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

Abstract

Objectives 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. Materials and methods 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. Results 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. Conclusions 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.

Emerging Role of Epitranscriptomics in Diabetes Mellitus and Its Complications

“…Meanwhile, the increasing interest in epitranscriptomics has also prompted the progresses in developing new detecting tools. It is recognized that total level of m 6 A modification in RNA can be detected by liquid chromatography-tandem mass spectrometry (LC-MS/MS) and colorimetric method (90,91). Many high-throughput sequencing methods have been developed to detected m 6 A modification based on different mechanisms, including antibody-dependent single-nucleotide resolution sequencing (PA-m 6 A-seq, m 6 A-CLIP and miCLIP), chemical labeling sequencing (m 6 A-labelseq and m 6 A-SEAL), MeRIP-m 6 A-seq (the most widely used method so far), endoribonuclease-based sequencing (m 6 A-REFseq and MASTER-seq), and other methods (such as m 6 A-LAICseq, DART-seq and Nanopore-seq), endoribonuclease-based sequencing (m 6 A-REF-seq and MASTER-seq), and other methods (such as m 6 A-LAIC-seq, DART-seq and Nanoporeseq) (11,(92)(93)(94)(95)(96).…”

Epigenetics: Novel crucial approach for osteogenesis of mesenchymal stem cells

“…Further, 1145 differentially methylated peaks, 2261 differentially expressed genes, and 671 differentially methylated and expressed genes were identified. 69 Intriguingly, another study demonstrated that FTO was downregulated during osteogenic differentiation and upregulated during adipogenic differentiation in BMSCs. FTO could bind to the fat-related gene PPARγ and demethylate it, resulting in an increased level of PPARγ and promoting the shift of BMSCs fate to adipocytes.…”

The m6A Reader YTHDF1 Accelerates the Osteogenesis of Bone Marrow Mesenchymal Stem Cells Partly via Activation of the Autophagy Signaling Pathway

“…m 6 A mediates almost every aspect of mRNA metabolism, including splicing, export, turnover, translation, and regulates mRNA stability [4][5][6]. Recently, studies have shown that m 6 A modification plays a crucial role in bone metabolism [7]. For example, conditional knockout m 6 A methyltransferase Mettl3 in mice induces the pathological characteristics of osteoporosis and resulted in impaired bone formation, incompetent osteogenic differentiation potential, and increased morrow adiposity.…”