RNautophagy依赖性表达lncrna-Linc00942通过抑制MSI2蛋白降解促进胃癌化疗耐药的分子机制

Chemoresistance remains the uppermost disincentive for cancer treatment. However, the functions of long non-coding RNAs (lncRNAs) in chemoresistance are largely unclarified. We screened chemoresistance-related lncRNAs in preliminary investigations and identified Linc00942, as one of the most upregulated lncRNA. Knockdown of its expression restored the sensitivity to multiple chemotherapeutic drugs such as cisplatin, doxorubicin and 5-fluorouracil. As we previously reported, autophagy was blocked in chemoresistant gastric cancer cells. Interestingly, autophagy activation reduced Linc00942 expression. SQSTM1/p62, the critical adaptor protein to transport cargoes to-be-degraded to autophagosomes, directly bound to Linc00942 and promoted its RNautophagy-dependent degradation. Moreover, we identified MSI2 as one downstream protein target interacted with Linc00942 by antibody array-based screening. Knockdown of Linc00942 expression activated ubiquitination-dependent proteasomal degradation of MSI2 protein, eventually promoting chemoresistance. Therefore, we postulated that.RNautophagy-dependent degradation of Linc00942 restores chemoresistance by derepressing ubiquitination dependent degradation of MSI2 protein. To prove it, we will perform a serial of experiments to analyze the regulation of linc00942 homeostasis by autophagy, the influence of Linc00942 on ubiquitination dependent degradation of MSI2 protein and their relevance to chemoresistance. By doing so, we believe that the project will provide a deeper insight to understand chemoresistance and novel rational foundation to improve the clinical efficacy of chemotherapy for gastric and probably other cancers.

化疗耐药仍是胃癌等恶性肿瘤临床治疗失败的主要原因，长链非编码RNA(lncRNA)参与调控基因表达及代谢重编程等诸多分子事件，但其在耐药中的作用及其机制仍需深入探讨。Linc00942在胃癌耐药细胞中高表达；活化细胞自噬可下调其表达；自噬调控蛋白p62结合并招募linc00942进行自噬依赖性降解；敲减Linc00942的表达可促进MSI2蛋白的蛋白酶体依赖性降解，并逆转胃癌化疗耐药。因此，我们推测受细胞自噬性降解调控的linc00942可能通过结合并抑制MSI2的泛素化依赖性降解来促进胃癌化疗耐药。本项目拟通过分析细胞自噬调节linc00942代谢稳态平衡的作用及其机制，探索linc00942影响MSI2蛋白表达的分子机理，解析linc00942在胃癌化疗耐药中的调控功能及机制，从而揭示非编码RNA代谢稳态的新调控方式及胃癌化疗耐药的本质和规律，为改善胃癌临床诊治提供科学依据和理论支撑。

顺铂 (Cisplatin , DDP) 引起的化疗耐药仍然是晚期胃癌治疗的主要制约因素。尽管越来越多的证据显示长链非编码RNA (Long noncoding RNA , LncRNA) 参与调控肿瘤化疗耐药，其在胃癌化疗耐药中发挥的调节作用和具体机制仍有待进一步研究。项目旨在寻找胃癌中与化疗耐药相关的关键lncRNA，探究其在耐药中的作用机制，从而为化疗耐药后胃癌患者提供新的治疗策略。本研究发现LncRNA Linc00942 （以下简称LNC942）在化疗耐药的胃癌细胞中显著高表达并与胃癌患者的不良预后呈正相关。我们通过构建体内外胃癌耐药模型解析了LNC942促进胃癌化疗耐药的机制：(1) LNC942与MSI2蛋白特异性地结合并调控其蛋白的稳定性；(2) LNC942通过阻止MSI2与SCFβ-TRCP E3泛素连接酶的相互作用以及随后的蛋白泛素化依赖性降解来上调MSI2表达；(3) LNC942以N6-甲基腺苷 (N6-methyladenosine, m6A) 依赖性方式稳定c-Myc mRNA，其中MSI2作为一个潜在的m6A阅读蛋白识别和结合具有m6A修饰的c-Myc mRNA来上调c-Myc表达；(4) LNC942通过调控下游MSI2-c-Myc轴抑制细胞凋亡和诱导干细胞干性使胃癌细胞产生化疗耐药性，使用小分子抑制剂-罗米地辛 (Romidepsin, FK228) 特异性结合并抑制MSI2活性后，增加了胃癌细胞的化疗敏感性。本研究首次发现LNC942是与胃癌耐药相关的关键lncRNA, 并揭示了其上调MSI2而促进胃癌化疗耐药的作用机制；(2) 鉴定出MSI2具有m6A阅读蛋白的新功能，可结合并稳定m6A修饰后的c-Myc mRNA; (3) 发现了MSI2的特异性小分子抑制剂-FK228， FK228与DDP联合使用在逆转胃癌化疗耐药中具有一定的转化价值。综上，LNC942可抑制MSI2蛋白的泛素化依赖性降解，而表达上调的MSI2进一步通过识别和结合具有m6A修饰的c-Myc mRNA 上调c-Myc表达，进而抑制细胞凋亡和诱导干细胞干性使胃癌细胞产生化疗耐药性。此外，使用MSI2特异性小分子抑制剂可以恢复胃癌细胞的化疗敏感性，因此抑制LNC942-MSI2-c-Myc轴有望为化疗耐药后胃癌患者提供新的治疗策略。