MiR-223 Attenuates ODAM Gene Expression Induced by TNF-α and IL-1β inHuman Gingival Epithelial Cells

Odontogenic ameloblast-associated protein（ODAM）is a secretory protein that contributes to cell adhesion and is expressed by ameloblasts and junctional epithelium（JE）. MicroRNAs（miRNAs）are non-coding small RNAs which silence gene expression in post transcriptional regulation. In our previous study, we used miRNA and DNA arrays to demonstrate that expressions of miR-223 and ODAM were induced in the inflamed gingiva of periodontitis patients. To verify the effect of mir-223 on ODAM expression in Ca9-22 human gingival epithelial cells, we purified total RNA and protein from the cells transfected with miR-223 expression plasmid or miR-223 inhibitor and treated with TNF-α or IL-1β. ODAM, inhibitor of κB kinase α（IKKα）, inhibitor of κB kinase β（IKKβ）, and mitogen-activated protein kinase phosphatase 5（MKP-5）protein and mRNA levels were studied by Western blot and qPCR. -480ODAM 3’-untranslated region（3’-UTR）luciferase（LUC）construct and miR-223 expression plasmid were transfected in Ca9-22 cells, and LUC activities were measured with or without TNF-α or IL-1β stimulation. ODAM mRNA expressions increased by TNF-α or IL-1β stimulation, but were suppressed by miR-223 overexpression, and conversely increased with a miR-223 inhibitor. MKP-5 mRNA and protein expressions increased with TNF-α or IL-1β but were inhibited by miR-223. IKKα mRNA levels were unchanged with TNF-α or IL-1β but were suppressed by miR-223 overexpression. The activities of -480ODAM 3’-UTR LUC were induced by TNF-α or IL-1β, whereas suppressed by miR-223. SB203580（P38 MAPK inhibitor）, U0126（MEK1/2 inhibitor）, IMD0354（IKKβ inhibitor）, BMS345541（IKKβ and IKKα inhibitor）, and triptolide（NF-κB inhibitor）decreased TNF α and IL-1β-induced -480ODAM 3’-UTR LUC activity. These data suggest that miR-223 targets ODAM, IKKα and MKP-5 mRNAs expressed in human gingival epithelial cells to suppress ODAM expression.