MiR-200b attenuates IL-6 production through IKKβ and ZEB1 in human gingival fibroblasts.

MicroRNAs (miRNAs) play important roles in biological processes such as cell differentiation, development, infection, immune response, inflammation and tumorigenesis. We previously reported that the expression of miR-200b was significantly increased in inflamed gingiva compared with non-inflamed gingiva. To elucidate the roles of miR-200b in the inflamed gingiva, we have analyzed the effects of miR-200b on the expression of IL-6 in human gingival fibroblasts (HGF). Total RNA and protein were extracted from HGF after stimulation by interleukin-1β (IL-1β; 1 ng/ml) or tumor necrosis factor-α (TNF-α; 10 ng/ml) and transfected with miR-200b expression plasmid or miR-200b inhibitor. IL-6, IL-1β, inhibitor of nuclear factor kappa-B kinaseβ (IKKβ), Zinc-finger E-box-binding homeobox 1 (ZEB1) and E-cadherin mRNA and protein levels were analyzed by real-time PCR and Western blot. IL-1β and TNF-α increased IL-6 mRNA and protein levels, and they were significantly suppressed by miR-200b overexpression, whereas they were further increased by miR-200b inhibitor in HGF. IKKβ and ZEB1 which are target genes of miR-200b negatively regulate E-cadherin. MiR-200b suppressed the expression of IKKβ and ZEB1 and increased E-cadherin mRNA and protein levels in HGF. These results suggest that miR-200b attenuates inflammatory response via IKKβ and ZEB1 in periodontal tissue.

微小RNA（miRNAs）在细胞分化、发育、感染、免疫应答、炎症和肿瘤发生等生物学过程中发挥着重要作用。我们先前报道，与未发炎的牙龈相比，发炎牙龈中miR - 200b的表达显著增加。为了阐明miR - 200b在发炎牙龈中的作用，我们分析了miR - 200b对人牙龈成纤维细胞（HGF）中白细胞介素 - 6（IL - 6）表达的影响。 在用白细胞介素 - 1β（IL - 1β；1 ng/ml）或肿瘤坏死因子 - α（TNF - α；10 ng/ml）刺激后，从HGF中提取总RNA和蛋白质，并用miR - 200b表达质粒或miR - 200b抑制剂进行转染。通过实时定量PCR和蛋白质印迹法分析IL - 6、IL - 1β、核因子κB激酶β抑制剂（IKKβ）、锌指E盒结合同源框1（ZEB1）和E - 钙黏蛋白的mRNA和蛋白质水平。 IL - 1β和TNF - α增加了IL - 6的mRNA和蛋白质水平，并且它们在HGF中被miR - 200b过表达显著抑制，而被miR - 200b抑制剂进一步增加。作为miR - 200b靶基因的IKKβ和ZEB1对E - 钙黏蛋白起负调控作用。miR - 200b抑制了HGF中IKKβ和ZEB1的表达，并增加了E - 钙黏蛋白的mRNA和蛋白质水平。 这些结果表明，miR - 200b通过IKKβ和ZEB1减轻牙周组织中的炎症反应。