Transcriptome-wide m⁶A methylome during osteogenic differentiation of human adipose-derived stem cells

Adipose-derived stem cells are frequently used for bone regeneration both in vitro and in vivo. N⁶-methyladenosine (m⁶A) is the most abundant post-transcriptional modification on eukaryotic RNAs and plays multifaceted roles in development and diseases. However, the regulatory mechanisms of m⁶A in osteogenic differentiation of human adipose-derived stem cells (hASCs) remain elusive. The present study aimed to build the transcriptome-wide m⁶A methylome during the osteogenic differentiation of hASCs.

hASCs were harvested after being cultured in a basic or osteogenic medium for 7 days, and the osteogenic differentiation was validated by alkaline phosphatase (ALP) and Alizarin Red S staining, ALP activity assay, and qRT-PCR analysis of ALP, RUNX2, BGLAP, SPP1, SP7, and COL1A1 genes. The m⁶A level was colorimetrically measured, and the expression of m⁶A regulators was confirmed by qRT-PCR and western blot. Moreover, m⁶A MeRIP-seq and RNA-seq were performed to build the transcriptome and m⁶A methylome. Furthermore, bioinformatic analyses including volcano plots, Venn plots, clustering analysis, Gene Ontology (GO), Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway, gene sets enrichment analysis, and protein-protein interaction analysis were conducted.

In total, 1145 differentially methylated peaks, 2261 differentially expressed genes, and 671 differentially methylated and expressed genes (DMEGs) were identified. GO and KEGG pathway analyses conducted for these DMEGs revealed extensive and osteogenic biological functions. The "PI3K-Akt signaling pathway"; "MAPK signaling pathway"; "parathyroid hormone synthesis, secretion, and action"; and "p53 signaling pathway" were significantly enriched, and the DMEGs in these pathways were identified as m⁶A-specific key genes. A protein-protein interaction network based on DMEGs was built, and VEGFA, CD44, MMP2, HGF, and SPARC were speculated as the hub DMEGs.

The total m⁶A level was reduced with osteogenic differentiation of hASCs. The transcriptome-wide m⁶A methylome built in the present study indicated quite a few signaling pathways, and hub genes were influenced by m⁶A modification. Future studies based on these epigenetic clues could promote understanding of the mechanisms of osteogenic differentiation of hASCs.