Transcriptome-Wide N6-Methyladenosine Methylome in the Endothelial Differentiation Process of Bone Marrow Mesenchymal Stem Cells

Abstract

Background: Mesenchymal stem cell (MSC)-derived cells with endothelial phenotypes have demonstrated more potent angiogenic effects than ordinary MSCs. The roles of N6-methyladenosine (m6A) RNA methylation in the endothelial differentiation of MSCs are unknown.

Methods: Human BM-MSCs were isolated from the bone marrow by density gradient centrifugation. Inducing medium containing a few cytokines was applied to induce the endothelial differentiation of MSCs. Endothelial differentiation was evaluated using flow cytometry, Western blot, immunofluorescence, Matrigel tube formation assay and Dil-labeled acetylated low-density lipoprotein uptake assay. The expression of m6A regulators were evaluated by quantitative real-time polymerase chain reaction (qRT–PCR) and western blot. A combined analysis of high-throughput methylated RNA immunoprecipitation sequencing (MeRIP-seq) and RNA sequencing (RNA-seq) data was performed. Bioinformatic analyses including volcano plots, Venn plots, clustering analysis, Gene Ontology (GO), Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway and protein-protein interaction analysis were conducted.

Results: In brief, 16355 differentially methylated peaks, 2732 differentially expressed genes, and 1204 differentially methylated and expressed genes (DMEGs) were identified during this process. Functional enrichment clustering analysis of DMEGs revealed pathways including ‘mesenchymal cell differentiation’, ‘epithelial cell migration’ and ‘ECM-receptor interaction’ . The PPI network was constructed and the hub genes were identified.

Conclusions: The present study reveals the transcriptome-wide m6A methylome of endothelial differentiation in MSCs and identified m6A regulators, key pathways and hub genes that may regulate the endothelial differentiation process. Future studies based on these epigenetic analyses could promote understanding of the role of methylation in the process of endothelial differentiation of BM-MSCs.

Funding Information: This work was supported by the National Key R&D Program of China (grant No. 2017YFC0109105), the Scientific Research Seed Fund of Peking University First Hospital (grant No. 2018SF023), Youth Clinical Research Project of Peking University First Hospital (grant No. 2018CR16), and the Interdisciplinary Clinical Research Project of Peking University First Hospital (grant No. 2018CR33).

Declaration of Interests: The authors declare that they have no competing interests.

Ethics Approval Statement: Donors provided written informed consent. The present study was approved by the Ethics Committee of Peking University First Hospital.

Keywords: mesenchymal stem cells; endothelial; differentiation; m6A;epigenetic