mRNA methylation level GSEA-GOBP.

Background Haemoglobin H (HbH) disease is caused by a disorder of α-globin synthesis, and it results in a wide range of clinical symptoms. M6A methylation modification may be one of the mechanisms of heterogeneity. Therefore, this article explored the role of methyltransferase like 16 (METTL16) in HbH disease. Method The results of epigenetic transcriptome microarray were analysed and verified through bioinformatic methods and qRT-PCR, respectively. The overexpression or knock down of METTL16 in K562 cells was examined to determine its role in reactive oxygen species (ROS), cell cycle processes or iron overload. YTH domain family protein 3 (YTHDF3) was knocked down in K562 cells and K562 cells overexpressing METTL16 via siRNA to investigate its function. In addition, haemoglobin expression was detected through benzidine staining. qRT-PCR, WB, methylated RNA Immunoprecipitation (MeRIP) and (RNA Immunoprecipitation) RIP experiments were conducted to explore the mechanism of intermolecular interaction. Results METTL16, YTHDF3 and solute carrier family 5 member 3 (SLC5A3) mRNA and the methylation level of SLC5A3 mRNA were downregulated in HbH patients. Insulin-like growth factor 2 mRNA-binding protein 3 (IGF2BP3) mRNA expression was negatively correlated with HGB content among patients with HbH-CS disease. Overexpression of METTL16 increased ROS and intracellular iron contents in K562 cells, changed the K562 cell cycle, reduced hemin-induced haemoglobin synthesis, increased the expressions of SLC5A3 and HBG and increased SLC5A3 mRNA methylation levels. Knockdown of METTL16 reduced ROS and intracellular iron contents in K562 cells. Hemin treatment of K562 cells for more than 14 days reduced the protein expressions of METTL16 and SLC5A3 and SLC5A3 mRNA methylation levels. Knockdown of YTHDF3 rescued the intracellular iron content changes induced by the overexpression of METTL16. The RIP experiment revealed that SLC5A3 mRNA can be enriched by METTL16 antibody. Conclusion METTL16 may affect the expression of SLC5A3 by changing its m6A modification level and regulating ROS synthesis, intracellular iron and cycle of red blood cells. Moreover, METTL16 possibly affects the expression of haemoglobin through IGF2BP3, which regulates the clinical phenotype of HbH disease.背景 血红蛋白H（HbH）病是由α-珠蛋白合成障碍引发的疾病，其临床症状表现出广泛的异质性。N6-甲基腺嘌呤（m6A）甲基化修饰可能是导致这种异质性的机制之一。因此本研究探讨甲基转移酶样16（METTL16）在HbH病中的作用。 方法 本研究通过生物信息学分析与实时荧光定量聚合酶链反应（qRT-PCR）分别对表观转录组芯片结果进行分析与验证。在K562细胞中过表达或敲低METTL16，以探究其对活性氧（ROS）、细胞周期进程或铁过载的影响。通过小干扰RNA（siRNA）分别在K562细胞以及过表达METTL16的K562细胞中敲低YTH结构域家族蛋白3（YTHDF3），以研究其功能。此外，采用联苯胺染色检测血红蛋白表达情况。通过qRT-PCR、蛋白质印迹（WB）、甲基化RNA免疫沉淀（MeRIP）以及RNA免疫沉淀（RIP）实验，探究分子间相互作用的机制。 结果 在HbH病患者体内，METTL16、YTHDF3以及溶质载体家族5成员3（SLC5A3）的mRNA表达水平，以及SLC5A3 mRNA的甲基化水平均出现下调。在HbH-CS病患者中，胰岛素样生长因子2 mRNA结合蛋白3（IGF2BP3）的mRNA表达水平与血红蛋白（HGB）含量呈负相关。在K562细胞中过表达METTL16可提升ROS水平与细胞内铁含量，改变K562细胞周期，抑制血红素诱导的血红蛋白合成，上调SLC5A3与HBG的表达，并提升SLC5A3 mRNA的甲基化水平。敲低METTL16则会降低K562细胞内的ROS水平与细胞内铁含量。用血红素处理K562细胞超过14天，可降低METTL16与SLC5A3的蛋白表达水平，以及SLC5A3 mRNA的甲基化水平。敲低YTHDF3可挽救由METTL16过表达诱导的细胞内铁含量变化。RNA免疫沉淀实验结果显示，METTL16抗体可富集SLC5A3 mRNA。 结论 METTL16可能通过改变SLC5A3的m6A甲基化修饰水平，调控ROS合成、细胞内铁稳态以及红细胞周期，进而影响SLC5A3的表达。此外，METTL16或可通过IGF2BP3调控血红蛋白的表达，从而影响HbH病的临床表型。