Although the innate immune response can protect an organism against various pathogens, an excessive or prolonged immune response can be harmful and can result in acute or chronic inflammatory disorders. Thus, the cells involved in the innate immune response must be regulated to inhibit the occurrence of inflammation. It has been demonstrated that miRNA-146 is involved in inflammatory processes . Although the negative regulation of miRNA-146 has been reported in THP-1 monocytes and macrophages [8, 10], the effects of miRNA-146 in P.g LPS-stimulated HGFs remain unknown.
P.g is an important pathogenic organism in human periodontitis, which is a chronic inflammatory disease [15, 17, 18]. P.g LPS is a potent stimulator of inflammatory cytokine production and bone resorption. Lee et al. (2011) compared healthy tissues with tissues infected with periodontitis and found that miRNA-146 was involved in periodontal inflammation . Additionally, in our previous study, we screened the expression of miRNAs in healthy and periodontal-diseased gingiva and found that miRNA-146 expression increased in periodontal-diseased gingiva . In this study, miRNA-146 expression increased in HGFs after stimulation with P.g LPS, which further confirmed the previous studies [6, 7].
Wang et al. (1999)  and Imatani et al. (2001)  have shown that HGFs function as regulators of the cytokine network in periodontal tissues and produce inflammatory cytokines in response to the stimulation with bacterial cell components, such as P.g LPS. Thus, we further explored whether miRNA-146 regulated the secretion of inflammatory cytokines. After stimulation with P.g LPS, HGFs secreted IL-1β, IL-6 and TNF-α, which is consistent with other studies [15, 18]. IL-1β, IL-6 and TNF-α are important inducers of inflammation. In the present study, IL-1β, IL-6 and TNF-α levels increased after the inhibition of miRNA-146a and/or miRNA-146b-5p. These data indicate that miRNA-146 negatively regulates the secretion of pro-inflammatory cytokines and prevents aggressive inflammation. However, the mechanism of the miRNA-146-mediated increase in pro-inflammatory cytokines after P.g LPS stimulation is still unclear. Nakasa et al. reported that miRNA-146 was induced by TNF-α and IL-1β in the synovial tissues of patients with rheumatoid arthritis . We believe that miRNA-146 could be induced as a result of an increase in TNF-α and IL-1β after stimulation with P.g LPS; however, further confirmation is necessary. In this context, miRNA-146 would regulate the immune response in P.g LPS-stimulated HGFs.
IL-10 plays a major role in suppressing the immune and inflammatory responses by inhibiting the activity of Th1 cells, NK cells and macrophages . Moreover, Wang et al.  reported that LPS-induced IL-6 production was inhibited when HGFs were pretreated with IL-10, suggesting that the anti-inflammatory effects of IL-10 could affect LPS-induced pro-inflammatory cytokine production in vivo. In our study, IL-10 levels increased in P.g LPS-stimulated HGFs, indicating that the cells secrete cytokines that negatively regulate inflammation. However, IL-10 levels did not change after the inhibition of miRNA-146a and/or miRNA-146b-5p. The mechanism of IL-10 secretion warrants further investigation.
miRNA-146, like many mammalian miRNAs, may target a wide spectrum of genes . It is noteworthy that miRNA-146 has already been implicated in a number of cellular processes [21–23]. Additionally, it has been demonstrated that miRNA-146 is involved in TLR signaling pathways [9, 10]. TLR pathways are involved in innate immunity . Two key adapter molecules in the TLR pathways, TRAF6 and IRAK1, were identified as the target genes of miRNA-146 [10, 25]. TRAF6 and IRAK1 yielded high scores on the computational miRNA target prediction algorithms. Our results indicate that miRNA-146a and/or miRNA-146b-5p inhibitors increase IRAK1, but not TRAF6, levels. miRNA-146b-5p had no statistical effect on mRNA level of IRAK1, however, it could negatively regulate translation process of IRAK1, protein level had been tested significantly changed. This indicates miRNA-146b-5p may not involve in IRAK1 binding and mRNA regulation. But based on IRAK1 protein level decreased after miRNA-146b-5p inhibition, it suggests miRNA-146b-5p may regulate another target genes and indirectly affect IRAK1 protein level.
These data indicate that miRNA-146a and miRNA-146b-5p have different effects on IRAK1 expression. miRNAs regulate gene expression by binding to the 3’-UTR of target genes . Our results indicate that miRNA-146a and miRNA-146b-5p bind to the 3’-UTR of IRAK1, suggesting that miRNA-146a and miRNA-146b-5p directly regulate the expression of IRAK1. However, in our study, miRNA-146a and miRNA-146b-5p inhibitors did not result in an increase in TRAF6, which is inconsistent with previous studies . We believe that there are other regulatory mechanisms that control the expression of TRAF6 in HGFs, which is a topic that warrants further investigation. Thus, miRNA-146a and miRNA-146b-5p negatively regulate immune responses by inhibiting IRAK1, but not TRAF6, expression.