的耐药。

Objective To filtrate out genes related to lenvatinib resistance in hepatocellular carcinoma(HCC)based on CRISPR/Cas9 technology and explore their potential relationship.Methods CRISPR/Cas9 sgRNA genome-wide library technology combined with Affymetrix expression profiling microarray were applied to screen genes involved in lenvatinib resistance in HCC.TCGA database was used to analyze expression of those genes in HCC and its relation with prognosis of HCC patients,and THOC2 was finally identified as the lenvatinib resistance gene and studied in the follow-up research.The expression of THOC2 in HCC cell lines(MHCC-97H,MHCC-LM3,SMMC-7721)and lenvatinib-resistant/sensitive tissues were detected by immunohistochemical(IHC)staining,Western blotting,and RT-PCR.SMMC-7721-THOC2 cells with high expression of THOC2 were constructed by lentiviral infection,and co-immunoprecipitation(Co-IP)was used to investigate the probable mechanism of THOC2 mediated resistance to lenvatinib in HCC.Results A total of seven genes intimately associated with lenvatinib resistance in HCC were found,including THOC2,CTNNB1(β-catenin),MET(c-Met/HGFR),HTATIP2,C10orf11,PRDX3 and MAST4.TCGA analysis indicated that there was statistical difference in the expression of THOC2 between HCC and para-carcinoma tissues,and THOC2 was closely correlated with clinical stage and overall survival.IHC staining,Western blotting,and RT-PCR revealed that THOC2 was mainly accumulated in the nucleus and was more highly expressed in tumor tissue of lenvatinib-resistant patients than that in the sensitive group,and the expression of THOC2 in the high metastatic potential HCC lines(MHCC-97H,MHCC-LM3)was more significant than that of SMMC-7721.Western blotting and Co-IP further assayed that the protein levels of the downstream targets of the Wnt/β-catenin pathway(BAX,cyclinD1,and c-myc)were considerably increased after THOC2 upregulation and there was an interaction between THOC2 andβ-catenin.Conclusion High THOC2 gene expression may be engaged in regulating lenvatinib resistance in HCC through Wnt/β-catenin pathway.