He Xin, Zhang Jia'nan, Guo Yunshan, Yang Xiaowei, Huang Yunfei, Hao Dingjun
Department of Spine Surgery, Hong Hui Hospital, Xi'an Jiaotong University Health Science Center, No. 555, Youyi East Road, Beilin District, Xi'an, 710054, China.
Mol Neurobiol. 2023 May;60(5):2413-2425. doi: 10.1007/s12035-023-03222-0. Epub 2023 Jan 19.
Previous reports showed that LncRNA D26496 was downregulated and N6-methyladenosine (m6A) methyltransferase METTL3 was upregulated in sciatic nerve injury (SNI). YTH-Domain Family Member 2 (YTHDF2) regulated RNA degradation through recognizing m6A sites. However, whether METTL3-mediated m6A of D26496 plays a role in development of SNI is unknown. Therefore, in this study, we established a rat SNI model and a HO-induced Schwann cell injury model to investigate the role of D26496 in modulating SNI and how the expression of D26496 was regulated during this process. D26496 expression was downregulated in both models. Rats with SNI displayed severe oxidative stress, manifested as increased MDA production and decreased SOD and GSH activity. Moreover, overexpression of D26496 alleviated HO-induced Schwann cell injury likely by promoting cell proliferation and migration and suppressing cell apoptosis and oxidative stress. Mechanism studies found that METTL3 expression was upregulated after SNI, and silencing METTL3 reduced the D26496 m6A level, but upregulated D26496 expression. Subsequent studies found that YTHDF2 was upregulated after SNI, and abundant m6A modified D26496 in the precipitated protein-RNA complexes by anti-YTHDF2 antibody, whereas silencing YTHDF2 promoted D26496 expression but had no effect on m6A levels of D29496. Silencing D26496 reversed the protective effect of knocking down METTL3 or knocking down YTHDF2 on HO-induced cell damage. In vivo, D26496 overexpression alleviated SNI-induced neuropathic pain and oxidative stress. In conclusion, our results suggested that D26496 m6A modification mediated by METTL3 and recognition of D26496 m6A sites by YTHDF2 induced D26496 degradation, thereby participating in the progression of SNI.
先前的报道显示，在坐骨神经损伤（SNI）中，长链非编码RNA D26496表达下调，而N6-甲基腺苷（m6A）甲基转移酶METTL3表达上调。YTH结构域家族成员2（YTHDF2）通过识别m6A位点来调节RNA降解。然而，METTL3介导的D26496的m6A修饰是否在SNI的发展中起作用尚不清楚。因此，在本研究中，我们建立了大鼠SNI模型和过氧化氢（HO）诱导的雪旺细胞损伤模型，以研究D26496在调节SNI中的作用以及在此过程中D26496的表达是如何被调控的。在这两种模型中，D26496的表达均下调。SNI大鼠表现出严重的氧化应激，表现为丙二醛（MDA）生成增加，超氧化物歧化酶（SOD）和谷胱甘肽（GSH）活性降低。此外，D26496的过表达可能通过促进细胞增殖和迁移以及抑制细胞凋亡和氧化应激来减轻HO诱导的雪旺细胞损伤。机制研究发现，SNI后METTL3表达上调，沉默METTL3可降低D26496的m6A水平，但上调D26496的表达。随后的研究发现，SNI后YTHDF2上调，抗YTHDF2抗体沉淀的蛋白质-RNA复合物中存在大量m6A修饰的D26496，而沉默YTHDF2可促进D26496的表达，但对D29496的m6A水平没有影响。沉默D26496可逆转敲低METTL3或敲低YTHDF2对HO诱导的细胞损伤的保护作用。在体内，D26496的过表达减轻了SNI诱导的神经性疼痛和氧化应激。总之，我们的结果表明，METTL3介导的D26496的m6A修饰以及YTHDF2对D26496的m6A位点的识别诱导了D26496的降解，从而参与了SNI的进展。
