脊髓损伤后m6A甲基化修饰在神经干细胞自发分化中的调控作用

目的探讨脊髓损伤后mRNA m6A甲基化修饰对神经干细胞自发分化的调控作用。方法8周龄C57小鼠构建脊髓钳夹损伤模型，采用后肢运动功能评分（basso mouse scale, BMS）评价小鼠脊髓钳夹损伤模型构建情况；脊髓组织冰冻切片免疫荧光染色检测脊髓损伤1、3d后，损伤位点周围神经干细胞的数量及m6A甲基化水平；microRNA干扰技术敲低神经干细胞内的METTL14蛋白表达，并利用细胞免疫荧光检测神经干细胞自发分化后，神经元细胞、少突胶质细胞及星形胶质细胞数量；Western印迹法及Northern印迹法检测髓鞘碱性蛋白（myelin basic protein, MBP）与神经干细胞共培养后神经干细胞内METTL3、METTL14、FTO及ALKBH5蛋白表达情况及细胞内m6A甲基化水平。结果BMS评分结果表明小鼠脊髓钳夹损伤模型构建成功。免疫蛋白印迹结果显示，脊髓损伤后组织内脊髓损伤后星形胶质细胞的细胞标志蛋白GFAP显著增加，神经元标志蛋白NeuN和少突胶质细胞标志蛋白MBP表达显著降低。组织免疫荧光结果表明，损伤早期受损位点周围神经干细胞标志蛋白NESTIN达量增加，但m6A水平减少。细胞免疫荧光结果表明，m6A甲基化水平降低可促进神经干细胞神经向星形胶质细胞的自发分化。免疫蛋白印迹结果显示，MBP处理神经干细胞可使细胞内METTL3/METTL14表达量及细胞整体m6A甲基化水平降低。结论脊髓损伤后mRNA m6A甲基化降低可促进神经干细胞向星形胶质细胞分化。

mRNA m6A methylation modification plays crucial roles in spontaneous neural differentiation following spinal cord injury

ObjectiveTo investigate mRNA m6A methylation(m6A) in regulation of spontaneous neural stem cells(NSCs) differentiation after spinal cord injury(SCI). MethodsThe model of spinal cord clamp injury was established and verified by basso-mouse-scale(BMS) in 8-week-old C57 mice. Immunoblotting was applied to detect the expression of marker proteins of neural stem cells, neurons, microglia, oligodendrocytes and astrocytes. The number of NSCs and the m6A level around the injury site were detected by immunofluorescence staining in frozen tissue sections at 1 and 3 days after SCI. The expression of METTL14 protein in NSCs was knocked down by small RNA interference technique, and the number of neuronal cells, oligodendrocytes and astrocytes after spontaneous differentiation of NSCs was counted by cellular immunofluorescence. Immunoblotting and RNA dot blotting were applied to detect the expression levels of METTL3, METTL14, FTO and ALKBH5 and m6A in neural stem cells co-cultured with myelin basic protein. ResultsThe BMS score showed that the model of spinal cord clamp injury was established successfully. Immunoblotting results showed that the expression of GFAP was significantly increased, while NeuN and MBP were significantly decreased following SCI. Immunofluorescence results showed that the expression of NESTIN around the damaged sites was increased but the level of m6A decreased. The results of immunofluorescence showed that the reduction of m6A methylation promoted the spontaneous differentiation of NSCs into astrocytes. Immunoblotting results showed that MBP treatment of neural stem cells decreased the expression levels of METTL3/METTL14 and the global m6A. ConclusionDecreased mRNA m6A methylation can promote the differentiation of NSCs into astrocytes after SCI.