m6A去甲基化酶ALKBH5通过维持MACF1的表达促进胶质母细胞瘤侵袭的机制研究

The invasion of GBM to the surrounding and distant brain tissue is an important cause for its poor prognosis. Our previous experiments confirmed that: ALKBH5 is negatively correlated with the prognosis of GBM patients, and is positively correlated with tumor aggressiveness and malignancy; ALKBH5 mediates invasion of GBM caused by hypoxia, hypoglycemia, and TMZ; ALKBH5 positively regulates MACF1 expression; invasion of GBM can be induced by MACF1. It has been reported that ALKBH5 can bind to the pre-mRNA of target gene and remove the methyl group of the latter at m6A site; HuR can maintain RNA stability and translation after binding to the target RNA. The database predicts that: HuR can bind to the demethylated m6A site of MACF1 pre-mRNA. Based on the above, we proposed the following hypothesis: " ALKBH5 induced by hypoxia, hypoglycemia, and TMZ promotes the invasiveness of GBM by removing the m6A methylation of MACF1 pre-mRNA and then promoting the binding of HuR to MACF1 pre-mRNA, thereby stabilizing MACF1 expression. "This project intends to verify this hypothesis from the levels of molecular, cellular, animal and clinical specimens, and medical records to provide new ideas and candidate targets for the treatment of GBM.

GBM向周边及远处脑组织侵袭是导致其预后极差的重要原因。我们前期实验中证实：ALKBH5与GBM患者预后负相关，与肿瘤侵袭性及恶性程度正相关；ALKBH5介导低氧、低糖、TMZ诱导的GBM侵袭；ALKBH5可正向调节MACF1的表达；MACF1可诱导GBM侵袭。文献指出：ALKBH5能够与靶标基因的pre-mRNA结合，去除后者m6A位点的甲基；HuR与靶标RNA结合后能够维持RNA稳定和翻译。数据库预测发现：HuR能够与MACF1 pre-mRNA去甲基化后的m6A位点结合。据此，我们提出以下假说：“低氧、低糖、TMZ诱导的ALKBH5通过去除MACF1 pre-mRNA的m6A甲基化，促进HuR与MACF1 pre-mRNA结合，进而稳定MACF1表达，维持GBM的侵袭性。”本课题拟从分子、细胞、动物及临床标本、病历资料水平验证这一假说，为GBM的综合治疗提供新的思路及候选靶点。

本研究通过倍比稀释的方法建立同一患者来源的原代GBM单克隆细胞株，显微观察及体外、体内功能实验结果提示，同一患者来源具有相同遗传背景的原代单克隆细胞株具有高度异质性，其中DCD7#增殖最快，而DCD4#增殖最慢。细胞功能实验结果提示干扰ALKBH5表达后GBM细胞增殖受到抑制。分别将DCD4#及7#细胞中的ALKBH5富集下来后进行蛋白质组学测序，发现在增殖快的DCD7#中ALKBH5可与TARDBP相互结合成蛋白复合物。接下来体外、体内功能试验结果证实，干扰TARDBP后GBM细胞增殖受到抑制，致瘤性减弱。利用DCD4#及7#细胞进行RNA-seq测序和MERIP-seq测序筛选出下游靶基因ACAD8，RIP-PCR及meRIP-PCR实验结果证实ALKBH5与TARDBP形成的蛋白复合物通过下调ACAD8的m6A甲基化水平从而上调其mRNA表达水平。并且，细胞功能实验结果提示干扰ACAD8表达后GBM细胞增殖能力减弱。本课题的研究结果提示，在GBM细胞中ALKBH5与TARDBP结合形成复合物后共同调控ACAD8的前体mRNA m6A甲基化水平，以调控其剪切和成熟，促进ACAD8的表达，从而促进GBM细胞的增殖和肿瘤的进展。这一发现揭示了RNA m6A甲基化介导GBM恶性进展新的分子机制，未来将可应用于分子病理诊断甚至临床靶点治疗，以改善胶质母细胞瘤的诊断及治疗现状。