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.