LncRNA-AP006222.2/TGFβ/SMAD正反馈环路促进肝癌生长及增加多激酶抑制剂耐药的机制研究

Hepatocellular carcinoma（HCC）grows rapidly with serious adverse prognosis. Multi-kinase inhibitor can improve the survival of patients with advanced HCC. As for it acquiring chemo-resistance easily, multi-kinase inhibitor is limited in clinical application. Recently, many studies have shown that lncRNAs are frequently deregulated invarious diseases and have multiple functions in a wide range of biological processes, such as proliferation, chemo-resistance, but the mechanism is still unclear. We analyzed the expression profile of lncRNAs in 3 pairs of human HCC samples using microarray analysis. LncRNA-AP006222.2, a liver-enriched lncRNA, was selected and expressed highly in HCC. According to our earlier work, LncRNA-AP006222.2 enhances chemo-resistance of multi-kinase inhibitors HCC and promotes cell proliferation. Overexpression of lncRNA-AP006222.2 can increase the level of TGFβR1. On the contrary, TGFβ promotes Smad2/3 to bind the promoter of lncRNA-AP006222.2, and subsequently increases its expression. So we assume that lncRNA-AP006222.2/ TGFβ / SMAD positive feedback loop promotes HCC growth and increases chemo-resistance of multi-kinase inhibitors. This project plans to confirm the interaction in lncRNA-AP006222.2，TGFβ and SMAD, employing the methods of bioinformatics, EGFP reporter assays, and ChIP etc. Through the research of this subject, we try to provide additional proof for the molecular mechanisms of HCC growth and increases chemo-resistance of multi-kinase inhibitors, we also hope we could provide more theoretical evidence for developing new technology of the diagnosis and treatment of HCC.

肝癌生长迅速，多预后不良。而多激酶抑制剂能够改善晚期肝癌患者生存，但因易产生耐受而使用受限。研究表明长链非编码RNA参与包括肿瘤生长、耐药在内多种疾病过程调控，但作用机制尚不清楚。我们前期应用lncRNA芯片检测，筛选出肝脏富集且在癌组织中高表达的lncRNA-AP006222.2。实验证实此lncRNA可以增加肝癌多激酶抑制剂耐药及促进肝癌生长。上调此lncRNA可以促进TGFβR1的表达，反之TGFβ可以促进Smad2/3直接结合在此lncRNA的基因启动子序列并促进此lncRNA转录上调。因此，我们提出假设：lncRNA-AP006222.2/TGFβ/SMAD正反馈环路可以促进肝癌生长及增加多激酶抑制剂耐药。拟通过生物信息学、染色质免疫共沉淀、荧光报告基因等技术，研究lncRNA-AP006222.2，TGFβ，SMAD之间相互关系，旨在为研发肝癌诊疗新技术提供客观的依据。

肝癌生长迅速，虽然多激酶抑制剂对其治疗有一定效果，但易出现耐药，故肝癌大多预后较差。我们应用lncRNA芯片检测、生物信息分析等方法，筛选出肝脏富集且在癌组织中高表达的lncRNA-AP006222.2，结合患者临床资料，lncRNA-AP006222.2可以作为预测患者预后的一项指标。体内体外实验证实lncRNA-AP006222.2可以增加肝癌多激酶抑制剂耐药及促进肝癌生长。本项目深入分析了其中的调控机制，首先lncRNA-AP006222.2可以通过调控经典凋亡途径Bax及PARP途径影响肝癌的增殖及多激酶抑制剂耐药。更加深入研究发现过表达lncRNA-AP006222.2可以增加TGFβR1的表达，反之TGFβ可以促进Smad2/3直接结合在lncRNA-AP006222.2的基因启动子序列并促进lncRNA-AP006222.2转录上调，因此lncRNA-AP006222.2/TGFβ/SMAD正反馈环路可以促进肝癌生长及增加多激酶抑制剂耐药。另一方面，通过质谱分析等试验显示lncRNA-AP006222.2可以与ENO1特异性结合，深入研究揭示lncRNA-AP006222.2可以上调ENO1的表达，促进肝癌生长，进一步，本研究将lncRNA-AP006222.2分成3部分，通过体外转录及标记生物素的方法，对不同部分进行实验，找到lncRNA-AP006222.2与ENO1结合部位。本项目的研究结果解析了lncRNA-AP006222.2在肝癌的增殖及多激酶抑制剂耐药中的功能意义，完善了对肝癌的增殖及多激酶抑制剂耐药发生机制的认识，为抗肿瘤治疗和预防提供新的路径。