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

Mizuho Yamazaki-Takai, Hideki Takai, Yasunobu Iwai, Keisuke Noda, Masaru Mezawa, Yuto Tsuruya, Arisa Yamaguchi, Yohei Nakayama, Yorimasa Ogata

Odontology 2021 Apr

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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.

Citation

Mizuho Yamazaki-Takai, Hideki Takai, Yasunobu Iwai, Keisuke Noda, Masaru Mezawa, Yuto Tsuruya, Arisa Yamaguchi, Yohei Nakayama, Yorimasa Ogata. MiR-200b suppresses TNF-α-induced AMTN production in human gingival epithelial cells. Odontology. 2021 Apr;109(2):403-410