N6-methyladenosine promotes osteogenic differentiation of PDLSCs from periodontitis patients.

This study aimed to investigate the mechanism of N6-methyladenosine (m6A) in the osteogenic differentiation of periodontal ligament stem cells (PDLSCs) from periodontitis patients. Differentially m6A-methylated lncRNA/mRNA profiles were detected by a m6A epitranscriptomic microarray. Bioinformatics analysis was performed by Gene Ontology and Kyoto Encyclopedia of Genes and Genomes pathway analysis. The transfection efficiency of the lentivirus was detected. The osteogenic activity of PDLSCs from periodontitis patients (PPDLSCs) was assessed. The microarray results showed that 275 lncRNAs and 1292 mRNAs were significantly differentially methylated between PPDLSCs and PDLSCs from healthy people. Among those lncRNAs, lncRNA4114 (transcript_ID: ENST00000444114) showed both reduced m6A methylation levels and expression levels in PPDLSCs. Further bioinformatics analysis predicted that the differentially methylated mRNAs were mainly involved in cell interaction, stem cell pluripotency, and osteogenic differentiation signals. Then, overexpression of methyltransferase like 3 (METTL3) promoted the osteogenic differentiation of PPDLSCs, while knocking down METTL3 showed an inhibitory effect. Furthermore, METTL3 overexpression promotes the stability of lncRNA4114 to upregulate the expression level. Moreover, lncRNA4114 overexpression promoted the osteogenic differentiation of PPDLSCs. METTL3 promotes the osteogenic differentiation of PPDLSCs by regulating the stability of lncRNA4114.

Zhang X ，Liu J ，Gao J ，Sun W ，Chen X ，Wang X ，Qin W ，Jin Z ... -  《-》

Long Noncoding RNA and mRNA m6A Modification Analyses of Periodontal Ligament Stem Cells from the Periodontitis Microenvironment Exposed to Static Mechanical Strain.

Periodontal ligament stem cells (PDLSCs) play important roles in orthodontic tooth movement (OTM) and can respond to mechanical stress. Our previous study demonstrated that periodontal ligament stem cells derived from periodontitis tissue (pPDLSCs) are more sensitive to static mechanical strain (SMS) than those derived from healthy tissue (hPDLSCs) and reported the long noncoding RNA (lncRNA) expression profiles of pPDLSCs exposed to SMS. An increasing number of lncRNAs have been reported by various studies to be associated with the osteogenic differentiation of mesenchymal stem cells. Many studies have demonstrated that the n6-methyladenosine (m6A) modification exerts important effects on lncRNA and mRNA regulation of cell behaviors. However, the regulatory effects of lncRNA and mRNA m6A modification on PDLSCs have not been studied. Therefore, we performed an m6A microarray assay with pPLDSCs and hPDLSCs exposed to 12% SMS and found that 143 lncRNAs and 739 mRNAs were differentially methylated. These RNAs were thought to be involved in multiple differentiation and inflammatory responses. Moreover, we found that METTL3, an essential protein in the m6A system, was expressed at lower levels in the strain-exposed pPDLSCs than in strain-exposed hPLDSCs, and METTL3 promoted the osteogenic differentiation of pPDLSCs.

Sun W ，Liu J ，Zhang X ，Zhang X ，Gao J ，Chen X ，Wang X ，Qin W ，Jin Z ... -  《-》

Down-regulation of long non-coding RNA MEG3 suppresses osteogenic differentiation of periodontal ligament stem cells (PDLSCs) through miR-27a-3p/IGF1 axis in periodontitis.

Liu Y ，Liu C ，Zhang A ，Yin S ，Wang T ，Wang Y ，Wang M ，Liu Y ，Ying Q ，Sun J ，Wei F ，Liu D ，Wang C ，Ge S ... -  《Aging-US》

Long noncoding RNA related to periodontitis interacts with miR-182 to upregulate osteogenic differentiation in periodontal mesenchymal stem cells of periodontitis patients.

Wang L ，Wu F ，Song Y ，Li X ，Wu Q ，Duan Y ，Jin Z ... -  《Cell Death & Disease》

METTL3-Mediated lncRNA m(6)A Modification in the Osteogenic Differentiation of Human Adipose-Derived Stem Cells Induced by NEL-Like 1 Protein.

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). 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. 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. 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.

Song Y ，Pan Y ，Wu M ，Sun W ，Luo L ，Zhao Z ，Liu J ... -  《-》