Abstract
Bone mesenchymal stem cells (BMSCs) can be a useful cell resource for developing biological treatment strategies for bone repair and regeneration, and their therapeutic applications hinge on an understanding of their physiological characteristics. N6-methyl-adenosine (m6A) is the most prevalent internal chemical modification of mRNAs and has recently been reported to play important roles in cell lineage differentiation and development. However, little is known about the role of m6A modification in the cell differentiation of BMSCs. To address this issue, we investigated the expression of N6-adenosine methyltransferases (Mettl3 and Mettl14) and demethylases (Fto and Alkbh5) and found that Mettl3 was upregulated in BMSCs undergoing osteogenic induction. Furthermore, we knocked down Mettl3 and demonstrated that Mettl3 knockdown decreased the expression of bone formation-related genes, such as Runx2 and Osterix. The alkaline phosphatase (ALP) activity and the formation of mineralized nodules also decreased after Mettl3 knockdown. RNA sequencing analysis revealed that a vast number of genes affected by Mettl3 knockdown were associated with osteogenic differentiation and bone mineralization. Kyoto encyclopedia of genes and genomes (KEGG) pathway analysis revealed that the phosphatidylinositol 3-kinase/AKT (PI3K-Akt) signaling pathway appeared to be one of the most enriched pathways, and Western blotting results showed that Akt phosphorylation was significantly reduced after Mettl3 knockdown. Mettl3 has been reported to play an important role in regulating alternative splicing of mRNA in previous research. In this study, we found that Mettl3 knockdown not only reduced the expression of Vegfa but also decreased the level of its splice variants, vegfa-164 and vegfa-188, in Mettl3-deficient BMSCs. These findings might contribute to novel progress in understanding the role of epitranscriptomic regulation in the osteogenic differentiation of BMSCs and provide a promising perspective for new therapeutic strategies for bone regeneration.
Highlights
N6-methyl-adenosine (m6A) is a methylation modification at the N6 position of adenosine in both coding and noncoding RNAs and has been identified as the most prevalent internal chemical modification of mRNAs in eukaryotes [1,2]
The results showed that Mettl3 knockdown reduced the mRNA levels of Alp and Ocn in Bone mesenchymal stem cells (BMSCs) after osteogenic differentiation induction for 7 and 14 days (Figure 3C)
The results indicated that Mettl3 knockdown decreased the mRNA expression of Vegfa-164 and Vegfa-188 but did not significantly alter Vegfa-120 mRNA level (Figure 5A,C–E)
Summary
N6-methyl-adenosine (m6A) is a methylation modification at the N6 position of adenosine in both coding and noncoding RNAs and has been identified as the most prevalent internal chemical modification of mRNAs in eukaryotes [1,2]. Recent evidence has illustrated that epigenetic modifications, such as histone acetylation and DNA methylation, are involved in the cell differentiation process of BMSCs during bone regeneration [62,63]. As another layer of epigenetic regulation (RNA epigenetics), m6A modification has recently been reported to play important roles in cell function and the differentiation of embryonic stem cells (ESCs) and various cancer cell types [64,65,66]. The results showed that Mettl knockdown reduced the mRNA levels of Alp and Ocn in BMSCs after osteogenic differentiation induction for 7 and 14 days (Figure 3C). Significant difference compared with the control (* p < 0.05; ** p < 0.01; *** p < 0.001)
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