lncRNA00601在奥沙利铂诱导的肝癌干细胞重编程过程中的表观遗传学调控机制

Despite improvements in the management of hepatocellular carcinoma(HCC), the current routine treatments, such as surgical resection, transcatheter arterial chemoembolization and systemic chemotherapy, are frequently complicated by tumor recurrence, metastasis and drug resistance,which significantly compromise HCC patients’ prognosis. In our previous study,we demonstrated that after repeated chemotherapy, Oct4 and Nanog, the two key reprogramming genes, were activated by promoters demethylation.This process initiated HCC “stem-cell reprogramming” and empowered HCC cells with stem cell properties, including self-renewal and drug resistance.Further lncRNA microarray study revealed a new gene lncRNA00601,may regulate Oct4 and Nanog promoters demethylation through modulating activation-induced cytidine deaminase(AID),a key demethylaion enzyme in stem cell reprogramming.The aim of this project is to investigate the epigenetic regulation mechanics of lncRNA00601 in the process of HCC “stem-cell reprogramming” induced by oxaliplatin.This is a piloted study involving the role of long non-coding RNA on HCC “stem-cell reprogramming”, which will contribute potential therapeutic targets to the treatment of drug resistance for HCC．

近年来，肝癌的临床治疗虽已经取得了长足的进步，但传统的治疗策略仍难以达到根治的目的。尤其是中晚期肝癌患者接受反复的肝动脉化疗栓塞或全身化疗后，残癌细胞出现耐药抵抗，严重影响了患者的预后。课题组前期研究提示，肝癌细胞经历反复的化疗打击后，维持干细胞自我更新的重编程基因Oct4及Nanog启动子出现去甲基化改变，诱导癌细胞发生“干细胞重编程”，重编程后残癌细胞干性增加，化疗耐药性增强。通过高通量的lncRNA 芯片发现，一个功能未知的长链非编码RNA lncRNA00601可能通过调控活化诱导胞苷脱氨酶AID蛋白复合物，促进Oct4及Nanog启动子去甲基化，启动肝癌“干细胞重编程"。本课题拟以奥沙利铂耐药细胞为研究对象，通过体外及体内研究探讨lncRNA00601对肝癌干细胞重编程的表观遗传学调控机制及其对肝癌干性转化、获得性耐药的作用，为逆转肝癌耐药的靶向治疗提供新的理论基础及实验室依据。

课题组前期的基础研究发现，化疗诱导的奥沙利铂耐药细胞干性增加，发生“干性重编程”，源于重编程基因Oct4及Nanog启动子去甲基化改变。 通过LncRNA基因芯片筛选及进一步PCR、Western-Blot验证发现，长链非编码RNA LncRNA00601在肝癌奥沙利铂耐药细胞中高表达，并可能参与肿瘤细胞干性转化及奥沙利铂化疗耐药的重要过程。据此，本课题针对人肝癌细胞株97H和3B，分别建立了奥沙利铂耐药细胞株和LncRNA00601过表达细胞株，进行对其分子生物学机制的研究和分析。实验结果显示，LncRNA00601一方面通过调控去甲基化信号AID/TDG/GADD45a，促进干性基因Oct4及Nanog启动子去甲基化，促进奥沙利铂耐药肝癌细胞发生“干性重编程”。另一方面，LncRNA00601的过表达不仅能够显著降低GADD45A基因的mRNA水平，同时也能增加Gadd45a蛋白在细胞质中的分布。LncRNA00601通过调控GADD45A基因的mRNA水平以及Gadd45a蛋白的细胞核/质分布，对DNA损伤-Gadd45a-细胞凋亡这一信号通路进行负调控，进而导致肿瘤细胞奥沙利铂耐药。本研究揭示了LncRNA00601促进肝癌细胞奥沙利铂耐药及干性转化的分子生物机制，为肝癌的靶向治疗提供了新的分子标志物及新思路。