Eye Institute, Eye & ENT Hospital, Shanghai Medical College, Fudan University, Shanghai, China.
The Affiliated Eye Hospital, Nanjing Medical University, Nanjing, China.
Theranostics. 2022 Jan 1;12(1):277-289. doi: 10.7150/thno.63441. eCollection 2022.
Microvascular complication is a major cause of morbidity and mortality among the patients with diabetes. Pericyte dysfunction is the predominant pathological manifestation of microvascular complication. -methyladenosine (mA) serves as the most prevalent modification in eukaryotic mRNAs. However, the role of mA RNA modification in pericyte dysfunction is still unclear. Quantitative polymerase chain reactions and western blots were conducted to detect the change of mA RNA modification in pericytes and mouse retinas following diabetic stress. MTT assay, transwell migration assay, caspase 3/7 activity assay, calcein-AM/propidium iodide (PI) staining, and TUNEL staining were conducted to determine the role of METTL3 in pericyte biology . Retinal trypsin digestion, vascular permeability assay, and IB4-NG2 double immunofluorescent staining were conducted to determine the role of METTL3 in retinal pericyte dysfunction and vascular complication. RNA sequencing, RNA pull-down assays and immunoblots were conducted to clarify the mechanism of METTL3-mediated pericyte dysfunction and vascular complication. The levels of mA RNA methylation were significantly up-regulated in pericytes and mouse retinas following diabetic stress, which were caused by increased expression of METTL3. METTL3 regulated the viability, proliferation, and differentiation of pericytes . Specific depletion of METTL3 in pericytes suppressed diabetes-induced pericyte dysfunction and vascular complication . METTL3 overexpression impaired pericyte function by repressing PKC-η, FAT4, and PDGFRA expression, which was mediated by YTHDF2-dependent mRNA decay. METTL3-mediated mA methylation epigenetically regulates diabetes-induced pericyte dysfunction. METTL3-YTHDF2-PKC-η/FAT4/PDGFRA signaling axis could be therapeutically targeted for treating microvascular complications.
微血管并发症是糖尿病患者发病率和死亡率的主要原因。周细胞功能障碍是微血管并发症的主要病理表现。 -甲基腺苷（mA）是真核 mRNA 中最普遍的修饰。然而，mA RNA 修饰在周细胞功能障碍中的作用尚不清楚。 定量聚合酶链反应和蛋白质印迹用于检测糖尿病应激后周细胞和小鼠视网膜中 mA RNA 修饰的变化。MTT 测定、Transwell 迁移测定、caspase 3/7 活性测定、钙黄绿素 AM/碘化丙啶（PI）染色和 TUNEL 染色用于确定 METTL3 在周细胞生物学中的作用。视网膜胰蛋白酶消化、血管通透性测定和 IB4-NG2 双重免疫荧光染色用于确定 METTL3 在视网膜周细胞功能障碍和血管并发症中的作用。RNA 测序、RNA 下拉测定和免疫印迹用于阐明 METTL3 介导的周细胞功能障碍和血管并发症的机制。 在糖尿病应激后，周细胞和小鼠视网膜中的 mA RNA 甲基化水平显著上调，这是由 METTL3 表达增加引起的。METTL3 调节周细胞的活力、增殖和分化。周细胞中 METTL3 的特异性耗竭抑制了糖尿病诱导的周细胞功能障碍和血管并发症。METTL3 通过抑制 PKC-η、FAT4 和 PDGFRA 表达来损害周细胞功能，这是由 YTHDF2 依赖性 mRNA 降解介导的。METTL3 介导的 mA 甲基化通过表观遗传调控糖尿病诱导的周细胞功能障碍。METTL3-YTHDF2-PKC-η/FAT4/PDGFRA 信号轴可作为治疗微血管并发症的治疗靶点。
