miR-29/33在mtDNA损伤的T2DM羟甲基化紊乱及线粒体功能障碍中的关键调控作用

Type 2 diabetes mellitus (T2DM) is a complex metablic disease and associated with insulin resistance and mitochondrial dysfunction. Our results have investigated that the common mutations of mitochondrial DNA (mtDNA) are G3316A and T3394C in T2DM patients, and the specific mutations include both T3593C and A4833G in one case. Moreover, we also confirmed that elevated plasma levels of total free fatty acids (FFA) and saturated fatty acids with decrease of mtDNA, which is related to HOMA-IR. Recent studies imply that lower mtDNA content is related to DNA methylation disorders; FFA metabolism, mtDNA methylation and insulin signaling are regulated by miR-29a/b and miR-33a/b. We suppose that microRNAs (such as miR-29a/b and -33a/b) as key regulators involve in the DNA methylation disorder and mitochondrial dysfunction in T2DM with mtDNA damages. To verify the hypothesis, we will analyze the levels of 5-methylcytosine and 5-hydroxylmethycytosine of mtDNA in the models of differently expressed miR-29a/b and -33a/b after treatment with different concentrations of glucose, fatty acids and insulin. These results will demonstrate that whether these mutations play a role in the pathogenesis of T2DM, and the effects of miR-29a/b and -33a/b expression on mitochondrial dysfunction and disorder of 5-Hydroxymethylcytosine in Type 2 diabetes with mtDNA damages. Our findings would provide new opportunities for early detection of biomarkers and personalized, targeted treatment of T2DM.

2型糖尿病(T2DM)是一种与胰岛素抵抗和线粒体功能障碍有关的复杂代谢性疾病。我们前期发现：①T2DM的mtDNA突变主要为G3316A和T3394C，T3593C及A4833G突变发生于同一病例；②外周血mtDNA含量下降，与HOMA-IR、血浆FFA及饱和FFA升高相关。最近研究报道mtDNA含量下降与DNA甲基化紊乱有关，miR-29a/b参与FFA代谢及mtDNA甲基化的调控，miR-33a/b调控脂肪酸代谢和胰岛素信号。因此，我们推测miR-29/33在mtDNA损伤的T2DM羟甲基化紊乱及线粒体功能障碍中起关键调控作用。本项目拟在细胞和动物模型中，通过miR-29/33过表达和干扰，以不同因素处理，检测mtDNA甲基化、羟甲基化水平，动态观察糖脂代谢指标、胰岛素分泌及线粒体氧化代谢标志物；明确上述突变是否具有致病性，为T2DM的早期分子诊断和个性化治疗提供新的策略和分子靶标。

线粒体功能障碍诱发的脂肪酸β氧化受损与胰岛素抵抗密切相关。2型糖尿病（Type 2 diabetes mellitus, T2DM）是一种由胰岛素抵抗和胰岛素分泌受损导致的复杂代谢疾病，表现为长期慢性血糖水平升高。遗传和非遗传因素均可促进T2DM的发生与发展。诸多研究表明，表观遗传改变与T2DM及其心血管并发症的发生发展密切相关。与正常人相比，T2DM患者多种组织的基因组DNA甲基化均发生改变，而且非编码RNA对转录后基因表达水平的调节也参与T2DM的发病，如miR33与胰岛素分泌和脂代谢相关。然而，研究T2DM外周血表观遗传学改变的报道仍很少。.为此，本项目开展了多项microRNA, SNP和核酸修饰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T2DM患者血液标本，另一方面在细胞水平及糖尿病动物模型进行研究，探索miR29, miR33, mtDNA, DNA, RNA表观遗传学及脂代谢关键基因SNP在T2DM、糖尿病脂代谢紊乱、糖尿病心血管疾病中的可能作用。具体完成的内容：①建立基于HRM技术高灵敏的DNA突变及SNP基因型检测方法学；②探索正常人群、T2DM、心血管疾病、T2DM合并心血管疾病的血浆脂肪酸亚组份特征谱；③脂肪酸去饱和酶基因SNP基因型与对脂肪酸水平的影响；④DNA甲基化水平与mtDNA拷贝数的相关性研究；⑤发展高灵敏在线富集的液质联用技术平台，准确定量分析DNA甲基化及羟甲基化、RNA甲基化水平；⑥应用液质联用技术平台和DNA-5hmc测序探索正常人群、T2DM、糖尿病合并心血管疾病的DNA，RNA修饰变化规律。.通过研究，成功建立基于HRM技术的DNA突变和SNP分型的高灵敏检测方法，并用于临床标本检测；成功建立高灵敏的液质联用技术平台，并用于组织和血液DNA 、RNA甲基化修饰检测；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及糖尿病心血管疾病早期预警的分子标志物价值。