腺嘌呤去甲基化酶FTO对2型糖尿病糖脂代谢关键基因的调控作用

N6-methyladenosine (m6A) is the most prevalent internal modification in mRNA, which has a fundamental role in regulation of gene expression. Recent studies demonstrated that the fat mass and obesity-associated (FTO) protein, as a risk factor for type 2 diabetes mellitus (T2DM) and obesity, is an m6A-demethylase. Our previous studies have already demonstrated that a significant higher expression level of FTO and lower m6A content were in peripheral blood of T2DM patients and diabetic rats, compared with the normal controls; our results further confirmed that m6A content was not only negatively correlated with fasting blood glucose and triglycerides (TG), but correlated with high-density lipoprotein cholesterol (HDL-C), which implied that decreased m6A content was a risk factor for T2DM. Thus, we hypothesize that FTO regulates m6A dynamic modification and affects the expression levels of the regulatory genes involved in glucose and lipid metabolism, which will lead to impaired functional glucose homeostasis and lipid disorder. To verify the hypothesis, we will analyze T2DM specific expression profiles with contained m6A and confirm FTO plays a key regulation function in those genes involved in glucose and lipid metabolism using m6A-seq and liquid chromatography-electrospray ionization-tandem mass spectrometry (LC-ESI-MS/MS) analysis. These results will provide new insights into the pathogenesis of impaired glucose homeostasis and lipid disorder induced by mRNA methylation and novel ideals for the prevention of T2DM and its complications.

N6-甲基腺嘌呤(m6A)是mRNA修饰中最常见类型，在调控基因表达中起重要作用。最新研究表明，与2型糖尿病(T2DM)和肥胖相关的FTO基因编码的蛋白是m6A的特异性去甲基化酶。我们前期发现：①T2DM患者外周血FTO基因高表达，m6A含量明显下降，在糖尿病大鼠血液中存在相同的现象；②m6A含量与空腹血糖、甘油三酯负相关，而与高密度脂蛋白胆固醇正相关；③m6A含量下降是T2DM的风险因子。据此假设：去甲基化酶FTO通过调控m6A修饰而影响糖脂代谢关键基因表达，导致T2DM糖脂代谢紊乱。本课题拟采用最新的m6A修饰的mRNA深度测序(m6A-seq)和液质联用(LC-ESI-MS/MS)技术，从个体、细胞及动物水平明确FTO对糖脂代谢关键基因的调控作用，发现T2DM特异性的差异甲基化基因。本研究将从RNA甲基化新角度探讨糖脂代谢紊乱的发生机制，为T2DM及其并发症的防治提供新思路。

糖尿病是全球性的卫生和经济问题，95%以上患者是2型糖尿病，在中国成年人中的发病率近11%。心血管事件是2型糖尿病就诊和死亡的主要因素，而糖脂代谢紊乱是主要诱因。N6-甲基腺嘌呤(m6A)是mRNA修饰中最常见类型，在调控基因表达中起重要作用。研究m6A对T2DM糖脂代谢关键基因的调控作用，为糖尿病合并心血管疾病的防治提供新的分子靶标，具有重要的社会、经济和科学意义。然而，目前研究T2DM外周血表观遗传学改变的报道仍很少。. 围绕项目的总体研究目标，开展m6A高通量测序（m6A-Seq）、DNA修饰、RNA修饰、脂肪酸、microRNA和SNP的高灵敏检测方法学研究，一方面应用这些新发展的方法检测临床糖尿病患者血液标本，另一方面在细胞水平及糖尿病动物模型中进行研究，探索DNA, RNA表观遗传学、miR-33及脂代谢关键基因SNP在糖尿病、糖尿病脂代谢紊乱、糖尿病心血管疾病中的可能作用。. 通过研究，成功进行糖尿病外周血m6A-Seq，数据正在分析中；成功建立了高灵敏的液质联用技术平台，检测人类外周血、DNA、RNA甲基化、羟甲基化修饰检测；完成细胞模型和动物模型的构建；建立HRM技术的DNA突变和SNP分型的高灵敏检测方法，并应用于临床标本检测；发现糖尿病合并冠心病患者的血浆脂肪酸特征谱，发现m6A和5hmC分别具有作为糖尿病诊断以早期预警的分子标志物价值，明确去甲基化酶FTO对m6A及糖脂代谢关键基因（G6PC，FASN，FOXO1，DGAT2）的调控。