Abstract
BackgroundAlthough various microRNAs (miRNAs) regulate immune and inflammatory responses, the function of miRNAs in periodontitis has not been clearly illuminated. In this study, we measured miRNA-146 (miRNA-146a and miRNA-146b-5p) expression and explored its regulatory function in the inflammatory response in human gingival fibroblasts (HGFs).MethodsmiRNA-146a and miRNA-146b-5p expression was measured by performing real-time polymerase chain reaction in HGFs after Porphyromonas gingivalis (p.g) lipopolysaccharide (LPS) stimulation. After the HGFs were transfected with miRNA-146a and miRNA-146b-5p inhibitor, the expression levels of interleukin-1β (IL-1β), interleukin-6 (IL-6) and tumor necrosis factor-α (TNF-α) were measured by enzyme-linked immunosorbent assay (ELISA). Meanwhile, IL-1 receptor-associated kinase 1 (IRAK1) and TNF receptor-associated factor 6 (TRAF6) were detected by western blot and quantitative PCR. A luciferase assay was used to detect whether miRNA-146 could directly bind to the 3’-UTR of IRAK1.ResultsThe expression levels of miRNA-146a and miRNA-146b-5p significantly increased in the P.g LPS-stimulated HGFs compared to the non-stimulated HGFs. The inhibition of miRNA-146a and miRNA-146b-5p resulted in increased IL-1β, IL-6 and TNF-α secretion. The mRNA and protein levels of IRAK1, but not TRAF6, also increased. We further found that miRNA-146a and miRNA-146b-5p directly bound to the IRAK1 3’-UTR.ConclusionOur data suggest that miRNA-146 inhibits pro-inflammatory cytokine secretion through IRAK1 in HGFs, which indicates that miRNA-146 functions as a negative regulator of periodontal inflammation.
Highlights
Various microRNAs regulate immune and inflammatory responses, the function of miRNAs in periodontitis has not been clearly illuminated
We showed that miRNA-146 inhibition results in an increase in pro-inflammatory cytokines, such as IL-1β, IL-6 and tumor necrosis factor-α (TNF-α), through IL-1 receptor-associated kinase 1 (IRAK1) activation
Using real-time polymerase chain reaction (PCR), we confirmed that miRNA-146a and miRNA-146b-5p increased in pooled RNA samples and five individual RNA samples after P.g LPS stimulation (Figure 1C). miRNA146 up-regulation in P.g LPS-stimulated cells suggests that miRNA-146 may regulate the gingival inflammatory response
Summary
Various microRNAs (miRNAs) regulate immune and inflammatory responses, the function of miRNAs in periodontitis has not been clearly illuminated. We measured miRNA-146 (miRNA-146a and miRNA-146b-5p) expression and explored its regulatory function in the inflammatory response in human gingival fibroblasts (HGFs). Human gingival fibroblasts (HGFs), which are the major components of gingival connective tissue, directly interact with bacteria and bacterial products, including LPS, in periodontitis [2]. MicroRNAs (miRNAs) are an abundant class of short (20 to 25 nucleotides), non-coding RNA molecules. They function as post-transcriptional regulators that bind to complementary sequences in the 3' untranslated regions (3' UTRs) of target messenger RNA transcripts (mRNAs), usually resulting in gene silencing [3,4]. The function of miRNAs in HGFs during periodontitis remains unclear
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