口腔白斑病衍进过程中RNA结合蛋白DDX5的调控效应研究

A breakthrough was made recently in RNA-binding protein (RBP)-RNA interactions. RBP could regulate cell functions by m6A epigenetic modifications affecting RNA metabolism (Nat Immunol, 2017). Previous research has revealed that DDX5 and m6A methylase METTL3 were highly expressed in oral leukoplakia (OLK) tissues, and were positively correlated with degrees of abnormal proliferation. So the hypothesis was presented that DDX5 promotes cell proliferation by enhancing m6A-methylated proliferative transcripts exported from the nucleus, which is related to METTL3. To confirm that, firstly, expression level of DDX5 will be tested in a large-sample OLK cohort to verify the correlation between high expression of DDX5 in OLK and degrees of abnormal proliferation, its role in regulating abnormal proliferation was also investigated at the cellular level. Secondly, based on METTL3, the molecular mechanism of DDX5 regulating OLK abnormal proliferation will be studied. Finally, all those findings will be further confirmed by 4-nitroquinoline-1-oxide (4NQO) model of K5-cre; DDX5KI/KI mice and K5-cre; DDX5-/- mice. That can not only help us clarify the regulating effect and mechanism of RNA-binding proteins DDX5 in the development of oral leukoplakia, but also provide a new strategy for prevention and treatment of oral potentially malignant disorders.

RNA结合蛋白(RBP)通过m6A表观遗传修饰影响RNA代谢，进而调控细胞功能，是RBP-RNA相互作用领域新突破(Nat Immunol,2017)。申请人前期发现DDX5和m6A修饰酶METTL3在口腔白斑病(OLK)中高表达，与异常增生正相关；结合申请人既往OLK表观遗传机制研究，我们假定DDX5通过METTL3对增殖相关RNA进行m6A修饰，促进RNA出核翻译而导致细胞增殖失衡。为验证此假说，拟首先在大样本OLK队列中进一步验证DDX5与异常增生程度的关系，并在细胞水平考察其在调控口腔黏膜上皮异常增殖中的作用；然后以METTL3为切入点，研究DDX5调控OLK上皮异常增殖的分子机制；最后通过基因编辑小鼠4NQO饮水诱导口腔黏膜异常增生模型，从遗传学上印证体外研究结果。研究不仅有助于阐明m6A修饰影响RBP-RNA相互作用在OLK衍进中的效应及新机制，也为逆转其增殖表型提供新思路。

RNA结合蛋白(RBP)通过m6A表观遗传修饰影响RNA代谢，进而调控细胞功能，是RBP-RNA相互作用领域新突破(Nat Immunol, 2017)。根据前期数据及目前关于RBP-m6A修饰-RNA的研究进展，我们提出的研究假说是：在OLK发展衍进过程中，RNA结合蛋白DDX5可能招募METTL3，对增殖相关基因RNA进行m6A甲基化修饰，从而促进基因RNA出核翻译而导致细胞增殖失衡。以其中关键基因为靶点，可抑制并逆转OLK异常增殖的生物学表型。.本项目首先通过OLK组织样本验证DDX5与异常增生程度的关系，研究发现DDX5和METTL3在OLK组织中的染色强度显著高于正常组织，显著低于OSCC组织，且中重度异常增生组DDX5和METTL3的mRNA表达量明显高于正常对照组（P<0.05）。进一步在细胞水平考察其在调控口腔黏膜上皮异常增殖中的作用，结果发现沉默DDX5基因后DOK细胞和CAL-27细胞的存活率及增殖速度降低、迁移能力显著降低、细胞凋亡显著增加（P<0.05）。然后以METTL3为切入点，完成METTL3对DDX5调控OLK异常增殖影响的分子机制研究，结果显示沉默DDX5基因后p-p38及METTL3表达显著降低。采用IPA软件（Ingenuity Pathway Analysis）进行DDX5与METTL3相关性及通路分析，经细胞实验验证发现DDX5与METTL3之间可能通过TIMP1产生相互作用。最后通过组织样本m6A甲基化测序，显示OLK组织中甲基化程度高的基因主要参与FOXO信号通路，甲基化程度低的m6A基因主要参与PI3K/Akt信号通路、癌症、剪接体、内质网蛋白加工和内吞作用。将24个具有差异甲基化峰的基因和17个m6A调节因子进行IPA分析显示METTL14、METTL3、Akt、PI3K和SMAD2/3与WTAP形成相关性网络。.研究不仅有助于阐明m6A修饰影响RBP-RNA相互作用在OLK衍进中的效应及新机制，而且通过对细胞增殖失衡新机制的解析，也为逆转表型、靶向阻断提供了新思路。研究结果有助于将防控关口前移，通过阻断口腔白斑病的发展衍进，优化现有的诊疗策略并获得良好预后。同时，由于OLK病源丰富、病例集中、部位表浅、阶段明确且易于观察，也可为其他增殖失衡相关疾病临床防治新手段的开发提供参考。