m6A去甲基转移酶FTO调控自噬活化信号介导口腔鳞癌恶性进展的分子机制

Autophagy plays a pivotal role in the malignant progression of cancer. However, the exact molecular mechanisms which were involved in autophagy-mediated invasion and metastasis are still not clear. In our previous studies, we have confirmed that suppression of autophagic activity could enhance cancer malignant potentials in oral squamous cell carcinoma (OSCC). Interestingly, we also observed that the level of RNA methylation was increased while the m6A demethylase (FTO) was decreased in autophagy inducted by starvation and Rapamycin. Further studies showed that silence of FTO may enhance autophagic activity and inhibit cell proliferation, migration and invasion in OSCC. And the inhibitory effect of silencing FTO was partially rescued by 3-MA. These results suggested that FTO may regulate autophagy to promote malignant progression in OSCC. In this project, we aim to screen the key autophagy-related gene(s) which may be regulated by FTO based on meRIP-seq and RNA-seq technique. Then the key m6A binding protein of the target mRNA would be isolated and identified based on pull-down and High-performance liquid chromatography-mass spectrometry technology. In order to clarifying the regulatory role of FTO in autophagy and malignant progression of OSCC, the vivo and vitro experiment would be also involved. This project may illuminate the regulatory effect of RNA methylation on malignant progression of OSCC and may open new avenues for cancer therapy.

自噬与肿瘤恶性进展密切相关，但其确切分子调控机制仍不清楚。本课题组前期系列研究证实：抑制自噬活性可促进口腔鳞癌侵袭转移；在饥饿和雷帕霉素诱导自噬过程中，RNA甲基化水平上升，m6A去甲基转移酶FTO表达显著降低；沉默FTO可抑制口腔鳞癌细胞侵袭转移，增强自噬活性；若同时加入3-MA抑制自噬，则可部分解除FTO沉默对口腔鳞癌侵袭转移的抑制。基于上述发现，我们推测：FTO可通过调控自噬活性介导口腔鳞癌的恶性进展。本项目拟在上述研究基础上，利用meRIP-seq和RNA-seq技术分析在FTO影响口腔鳞癌恶性进程中发挥效应的自噬关键基因，同时采用pull-down技术和高效液相色谱-质谱联用仪分离和鉴定影响上述自噬关键基因mRNA稳定性的m6A结合蛋白，并结合体内外实验验证，阐明FTO调控自噬关键基因促进口腔鳞癌恶性进展的分子机制，为口腔鳞癌恶性进展的干预和治疗提供实验依据。

口腔鳞状细胞癌是头颈部最常见的恶性肿瘤。自噬是细胞通过溶酶体降解内源性底物的重要过程，自噬活性改变与口腔鳞癌恶性进展密切相关。N6-甲基腺嘌呤是真核生物mRNA上含量最丰富的甲基化修饰形式，对众多肿瘤的发生发展至关重要。在本项目中，我们试图阐明m6A去甲基转移酶FTO对口腔鳞癌自噬和恶性进展的功能，并探究FTO调控自噬的分子机制。我们发现口腔鳞癌组织中m6A水平低于癌旁非肿瘤组织，FTO mRNA水平高于癌旁非肿瘤组织。雷帕霉素处理的细胞m6A水平上调，FTO蛋白和mRNA水平均上调。沉默FTO的细胞m6A水平上调，自噬水平上调，迁移、侵袭、增殖能力下调，皮下移植瘤质量、体积下调，肺部转移瘤数量减少。分析meRIP-seq，eIF4G1的m6A修饰改变位点位于CDS第11外显子区域。雷帕霉素处理的细胞中eIF4G1 m6A修饰上调，mRNA表达下调。沉默FTO增强eIF4G1 mRNA稳定性，过表达YTHDF2降低eIF4G1 mRNA稳定性。FTO的催化位点和YTHDF2的结合位点均为CDS第11外显子1号m6A位点（1044）。沉默eIF4G1的细胞自噬水平上调，迁移、侵袭、增殖能力下调。口腔鳞癌组织中FTO和eIF4G1蛋白水平均高于癌旁非肿瘤组织，二者表达呈正相关。我们的研究可为口腔鳞癌恶性进展的干预和治疗提供新的思路和实验依据。