MZB1 targeted by miR-185-5p inhibits the migration of human periodontal ligament cells through NF-κB signaling and promotes alveolar bone loss

Abstract

Objective: To explore the role of Marginal Zone B and B-1 Cell-Specific Protein (MZB1), a novel molecule associated with periodontitis, in migration of human periodontal ligament cells (hPDLCs) and alveolar bone orchestration.

Background: MZB1 is an ER-localized protein and its upregulation has been found to be associated with a variety of human diseases. However, few studies have investigated the effect and mechanism of MZB1 on hPDLCs in periodontitis.

Methods: Gene expression profiles in human gingival tissues were acquired from the Gene Expression Omnibus (GEO) database, and candidate molecules were then selected through bioinformatic analysis. Subsequently, we identified the localization and expression of MZB1 in human gingival tissues, mice, and hPDLCs by immunofluorescence, RT-qPCR, and Western blot. Dual-luciferase reporter assay was applied to assess the binding of miR-185-5p to MZB1. Furthermore, the effects of MZB1 on cell migration, proliferation, and apoptosis in vitro were investigated by wound-healing assay, transwell assay, CCK-8 assay, and flow cytometry analysis. Finally, Micro-CT analysis and H&E staining were performed to examine the effects of MZB1 on alveolar bone loss in vivo.

Results: Bioinformatic analysis discovered that MZB1 was one of the most significantly increased genes in periodontitis patients. MZB1 was markedly increased in the gingival tissues of periodontitis patients, in the mouse models, and in the hPDLCs treated with lipopolysaccharide of Porphyromonas gingivalis (LPS-PG). Furthermore, in vitro experiments showed that MZB1, as a target gene of miR-185-5p, inhibited migration of hPDLCs. Overexpression of MZB1 specifically upregulated the phosphorylation of p65, while pretreatment of MZB1-overexpressed hPDLCs with PDTC (NF-κB inhibitor) notably reduced the p-p65 level and promoted cell migration. In addition, the mRNA expression levels of alkaline phosphatase (ALP) and Runt-related transcription factor 2 (Runx2) were inhibited in MZB1-overexpressed hPDLCs and miR-185-5p inhibitor treated hPDLCs, respectively. In vivo experiments showed that knockdown of MZB1 alleviated the loss of alveolar bone.

Conclusion: As a target gene of miR-185-5p, MZB1 plays a crucial role in inhibiting the migration of hPDLCs through NF-κB signaling pathway and deteriorating alveolar bone loss.

Keywords: MZB1; alveolar bone loss; human periodontal ligament cells; migration; periodontitis.