Transcriptome-wide m(6)A methylome during osteogenic differentiation of human adipose-derived stem cells.

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. 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. 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. 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.

Sun W ，Song Y ，Xia K ，Yu L ，Huang X ，Zhao Z ，Liu J ... -  《Stem Cell Research & Therapy》

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 ... -  《-》

microRNA expression profiles and the potential competing endogenous RNA networks in NELL-1-induced human adipose-derived stem cell osteogenic differentiation.

Studies have indicated that Nel-like molecule-1 (NELL-1) was an osteoblast-specific cytokine and some specific microRNAs (miRNAs) could serve as competing endogenous RNA (ceRNA) to partake in osteogenic differentiation of human adipose-derived stem cells (hASCs). The aim of this study was to explore the potential functional mechanisms of recombinant human NELL-1 protein (rhNELL-1) during hASCs osteogenic differentiation. rhNELL-1 was added to osteogenic medium to activate osteogenic differentiation of hASCs. High-throughput RNA sequencing (RNA-Seq) was performed and validated by real-time quantitative polymerase chain reaction. Gene ontology functional annotation and Kyoto Encyclopedia of Genes and Genomes pathway analysis were performed to detect the functions of differentially expressed miRNAs and genes. Coding-noncoding gene co-expression network and ceRNA networks were constructed to predict the potential regulatory role of miRNAs. A total of 1010 differentially expressed miRNAs and 1762 differentially expressed messenger RNAs (mRNAs) were detected. miRNA-370-3p, bone morphogenetic protein 2 (BMP2), and parathyroid hormone like hormone (PTHLH) were differentially expressed during NELL-1-induced osteogenesis. Bioinformatic analyses demonstrated that these differentially expressed miRNAs and mRNAs enriched in Rap1 signaling pathway, PI3K-Akt signaling pathway, p53 signaling pathway, Glucagon signaling pathway, and hypoxia-inducible factor-1 signaling pathway, which were important pathways related to osteogenic differentiation. In addition, miRNA-370-3p and has-miR-485-5p were predicted to interact with circ0001543, circ0002405, and ENST00000570267 in ceRNA networks. Based on the gain or loss of functional experiments by transfection, the results showed that miR-370-3p was a key regulator in osteogenic differentiation by targeting BMP2 and disturbing the expression of PTHLH, and participated in NELL-1-stimulated osteogenesis. The present study provided the primary data and evidence for further exploration on the roles of miRNAs and ceRNAs during NELL-1-induced ossification of hASCs.

Yu L ，Cen X ，Xia K ，Huang X ，Sun W ，Zhao Z ，Liu J ... -  《-》

Long non-coding RNA HIF1A-AS2 facilitates adipose-derived stem cells (ASCs) osteogenic differentiation through miR-665/IL6 axis via PI3K/Akt signaling pathway.

Wu R ，Ruan J ，Sun Y ，Liu M ，Sha Z ，Fan C ，Wu Q ... -  《Stem Cell Research & Therapy》

A signal-amplification circuit between miR-218 and Wnt/β-catenin signal promotes human adipose tissue-derived stem cells osteogenic differentiation.

Human adipose-derived stem cells (hASCs) have become a highly attractive source of seed cells in bone regenerative. It has become a key issue how to effectively improve osteogenic differentiation of hASCs in the bone tissue engineering. Numerous regulatory pathways dominate osteogenic differentiation of hASCs involve transcriptional factors and signaling molecules. However, how these factors combine with each other to regulate hASCs osteogenic differentiation still remain to be illustrated. The identification of microRNAs will illuminate this and might permit finely tuning the osteogenic differentiation process. Here, we present evidence that miR-218 acts as a positive regulator of hASCs osteogenesis. Real-time PCR shows that miR-218 was up-regulated during osteogenic differentiation. Overexpression of exogenous miR-218 enhanced osteogenic differentiation in vitro, whereas inhibition of miR-218 would suppress osteogenic differentiation. Furthermore, miR-218 directly targeted SFRP2 and DKK2, which is a WNT signaling pathway antagonist, and enhanced Wnt/β-catenin signaling activity. Finally, we found that mimicking Wnt/β-catenin signal strengthened the expression level of miR-218, while blocking the signal attenuated the expression level of miR-218. This feed-forward regulatory circuit provides additional insight into how miRNAs acting as a signal amplifier interact with signal molecules during hASCs osteogenic differentiation. Taken together, we have established a regulatory network with a central role for the miR-218 in hASCs osteogenic differentiation.

Zhang WB ，Zhong WJ ，Wang L 《-》