TRIM8调控胶质母细胞瘤干细胞干性的作用及分子机制研究

Glioblastoma (GBM) is the most deadly primary brain tumor, with a poor prognosis following compound therapy. Glioblastoma stem cells (GSCs) contribute to tumor recurrent and therapeutic resistance. Recently our team reported that TRIM8 participates in maintaining GBM stemness, though whose exact mechanism still remains unclear and deserves further explorations. Our preliminary experiment finds out that over-expression of TRIM8 results in decline of RNA-m6A methyltransferase METTL3. Plenty of reports show that METTL3 plays an important role in N6-methyladenosine (m6A) modification, which can also regulate GBM self-renewal capacity and stemness. So here we come up with the hypothesis that TRIM8 regulates GSC stemness and self-renewal capacity via METTL3 mediating m6A modification. First, our project focus on exploring METTL3 and stem cell markers’ change tendency upon up/down-regulation of TRIM8 in GSCs. Western blot and neuro-sphere formation assay will be allied for conformation. As is reported and known that TRIM8 can function as ubiquitin E3 ligase, protein co-immunoprecipitation technique and ubiquitination assay will be used to verify whether TRIM8 mediates METTL3 via ubiquitin-proteasomal degradation or not. Further, m6A-sequence analysis will be employed in searching for METTL3’s potential targets. Finally, this project will analyze those histological and clinical implications of TRIM8 and METTL3 based on patients’ clinical data.

胶质母细胞瘤（GBM）是预后极差的恶性脑肿瘤，胶质母细胞瘤干细胞（GSCs）在GBM治疗后复发起重要作用。我们既往研究发现三体结构域蛋白8(TRIM8)可影响GSC干性，具体机制不甚清楚。本研究预实验发现TRIM8可调控RNA甲基化供体酶METTL3表达。METTL3是mRNA N6-甲基腺苷化（m6A）修饰中重要的酶，可调控GSCs干性。据此，我们提出科学假设：TRIM8通过调控METTL3介导m6A修饰调节GSC干性。本项目拟首先在GSCs中上调/敲减TRIM8，通过WB、神经球增殖实验等确定TRIM8对METTL3及GSCs干性的影响。基于TRIM8有泛素化调节作用，遂用免疫共沉淀实验和泛素化检测探讨其是否通过泛素化-蛋白酶体途径降解METTL3。然后使用m6A-seq技术检测METTL3下游调控靶标mRNA的m6A修饰。最后结合临床资料分析TRIM8和METTL3临床肿瘤学意义。

胶质母细胞瘤（GBM）是预后极差的恶性脑肿瘤，胶质母细胞瘤干细胞（GSCs）在GBM治疗后复发起重要作用。既往研究发现三体结构域蛋白8(TRIM8)可影响GSC干性，具体机制不甚清楚。本项目成功培养胶质母细胞瘤干细胞（GSCs），并发现TRIM8 可调节GSCs 干性标记物表达并影响自我增殖能力，使用细胞增殖等功能实验观察与分析发现TRIM8可促进肿瘤干细胞生物学行为。本项目发现上调/下调TRIM8可导致METTL3表达下降/升高，蛋白酶体抑制剂MG132可抑制METTL3 的降解，提示TRIM8可能通过蛋白酶体降解途径参与调节METLL3的变化。使用m6A点印迹实验发现上调/下调TRIM8后致GSC细胞总m6A修饰水平下降/升高，提示其可能通过m6A修饰甲基化供体酶METTL3影响总m6A修饰。GBM病理标本发现GBM中TRIM8表达偏高，METTL3也有表达；而WB结果显示TRIM8高表达伴随METTL3相对表达偏低。提示TRIM8-METTL3表达与临床GBM肿瘤存在密切相关性。综上所述，TRIM8通过调控METTL3介导的m6A修饰调节GSCs干性维持。靶向以上通路将可能为今后诊治胶质母细胞瘤提供新思路。