Department of Orthopedics, The Provincial Hospital Affiliated To Shandong University, Shandong, 250014, China.
Calcif Tissue Int. 2020 May;106(5):486-493. doi: 10.1007/s00223-019-00654-6. Epub 2020 Jan 2.
Ossification of the ligamentum flavum (OLF) is characterized by a process of ectopic bone formation in the ligamentum flavum. The definitive pathophysiology of OLF still remains unclear, but the epigenetic mA modification plays an important role in OLF. In addition, no studies have reported the function of ALKBH5 in OLF development. In this study, we investigated the function of the mA demethylation enzyme ALKBH5 in OLF. To evaluate the function of ALKBH5, OLF tissues and normal ligamentum flavum tissues were collected. In vitro methods, including HE, IHC and western blotting assays, were used to evaluate the association of ALKBH5 with OLF. In addition, we verified the effects of ALKBH5 on osteogenesis using alizarin red and ALP staining. MeRIP q-PCR was performed to investigate the methylation level of BMP2. Moreover, the mechanism of ALKBH5-mediated regulation of the ossification of the ligamentum flavum cells through the AKT signaling pathway was also verified. The present study showed that the expression of ALKBH5 increased in OLF tissues. The overexpression of ALKBH5 increased the expression of osteogenic genes and promoted the ossification of ligamentum flavum cells. Furthermore, BMP2 was significantly enriched in the ligamentum flavum cells of the anti-mA group compared with those of the IgG group. The overexpression of ALKBH5 led to the activation of p-AKT, and BMP2 was regulated by ALKBH5 through the AKT signaling pathway. ALKBH5 promoted the osteogenesis of the ligamentum flavum cells through BMP2 demethylation and AKT activation. ALKBH5 was shown to be an important demethylation enzyme in OLF development.
黄韧带骨化（OLF）的特征是黄韧带中异位骨形成的过程。OLF 的明确病理生理学仍然不清楚，但表观遗传 mA 修饰在 OLF 中起着重要作用。此外，尚无研究报道 ALKBH5 在 OLF 发展中的作用。在这项研究中，我们研究了 mA 去甲基化酶 ALKBH5 在 OLF 中的功能。为了评估 ALKBH5 的功能，收集了 OLF 组织和正常黄韧带组织。体外方法，包括 HE、免疫组织化学和 Western blot 分析，用于评估 ALKBH5 与 OLF 的关联。此外，我们使用茜素红和 ALP 染色来验证 ALKBH5 对成骨的影响。MeRIP q-PCR 用于研究 BMP2 的甲基化水平。此外，还验证了 ALKBH5 通过 AKT 信号通路调节黄韧带细胞骨化的机制。本研究表明，ALKBH5 在 OLF 组织中的表达增加。ALKBH5 的过表达增加了成骨基因的表达，并促进了黄韧带细胞的骨化。此外，与 IgG 组相比，抗-mA 组的黄韧带细胞中 BMP2 明显富集。ALKBH5 的过表达导致 p-AKT 的激活，并且 BMP2 通过 AKT 信号通路被 ALKBH5 调节。ALKBH5 通过 BMP2 去甲基化和 AKT 激活促进黄韧带细胞的成骨作用。ALKBH5 被证明是 OLF 发展中的重要去甲基化酶。
