2型糖尿病患者m^(6)A腺苷的动态调节及其发生机制

Dynamic regulation of m^(6)A in patients with type 2 diabetes mellitus and its mechanism

目的观察高血糖因素对2型糖尿病(T2DM)患者外周血mRNA N6甲基腺苷(m^(6)A)的动态调节,并分析其可能发生机制。方法选择南京中医药大学附属医院2017年收治的60例T2DM患者和体检的65例与T2DM患者年龄、性别相匹配的健康人为研究对象,采集受试者空腹外周静脉血,采用红细胞裂解液处理后,分离收集白细胞,进一步提取RNA。首先选取了9例空腹血糖正常、高血糖或低血糖的患者,采用液相色谱-电喷雾电离质谱法(LC-ESIMS)检测患者血样中RNA的m^(6)A含量;同时qPCR测定甲基化酶和去甲基化酶的mRNA水平。另外对高糖处理的HepG2和FTO敲除或过表达的HepG2细胞,再一次证实相关的研究。结果T2DM患者的m^(6)A降低,FTO、METL3和METL14 mRNA表达水平升高,m^(6)A含量则显著降低。然而在HepG2细胞中,高糖上调FTO蛋白对METL3或METL14无显著影响,FOXO1、G6PC和DGAT2的mRNA表达水平均显著增多。结论T2DM患者高糖升高促进FTO mRNA表达,从而导致m^(6)A含量降低。而甲基转移酶上调的原因又极有可能是m^(6)A含量降低导致的。此外,FTO会诱导G6PC、FOXO1和DGAT2的mRNA表达改变,而这些基因同葡萄糖脂质的代谢紧密相关,由此显示m^(6)A的改变与糖代谢有很大的关系。

Objective To observe the dynamic regulation of hyperglycemia on the mRNA N6-methyladenosine(m^(6)A)in peripheral blood of patients with type 2 diabetes mellitus(T2DM),and to analyze the possible mechanism.Methods We selected a total of 60 patients with T2DM as the study subjects,who were admitted to the Affiliated Hospital of Nanjing University of Chinese Medicine in 2017.And we also selected 65 healthy subjects who had physical examination with matched age and gender of T2DM patients.The fasting peripheral venous blood of the subjects was collected.After being treated with erythrocyte lysate,white blood cells were separated and collected for further RNA extraction.First,9 patients with normal fasting glucose,hyperglycemia or hypoglycemia were selected for detecting the content of RNA m^(6)A in their blood samples by liquid chromatography-electrospray ionization mass spectrometry(LC-ESIMS).The mRNA levels of methylase and demethylase were measured by qPCR.In addition,relevant studies were further confirmed in HepG2 cells treated with high glucose and HepG2 cells with FTO knockout or over-expression.Results In T2DM patients,their m^(6)A decreased,while the mRNA expressions of FTO,METL3 and METL14 increased,and the content of m^(6)A significantly decreased.However,in HepG2 cells,the up-regulation of FTO protein by high glucose had no significant effect on METL3 or METL14,and the mRNA expressions of FOXO1,G6PC,and DGAT2 significantly increased.Conclusion In patients with T2DM,high glucose promotes the mRNA expression of FTO,leading to a decrease in m 6A.The up-regulation of methyltransferase is probably caused by the decrease of m^(6)A content.In addition,FTO induces changes in mRNA expressions of G6PC,FOXO1,and DGAT2 and these genes are closely related to glucose lipid metabolism.It indicates that changes in m^(6)A are highly correlated with glucose metabolism.