MiR-200b Regulates TNF-α and IL-1β Induced ODAM Gene Expression in Human Gingival Epithelial Cells

抄録

Odontogenic ameloblast-associated protein（ODAM）is a secreted protein produced by junctional epithelium（JE）and mature ameloblasts and is involved in odontogenesis and attachment of JE to the tooth. We previously demonstrated that miR-200b and ODAM expressions were increased in inflamed gingiva collected during periodontal surgery using miRNA and DNA arrays. The purpose of this study was to investigate how mir-200b affects the expression of ODAM in inflamed junctional epithelium, therefore an inflammatory state was expressed by stimulating Ca9-22 human gingival epithelial cells with TNF-α or IL-1β. Ca9-22 cells were transfected with a miR-200b expression plasmid with or without treatment of TNF-α or IL-1β and analyzed the ODAM, IKKα, IKKβ and MKP-5 mRNA and protein levels by real-time PCR and Western blot. -480ODAM+3́-UTR luciferase（LUC）construct was transfected together with miR-200b expression plasmid in Ca9-22 cells, and measured LUC activities after stimulation by TNF-α or IL-1β. TNF-α and IL-1β-induced mRNA and protein levels of ODAM, IKKβ and MKP-5 were inhibited by overexpression of miR-200b. TNF-α o r IL-1β increased LUC activities of -480ODAM+3́-UTR were inhibited by miR-200b overexpression. TNF-α or IL-1β-induced -480ODAM3́-UTR LUC activities were inhibited by p38 MAPK, MEK1/2, IKKβ and NF-κB inhibitors. In conclusion, miR-200b appears to suppress TNF-α or IL-1β-induced ODAM expressions in gingival epithelial cells by targeting ODAM, IKKβ and MKP-5.