MiR-200b suppresses TNF-α-induced AMTN production in human gingival epithelial cells

• Published in: Odontology 2021-04, Vol.109 (2), p.403-410
• Main Authors: Yamazaki-Takai, Mizuho, Takai, Hideki, Iwai, Yasunobu, Noda, Keisuke, Mezawa, Masaru, Tsuruya, Yuto, Yamaguchi, Arisa, Nakayama, Yohei, Ogata, Yorimasa
• Format: Article
• Language: English
• Subjects: 3' Untranslated regions; Dental enamel; Dental Enamel Proteins - genetics; Dentistry; Enzyme inhibitors; Epithelial Cells; Epithelium; Gene expression; Gingiva; Humans; Kinases; Medicine; MicroRNAs - genetics; miRNA; NF-κB protein; Non-coding RNA; Oral and Maxillofacial Surgery; Original Article; Post-transcription; Proteins; Triptolide; Tumor Necrosis Factor-alpha - genetics; Tumor necrosis factor-α
• ISSN: 1618-1247; 1618-1255
• DOI: 10.1007/s10266-020-00555-3

Amelotin (AMTN) is an enamel protein that is localized in junctional epithelium (JE) of gingiva and suggested to be involved in the attachment between JE and tooth enamel. MicroRNA is a small non-coding RNA that regulates gene expression at post-transcriptional level by binding to the 3′-untranslated region (3′-UTR) of target mRNAs. In this study, we have analyzed the effects of miR-200b on the expression of AMTN in human gingival epithelial (Ca9-22) cells. Total RNAs and proteins were extracted from Ca9-22 cells transfected with miR-200b expression plasmid or miR-200b inhibitor and stimulated by TNF-α (10 ng/ml, 12 h). AMTN and inhibitor of kappa-B kinase beta (IKKβ) mRNA and protein levels were measured by qPCR and Western blot. Human AMTN 3′-UTR that contains putative miR-200b target sites were cloned downstream of -353AMTN luciferase (LUC) plasmid. Ca9-22 cells were transfected with -353AMTN 3′-UTR LUC constructs and miR-200b expression plasmid, and LUC activities were measured with or without stimulation by TNF-α. TNF-α-induced AMTN mRNA levels were partially inhibited by miR-200b overexpression and enhanced by miR-200b inhibitor. TNF-α-induced IKKβ mRNA and protein levels were almost completely inhibited by miR-200b. Transcriptional activities of -353AMTN 3′-UTR LUC constructs were induced by TNF-α and partially inhibited by miR-200b. IKKβ inhibitor IMD0354 and NF-κB inhibitor triptolide decreased TNF-α-induced LUC activities. Furthermore, both inhibitors reduced AMTN mRNA levels in the presence or absence of TNF-α. These results suggest that miR-200b suppresses AMTN expression by targeting to AMTN and IKKβ mRNAs in the human gingival epithelial cells.