# METTL3 knockdown promotes the osteogenic differentiation of hPDLSCs by regulating CARD11 levels

## TL;DR

Reducing METTL3 levels helps human periodontal ligament stem cells become bone-like cells by lowering CARD11, which may help treat periodontitis.

## Contribution

This study reveals a novel regulatory mechanism involving METTL3 and CARD11 in osteogenic differentiation and periodontitis.

## Key findings

- METTL3 silencing reduced m6A and mRNA levels of CARD11 in hPDLSCs.
- METTL3 knockdown promoted osteogenic differentiation of hPDLSCs in vitro and in vivo.
- CARD11 overexpression reversed the effects of METTL3 silencing and inactivated the NF-κB pathway.

## Abstract

•METTL3 was upregulated in periodontitis.•METTL3 silencing promoted the osteogenic differentiation of hPDLSCs.•METTL3 regulated the m6A levels of CARD11.•CARD11 overexpression neutralized the role of si-METTL3 in hPDLSCs.

METTL3 was upregulated in periodontitis.

METTL3 silencing promoted the osteogenic differentiation of hPDLSCs.

METTL3 regulated the m6A levels of CARD11.

CARD11 overexpression neutralized the role of si-METTL3 in hPDLSCs.

Periodontitis is a multifactorial progressive disease predominantly related to bacterial infection. Human Periodontal Ligament Stem Cells (hPDLSCs) are important cell populations for repair and regeneration in periodontal tissue. Recently, Methyltransferase-Like-3 (METTL3) is demonstrated to be closely related to the osteogenic differentiation of stem cells by modulating m6A levels. Additionally, through the bioinformatic analysis, CARD11 was demonstrated to be closely related to periodontitis. Therefore, this study was performed to investigate the role of METTL3 in the osteogenic differentiation of hPDLSCs and the periodontitis rat model.

The biomarkers of hPDLSCs were analyzed by flow cytometry. qRT‒PCR was performed to measure METTL3 and CARD11 levels. The protein levels of Runx2, Osterix, Osteocalcin, p-p65, and p-IκBα were detected by western blotting. Alizarin red and alkaline phosphatase staining were carried out. The relationship between METTL3 and CRAD11 was confirmed by RIP and dual luciferase report.

The results showed that METTL3 and CARD11 were dramatically upregulated in periodontitis patients. In addition, METTL3 silencing dramatically decreased the m6A and mRNA levels of CARD11 and increased the Runx2, Osterix, and Osteocalcin levels. Alizarin red and alkaline phosphatase staining showed that METTL3 silencing promoted the osteogenic differentiation of hPDLSCs. Additionally, overexpression of CARD11 neutralized the role of CARD11 in hPDLSCs and inactivated the NF-κB signaling pathway. Finally, METTL3 knockdown relieved the periodontitis progression in vivo.

In conclusion, METTL3 participates in periodontitis progression by regulating CARD11 levels through the NF-κB signaling pathway.