ALKBH5 Regulates Osteogenic Differentiation via the lncRNA/mRNA Complex.

ABSTRACT

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.