ALKBH5通过下调LITAF增强骨肉瘤干细胞耐药性的机制研究

骨肉瘤的转移和复发是严重的临床问题。肿瘤干细胞（Osteosarcoma stem cells, OSC）的治疗抵抗是导致这一问题的根本原因，但其机制尚未被完全阐明。前期研究中发现，去甲基化酶ALKBH5通过下调LITAF增强OSC的耐药性；且LITAF mRNA的m6A修饰位点与hsa-miRNA-665的结合位点重合。因此，可推测表达上调的ALKBH5通过脱去LITAF的m6A修饰，从而促进hsa-miRNA-665与之结合并使其降解，最终导致OSC的凋亡通路受阻而耐药。本课题拟采用RIP技术和双荧光素酶报告系统验证ALKBH5蛋白和LITAF mRNA的互作关系以及机制，再利用原位模型探索通过基因操作和miRNA抑制剂逆转骨肉瘤耐药的可能性。通过上述研究，有望阐明ALKBH5增强OSC耐药性的机制，同时也将发现一种全新的机制用以阐释去m6A修饰降解mRNA的作用。

Metastases and postoperative relapse of osteosarcoma are serious clinical problems. Therapeutic resistance of cancer stem cells has been shown as the root cause of tumor metastasis and recurrence in emerging evidence, but the mechanism has not been fully elucidated. In previous studies, we found that m6A demethylase ALKBH5 enhanced the drug resistance of osteosarcoma stem cells by down-regulating the LITAF. Moreover, the m6A methylation site of LITAF mRNA overlapped with the binding site of hsa-miRNA-665.Therefore, we speculated that by removing the m6A modification of the LITAF, the upregulation of ALKBH5 in osteosarcoma stem cells could promote the binding of hsa-miRNA-665 with LITAF mRNA, which was further degraded, ultimately leading to the blocking of the apoptotic pathway of osteosarcoma stem cells and drug resistance. In this study, RNA binding protein immunoprecipitation and dual-luciferase reporter assay were used to verify the interaction as well as the mechanism between ALKBH5 protein with LITAF mRNA. Furthermore, the orthotopic xenograft models of osteosarcoma were used to explore the possibility of reversing the drug resistance of osteosarcoma through gene manipulation and miRNA antagomir. Through the above studies, it is expected to clarify the molecular mechanism of ALKBH5 enhancing the drug resistance of osteosarcoma stem cells, and provide a theoretical foundation for the study about reversing drug resistance. Simultaneously, a new molecular mechanism and a unique perspective will be exhibited to explain the RNA-degrading effects of m6A demethylation.