METTL3-Mediated lncRNA m⁶A Modification in the Osteogenic Differentiation of Human Adipose-Derived Stem Cells Induced by NEL-Like 1 Protein

Objectives: This study aimed to explore the regulatory mechanism of methyltransferase3 (METTL3) -mediated long non-coding RNA (lncRNA) N6-methyladenosine (m⁶A) modification in the osteogenic differentiation of human adipose-derived stem cells (hASCs) induced by NEL-like 1 protein (NELL-1).

Materials and methods: Methylated RNA immunoprecipitation sequencing (MeRIP-seq) and high- throughput sequencing for RNA (RNA-seq) were performed on hASCs. Osteogenic ability was detected by alkaline phosphatase (ALP) staining, Alizarin Red S(ARS) staining, ALP quantification and Quantitative real-time polymerase chain reaction analysis (qRT-PCR). Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis predicted the osteogenesis-related pathways enriched for the lncRNAs and identified the target lncRNAs. After overexpression and knockdown of METTL3, methylated RNA immunoprecipitation-qPCR (MeRIP-qPCR) and qRT-PCR were used to detect the levels of m⁶A modification and the expression of the target lncRNA, and the binding of both was confirmed by RNA binding protein immunoprecipitation (RIP) assay. The effects of lncRNA and METTL3 on phosphorylation of the key proteins of the pathway were detected by western blot analysis.

Results: In vitro experiments showed that METTL3 can promote osteogenic differentiation and that its expression level is upregulated. KEGG pathway analysis predicted that lncRNAs with differentially upregulated methylated peaks were enriched mostly in the mitogen-activated protein kinase (MAPK) signaling pathway, in which Serine/threonine protein kinase 3 (STK3) was the predicted target gene of the lncRNA RP11-44 N12.5. The m⁶A modification and expression of RP11-44 N12.5 were both regulated by METTL3. Subsequently, lncRNA RP11-44 N12.5 and METTL3 were found to regulate the phosphorylation levels of three key proteins in the MAPK signaling pathway, ERK, JNK and p38.

Conclusions: This study shows, for the first time, that METTL3 can activate the MAPK signaling pathway by regulating the m⁶A modification and expression of a lncRNA, thereby enhancing the osteogenic differentiation of hASCs.