Abstract

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 m⁶A 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 m⁶A 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-m⁶A 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.

中文翻译：

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通过AKT信号通路在黄韧带骨化中由m 6 A脱甲基酶ALKBH5修饰的BMP2

摘要

黄韧带骨化（OLF）的特征是黄韧带中异位骨形成的过程。OLF的确切病理生理学仍不清楚，但是表观遗传学m ⁶ A修饰在OLF中起重要作用。另外，没有研究报道ALKBH5在OLF发育中的功能。在这项研究中，我们研究了m ⁶的功能OLF中的脱甲基酶ALKBH5。为了评估ALKBH5的功能，收集了OLF组织和正常的黄韧带组织。体外方法，包括HE，IHC和western blotting分析，用于评估ALKBH5与OLF的关联。此外，我们使用茜素红和ALP染色验证了ALKBH5对成骨的影响。进行了MeRIP q-PCR研究BMP2的甲基化水平。此外，还证实了ALKBH5介导的通过AKT信号通路调节黄韧带骨化的机制。本研究表明ALKBH5在OLF组织中的表达增加。ALKBH5的过表达增加成骨基因的表达，并促进黄韧带细胞的骨化。此外，^6A组与IgG组相比。ALKBH5的过表达导致p-AKT的激活，而BMP2由ALKBH5通过AKT信号通路调节。ALKBH5通过BMP2脱甲基和AKT激活促进黄韧带细胞的成骨。已显示ALKBH5是OLF发育中的重要脱甲基酶。