Human adipose-derived stem cells (hASCs) are commonly used in bone tissue regeneration. The N6-methyladenosine (m6A) modification has emerged as a novel regulatory mechanism for gene expression, playing a critical role in osteogenic differentiation of stem cells. However, the precise role and mechanism of alkylation repair homolog 5 (ALKBH5) in hASC osteogenesis remain incompletely elucidated and warrant further investigation. Herein, we employed methylated RNA immunoprecipitation sequencing, RNA sequencing, and weighted gene coexpression network analysis to identify a key long noncoding RNA (lncRNA) in hASCs: lncRNA AK311120. Functional experiments demonstrated that lnc-AK311120 promoted the osteogenic differentiation of hASCs, while a mutation at the m6A central site A of lnc-AK311120 was found to decrease the level of m6A modification. The osteogenic effect of ALKBH5 was confirmed both in vitro and in vivo using a mandibular defect model in nude mice. Subsequent investigations revealed that knockdown of ALKBH5 resulted in a significant increase in the m6A modification level of lnc-AK311120, accompanied by a downregulation in the expression level of lnc-AK311120. Additional rescue experiments demonstrated that overexpression of lnc-AK311120 could restore the phenotype after ALKBH5 knockdown. We observed that AK311120 interacted with the RNA-binding proteins DExH-Box helicase 9 (DHX9) and YTH domain containing 2 (YTHDC2) to form a ternary complex, while mitogen-activated protein kinase kinase 7 (MAP2K7) served as the shared downstream target gene of DHX9 and YTHDC2. Knockdown of AK311120 led to a reduction in the binding affinity between DHX9/YTHDC2 and the target gene MAP2K7. Furthermore, ALKBH5 facilitated the translation of MAP2K7 and activated the downstream JNK signaling pathway through the AK311120–DHX9–YTHDC2 complex, without affecting its messenger RNA level. Collectively, we have investigated the regulatory effect and mechanism of ALKBH5-mediated demethylation of lncRNA in hASC osteogenesis for the first time, offering a promising approach for bone tissue engineering.

中文翻译：

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ALKBH5 通过 lncRNA/mRNA 复合物调节成骨分化


人脂肪来源的干细胞 （hASCs） 通常用于骨组织再生。N6-甲基腺苷 （m6A） 修饰已成为一种新的基因表达调节机制，在干细胞的成骨分化中起关键作用。然而，烷基化修复同源物 5 （ALKBH5） 在 hASC 成骨中的确切作用和机制仍未完全阐明，需要进一步研究。在此，我们采用甲基化 RNA 免疫沉淀测序、RNA 测序和加权基因共表达网络分析来鉴定 hASCs 中的关键长链非编码 RNA （lncRNA）：lncRNA AK311120。功能实验表明，lnc-AK311120 促进 hASCs 的成骨分化，而发现 lnc-AK311120 的 m6A 中心位点 A 突变降低了 m6A 修饰的水平。使用裸鼠的下颌骨缺损模型在体外和体内证实了 ALKBH5 的成骨作用。随后的研究表明，敲低 ALKBH5 导致 lnc-AK311120 的 m6A 修饰水平显着增加，伴随着 lnc-AK311120 表达水平的下调。其他挽救实验表明，lnc-AK311120 过表达可以恢复 ALKBH5 敲低后的表型。我们观察到AK311120与 RNA 结合蛋白 DExH-Box 解旋酶 9 （DHX9） 和包含 2 的 YTH 结构域 （YTHDC2） 相互作用形成三元复合物，而丝裂原活化蛋白激酶激酶 7 （MAP2K7） 作为 DHX9 和 YTHDC2 的共享下游靶基因。敲低 AK311120 导致 DHX9/YTHDC2 与靶基因 MAP2K7 之间的结合亲和力降低。 此外，ALKBH5 促进 MAP2K7 的翻译，并通过 AK311120-DHX9-YTHDC2 复合物激活下游 JNK 信号通路，而不影响其信使 RNA 水平。总的来说，我们首次研究了 ALKBH5 介导的 lncRNA 去甲基化在 hASC 成骨过程中的调节作用和机制，为骨组织工程提供了一种有前途的方法。