肿瘤耐药相关lncRNA的自噬依赖性表达调控及其意义

Chemoresistance is the major reason for the treatment failure and poor outcome of human cancers. The development of chemoresistance of human cancer is a very complicated process. Many genetic and epigenetic alterations contribute to chemoresistance. However, the regulation and function of long non-coding RNAs (lncRNAs) in the development of chemoresistance is largely unknown. In the preliminary investigations for this proposal, we applied lncRNA array to screen deregulated lncRNAs in our previously established drug-resistant cancer cell lines. Among 109 significantly deregulated lncRNAs, we identified ARHGAP5-AS1 as one of lncRNAs potentially related with chemoresistance. Its expression was increased in drug-resistant cells and knockdown of its expression restored sensitivity to chemotherapeutic drugs. Furthermore, its upregulation was associated with shorter overall survival (OS) and progression-free survival (PFS) of gastric cancer patients. In an effort to understand the mechanism underlying its regulation in chemoresistance, we found the expression of ARHGAP5-AS1 was decreased when autophagy was activated. However, its expression was restored once the autophagy flux was blocked by chloroquine (CQ), indicating a potential autophagy-dependent regulation of its RNA abundance. Interestingly, p62 (SQSTM1), the adaptor protein responsible for transporting proteins to-be-degraded to autophgosome, could interact with ARHGAP5-AS1 and other potential chemoresistance-related lncRNAs. Therefore, we postulated that autophagy-dependent degradation might be important to the homeostasis of chemoresistance-related lncRNAs, in a similar manner to regulate organelle and protein homeostasis. In the next, we found ARHGAP5-AS1 could upregulate the expression of ARHGAP5 and knocking-down ARHGAP5 also reversed drug resistance. Furthermore, ARHGAP5-AS1 localized in both nucleus and cytoplasm, indicating it might regulate ARHGAP5 mRNA expression in addition to ARHGAP5 gene transcription. Indeed, we found ARHGAP5-AS1 depletion reduced the stability of ARHGAP5 mRNA. Meanwhile, modification of ARHGAP5 mRNA namely N6-methyladenosine (m6A) was also reduced, accompanied with the reduced interaction of ARHGAP5 mRNA with RNA binding protein HuR. Since HuR is relevant to m6A-associated stabilization of ARHGAP5 mRNA, ARHGAP5-AS1 might stabilize ARHGAP5 mRNA by enhancing m6A modification. In summary, we proposed that chemoresistance-related lncRNAs like ARHGAP5-AS1 could be the substrate for autophagy-dependent degradation. Inhibition of autophagy resulted from the activation of Akt/mTOR signaling upreguated ARHGAP5-AS1 to stimulate ARHGAP5 mRNA and protein expression, eventually promoting chemoresistance. We would like to explore the autophagy-dependent degradation chemoresistance-related lncRNAs in chemoresistance. By doing so, this proposed work will clarify not only new regulations of lncRNA homeostasis, but also new roles of lncRNA on mRNA modifications such as m6A.

肿瘤耐药涉及基因表达和代谢重编程等诸多分子事件，但长链非编码RNA（lncRNA）在肿瘤耐药中的调控及作用仍知之甚少。经过前期筛选鉴定，我们发现ARHGAP5-AS1等耐药相关lncRNA都可结合自噬调控蛋白p62；ARHGAP5-AS1等lncRNA因自噬被抑制而在耐药肿瘤细胞中表达上升；敲低ARHGAP5-AS1的表达，可抑制ARHGAP5 mRNA甲基化(m6A)，下调其表达并逆转耐药，因此提出肿瘤耐药相关lncRNA表达水平受细胞自噬所调控的新假设。我们将在本项目中分析自噬对ARHGAP5-AS1等lncRNA表达的调控作用，探索ARHGAP5-AS1等耐药相关lncRNA对下游基因的转录及其mRNA稳定性等的调控作用，从而阐明自噬对lncRNA代谢稳态的调控，以及lncRNA在mRNA修饰等表达调控中的作用及其机制，为揭示肿瘤耐药和遗传信息传递等生命活动的本质和规律提供新的思路。

肿瘤耐药涉及表观遗传和代谢重编程等诸多分子事件，但长链非编码RNA（lncRNA）在肿瘤耐药中的调控及作用仍知之甚少。在本项目研究中，我们发现ARHGAP5-AS1、linc00942等耐药相关lncRNA都可结合自噬调控蛋白p62，并因自噬被抑制而在耐药肿瘤细胞中表达上升；敲低ARHGAP5-AS1、linc00942等的表达，可抑制ARHGAP5 、DNMT3a等下游mRNA甲基化(m6A)，下调其表达并逆转耐药。因此，自噬可能通过降解linc00942等耐药相关lncRNA而重塑DNA甲基化谱，重编程细胞代谢等，从而提高化疗敏感性。总体上，本项目研究通过阐明自噬对lncRNA代谢稳态的调控，以及lncRNA在mRNA修饰等表达调控中的作用及其机制，明确RNautophagy和RNA m6A修饰在retrograde信号通路重塑表观遗传密码中的作用，从而为揭示肿瘤耐药和遗传信息传递等生命活动的本质和规律提供了新的思路。以本项目研究为基础，近三年来，项目组在cell stem cell等SCI期刊共发表相关标注论文7篇。