METTL3 promotes SMSCs chondrogenic differentiation by targeting the MMP3, MMP13, and GATA3

Synovium-derived mesenchymal stem cells (SMSCs) are multipotential non-hematopoietic progenitor cells that can differentiate into various mesenchymal lineages in adipose and bone tissue, especially in chondrogenesis. Post-transcriptional methylation modifications are relative to the various biological development procedures. N⁶-methyladenosine (m⁶A) methylation has been identified as one of the abundant widespread post-transcriptional modifications. However, the connection between the SMSCs differentiation and m⁶A methylation remains unknown and needs further exploration.

SMSCs were derived from synovial tissues of the knee joint of male Sprague-Dawley (SD) rats. In the chondrogenesis of SMSCs, m⁶A regulators were detected by quantitative real-time PCR (RT-PCR) and Western blot (WB). We observed the situation that the knockdown of m⁶A "writer" protein methyltransferase-like (METTL)3 in the chondrogenesis of SMSCs. We also mapped the transcript-wide m⁶A landscape in chondrogenic differentiation of SMSCs and combined RNA-seq and MeRIP-seq in SMSCs by the interference of METTL3.

The expression of m⁶A regulators were regulated in the chondrogenesis of SMSCs, only METTL3 is the most significant factor. In addition, after the knockdown of METTL3, MeRIP-seq and RNA-seq technology were applied to analyze the transcriptome level in SMSCs. 832 DEGs displayed significant changes, consisting of 438 upregulated genes and 394 downregulated genes. DEGs were enriched in signaling pathways regulating the glycosaminoglycan biosynthesis-chondroitin sulfate/dermatan sulfate and ECM-receptor interaction via Kyoto Encyclopedia of genes and genomes (KEGG) pathway enrichment analysis. The findings of this study indicate a difference in transcripts of MMP3, MMP13, and GATA3 containing consensus m⁶A motifs required for methylation by METTL3. Further, the reduction of METTL3 decreased the expression of MMP3, MMP13, and GATA3.

These findings confirm the molecular mechanisms of METTL3-mediated m⁶A post-transcriptional change in the modulation of SMSCs differentiating into chondrocytes, thus highlighting the potential therapeutic effect of SMSCs for cartilage regeneration.