Zhu Mengqi, Tao Zhengyong, Duan XingYu, Feng Lijun, Ma Xiaojun, Wang Jiong, Shi ZhiYun, Zhang Xu, Niu NingKui
Department of Orthopedic, General Hospital of Ningxia Medical University, Ningxia Hui Autonomous Region, No.804 Shengli Street, Yinchuan, 750004, China.
Ningxia Medical University, Ningxia Hui Autonomous Region, Yinchuan, 750004, China.
J Orthop Surg Res. 2025 Aug 20;20(1):779. doi: 10.1186/s13018-025-06204-1.
N6-methyladenosine (m6A) is the most common and abundant internal modification in RNA. However, the role of m6A in spinal tuberculosis (STB) remains incompletely elucidated. In our previous study, miRNA-seq was performed on peripheral blood and tissues from STB patients, and miR-29a-3p was identified as differentially expressed in STB patients through screening. In this study, we mainly explored the regulation of miR-29a-3p by ALKB homolog 5 (ALKBH5) in STB.
Tissue specimens were obtained from 20 patients with lumbar degenerative disease and 20 patients with STB. The expression levels of ALKBH5 and miR-29a-3p in STB were assessed using qRT-PCR, immunohistochemistry, and immunofluorescence assays. MeRIP analyses were performed to investigate the role of ALKBH5 in regulating the m6A modification of miR-29a-3p. Additionally, Western blot, ELISA, and qRT-PCR techniques were employed to validate the regulatory mechanism of ALKBH5-mediated miR-29a-3p in the inflammatory response associated with STB.
ALKBH5 was upregulated in both spinal tuberculosis tissues and cellular models, whereas miR-29a-3p exhibited marked downregulation. Inhibition of miR-29a-3p expression led to increased levels of tumor necrosis factor-α (TNF-α), interleukin-1β (IL-1β), and interleukin-17 A (IL-17 A). Conversely, overexpression of miR-29a-3p effectively suppressed the production of inflammatory factors. Furthermore, ALKBH5 was found to directly target miR-29a-3p and regulate its methylation modification, thereby inhibiting the maturation of miR-29a-3p. Additionally, ALKBH5 suppressed the expression of miR-29a-3p, which in turn promoted the release of inflammatory factors associated with spinal tuberculosis.
N6-甲基腺苷（m6A）是RNA中最常见且含量丰富的内部修饰。然而，m6A在脊柱结核（STB）中的作用仍未完全阐明。在我们之前的研究中，对STB患者的外周血和组织进行了miRNA测序，并通过筛选确定miR-29a-3p在STB患者中差异表达。在本研究中，我们主要探讨了ALKB同源物5（ALKBH5）对STB中miR-29a-3p的调控作用。
从20例腰椎退行性疾病患者和20例STB患者中获取组织标本。采用qRT-PCR、免疫组织化学和免疫荧光分析评估STB中ALKBH5和miR-29a-3p的表达水平。进行MeRIP分析以研究ALKBH5在调节miR-29a-3p的m6A修饰中的作用。此外，采用蛋白质免疫印迹、酶联免疫吸附测定和qRT-PCR技术验证ALKBH5介导的miR-29a-3p在与STB相关的炎症反应中的调控机制。
ALKBH5在脊柱结核组织和细胞模型中均上调，而miR-29a-3p则显著下调。抑制miR-29a-3p表达导致肿瘤坏死因子-α（TNF-α）、白细胞介素-1β（IL-1β）和白细胞介素-17A（IL-17A）水平升高。相反，miR-29a-3p的过表达有效抑制了炎症因子的产生。此外，发现ALKBH5直接靶向miR-29a-3p并调节其甲基化修饰，从而抑制miR-29a-3p的成熟。此外，ALKBH5抑制miR-29a-3p的表达，进而促进与脊柱结核相关的炎症因子的释放。
