Luo Yunfei, Li Jin-E, Xu Shan, Zeng Haixia, Zhang Yuying, Yang Shiqi, He Xiaoju, Liu Jianping
Department of Endocrinology and Metabolism of the Second Affiliated Hospital, Jiangxi Medical College, Nanchang University, Nanchang City, Jiangxi Province, China; School of basic medicine, Nanchang Medical College, Nanchang City, Jiangxi Province, China.
Department of Endocrinology and Metabolism of the Second Affiliated Hospital, Jiangxi Medical College, Nanchang University, Nanchang City, Jiangxi Province, China.
Biochim Biophys Acta Mol Basis Dis. 2025 Aug;1871(6):167904. doi: 10.1016/j.bbadis.2025.167904. Epub 2025 May 13.
Generating mature β-cells from stem cells remains a significant challenge in diabetes cell therapy. Human amniotic epithelial stem cells (hAESCs) have made their mark in regenerative medicine, and provide several advantages compared to other stem cells. Methyltransferase-like 3 (METTL3), an essential RNA methyltransferase participating in N6-methyladenosine (m6A) mRNA methylation, plays a critical role in the normal development of β-cells, yet its deletion in β-cells leads to β-cell dysfunction and hyperglycemia.
In this study, we isolated and characterized hAESCs from human amniotic membranes, differentiated these hAESCs into insulin-producing cells (IPCs), and explored the role of METTL3 in such differentiation. We examined the expression of METTL3 and insulin like growth factor 2 mRNA binding protein 2 (IGF2BP2, a decodes m6A methylation "reader") in the generated IPCs. Subsequently, we suppressed METTL3 using an inhibitor (STM2457) and overexpressed METTL3 via plasmid transfection (METTL3-OE). The differentiated STM2457 and METTL3-OE IPCs were compared to normal induction (WT) IPCs regarding the expression of β-cell markers by RT-qPCR and western blotting, immunofluorescence, C-peptide release, and glucose-stimulated insulin secretion (GSIS). Methylated RNA immunoprecipitation (MeRIP)-qPCR was used to examine the molecular mechanism underlying METTL3/m6A signaling axis in MaFA (endocrine pancreatic β-cells marker) expression. We examined the potential therapeutic uses and efficacy of IPCs through streptozotocin (STZ)-induced C57BL/6 DM.
Isolated hAESCs displayed all characteristics of ESCs and could generate IPCs. METTL3 and IGF2BP2 were elevated during differentiation. Overexpressing METTL3 improved the expression of β-cell markers in the final differentiated IPCs, improved C-peptide release, and demonstrated increased insulin secretion upon challenging with high glucose conditions, whereas inhibiting METTL3 attenuated these effects. Moreover, METTL3 modulated the MaFA expression in an m6A-dependent manner.
These findings suggest METTL3 as a promoting factor of IPCs generation, with its up-regulation potentially generating more mature IPCs for hAESCs therapy of diabetes mellitus.
从干细胞生成成熟的β细胞仍然是糖尿病细胞治疗中的一项重大挑战。人羊膜上皮干细胞（hAESCs）在再生医学领域崭露头角，与其他干细胞相比具有若干优势。甲基转移酶样3（METTL3）是一种参与N6-甲基腺苷（m6A）mRNA甲基化的重要RNA甲基转移酶，在β细胞的正常发育中起关键作用，然而其在β细胞中的缺失会导致β细胞功能障碍和高血糖。
在本研究中，我们从人羊膜中分离并鉴定了hAESCs，将这些hAESCs分化为胰岛素生成细胞（IPCs），并探讨了METTL3在这种分化中的作用。我们检测了所生成的IPCs中METTL3和胰岛素样生长因子2 mRNA结合蛋白2（IGF2BP2，一种解码m6A甲基化的“读取器”）的表达。随后，我们使用抑制剂（STM2457）抑制METTL3，并通过质粒转染（METTL3-OE）过表达METTL3。通过RT-qPCR和蛋白质印迹、免疫荧光、C肽释放以及葡萄糖刺激的胰岛素分泌（GSIS），将分化的STM2457和METTL3-OE IPCs与正常诱导（WT）IPCs在β细胞标志物的表达方面进行比较。甲基化RNA免疫沉淀（MeRIP）-qPCR用于研究METTL3/m6A信号轴在MaFA（内分泌胰腺β细胞标志物）表达中的分子机制。我们通过链脲佐菌素（STZ）诱导的C57BL/6糖尿病模型研究了IPCs的潜在治疗用途和疗效。
分离出的hAESCs表现出胚胎干细胞的所有特征，并能生成IPCs。在分化过程中，METTL3和IGF2BP2表达升高。过表达METTL3可改善最终分化的IPCs中β细胞标志物 的表达，改善C肽释放，并在高糖条件刺激下显示胰岛素分泌增加，而抑制METTL3则减弱了这些作用。此外，METTL3以m6A依赖的方式调节MaFA的表达。
这些发现表明METTL3是IPCs生成的促进因子，上调其表达可能为hAESCs治疗糖尿病生成更多成熟的IPCs。
