METTL3-Mediated lncSNHG7 m6A Modification in the Osteogenic Differentiation of Human Dental Stem Cells

Abstract:

Background: Human dental pulp stem cells (hDPSCs) play an important role in endodontic regeneration, with self-renewal and pluripotency capacity. N6-methyladenosine (m⁶A) is the most common RNA modification, and noncoding RNAs have also been demonstrated to have regulatory roles in the expression of m⁶A regulatory proteins. This study aimed to explore the regulatory mechanism of methyltransferase 3 (METTL3)-mediated long noncoding RNA (lncRNA) m⁶A modification in the osteogenic differentiation of hDPSCs.

Methods: Single base site PCR (MazF) was used to detect the m⁶A modification site of lncSNHG7 before and after mineralization of hDPSCs, combined with the prediction information from the StarBase database and real-time quantitative polymerase chain reaction (qRT–PCR) to screen the target m⁶A modification protein, and bioinformatics analysis was used to analyse the related pathways rich in lncSNHG7. After knockdown of lncSNHG7 and METTL3, osteogenic ability was detected by alkaline phosphatase (ALP) staining, Alizarin Red S (ARS) staining, qRT–PCR and Western blotting. After METTL3 knockdown, the m⁶A modification level and its expression of lncSNHG7 were detected by MazF, and their binding was confirmed by RNA binding protein immunoprecipitation (RIP) analysis. Finally, Western blot analysis was used to detect the effects of lncSNHG7 and METTL3 on the Wnt/β-catenin pathway.

Results: MazF experiments revealed that lncSNHG7 had a m⁶A modification before and after mineralization of hDPSCs, and the occurrence site was 2081. The m⁶A-modified protein METTL3 was most significantly upregulated after mineralization of hDPSCs. Knockdown of lncSNHG7 and METTL3 inhibited the osteogenic differentiation of hDPSCs. The m⁶A modification and expression of lncSNHG7 were both regulated by METTL3. Subsequently, lncSNHG7 and METTL3 were found to regulate the key proteins in the Wnt/β-catenin signaling pathway, β-catenin and GSK-3β.

Conclusion: These results revealed that METTL3 can activate the Wnt/β-catenin signaling pathway by regulating the m⁶A modification and expression of lncSNHG7 in hDPSCs to enhance the osteogenic differentiation of hDPSCs. Our study provides new insight into stem cell-based tissue engineering.