lncRNA-TMPO-AS1调控TGFβ通路促进肝细胞癌根治术后复发转移的作用及分子机制研究

The relationship between lncRNAs and hepatocellular cancer recurrence after radical resection deserves further study. Our previous study found that lncRNA-TMPO-AS1 upregulated in HCC, and the factor showed significant correlation in recurrence after liver cancer radical resection. Meanwhile the knockdown of lncRNA-TMPO-AS1 was proved significantly inhibiting tumor cell colony formation and metastasis, as well as the abnormal expression of TMPO-AS1 is closely related to abnormal activation of TGFβ signal pathway. TGFβ pathway plays a two-way role of tumor suppressor and promoter in the development of liver cancer. We believe that the TMPO-AS1 upregulated TGFβ pathway in liver tissue to promote liver tumor progression, thus contributing to liver cancer recurrence and metastasis. Therefore, this study intends to further clear the role of TMPO-AS1 in HCC clinical relevance by using clinical samples and information; by positively and negatively intervening the TMPO-AS1 expression, as well as functional experiments to confirm and explore its tumor promotion. Then we intend to apply the TGFβ pathway activator and inhibitor compounded with EMSA, RNA-pull down, RIP technology to clear TMPO-AS1 molecular regulating mechanisms in TGFβ pathway. In summary, this study is designed to investigate the long non-coding RNA-TMPO-AS1 as a new molecule in the development of liver cancer, and providing it as a new targets for clinical diagnosis and treatment.

lncRNAs与肝细胞癌（HCC）术后复发转移的相关性值得亦有待深入研究。课题组前期研究发现HCC中lncRNA TMPO-AS1表达上调；与肝癌根治术后复发显著相关；基因敲减后显著抑制肿瘤细胞克隆形成及转移；异常表达的TMPO-AS1与TGFβ通路异常激活紧密相关，而TGFβ通路在肝癌发展中起抑癌和促癌双向作用。我们认为肝组织中表达上调的TMPO-AS1调控TGFβ通路向促癌方向转化，进而促进肝癌术后复发转移。为此，本研究拟进一步扩大病例数并结合临床表型，明确TMPO-AS1与HCC临床相关性；正负向干预TMPO-AS1表达，通过体内外功能实验证实并发掘促癌作用；应用TGFβ通路激活剂、抑制剂结合EMSA、RNA-pull down、RIP等技术明确TMPO-AS1调控TGFβ通路的分子机制。旨在探讨长链非编码RNA TMPO-AS1作为肝癌发展的新作用分子，为临床诊疗提供新靶点。

对原发性肝癌及癌旁组织中差异表达的lncRNAs 进行筛查发现：相较于原发性肝癌（HCC）癌旁组织，TMPO-AS1 在原发性肝癌组织中显著表达上调。此结论在TCGA数据库中得到进一步验证。生存曲线分析显示，HCC 组织中TMPO-AS1 高表达组其复发率显著高于低表达组，无疾病进展生存率（disease-free survival，DFS）和总体生存率(overall survival，OS)显著低于低表达组。课题组在TMPO-AS1 高表达的肝癌细胞株中基因敲减TMPO-AS1后发现：细胞株克隆形成能力明显减弱并伴细胞迁移侵袭能力显著下降，体内裸鼠成瘤的显著减弱。细胞株TMPO-AS1down与亲本细胞株行转录组测序分析测，并将所得差异表达基因行gene ontology 分析显示：TMPO-AS1 与TGFβ 信号传导途径显著相关；进一步研究发现TGFβ通路的异常激活进而促进上皮间质转化（EMT）,进而导致肿瘤复发转移。本课题明确反义长链非编码RNA TMPO-AS1 高表达与原发性肝细胞癌患者术后复发转移的关联性，为TMPO-AS1 作为能准确诊断、预测复发转移及化学耐药的新型分子标志物，指导患者个体化诊疗提供理论依据；明确TMPO-AS1在肝癌恶变进程的作用及其分子机制，为lncRNA TMPO-AS1 成为肝细胞癌治疗新靶点奠定实验基础。