MiRNAs have an important role in several biologic processes, including differentiation, proliferation, and apoptosis of the cells (19, 20). It is reported that changes their levels participate in the development of different diseases such as cancers, cardiovascular diseases, chronic hepatitis, and diabetes (21–24). MiR-146a controls inflammatory responses through down-regulating the expressions of IL-1 receptor-associated kinase-1 (IRAK-1) and tumor necrosis factor receptor-associated factor 6 (TRAF 6) (25, 26). Thus, dysfunction and/ or down-regulation of miR-146a results in the pathogenesis of inflammatory diseases, especially periodontitis (8). In our knowledge, there is no report pointing to anti-inflammatory effect of miR-146a on the pathogenesis of the disease through RANKL/OPG axis. This study was therefore focused on determining the possible contribution of miR-146a in this filed.
In previous study, we observed the level of miR-146a in gingival tissues of patients with chronic periodontitis, a form of the disease according to the update of the 1999 American academy of periodontology classification criteria (27, 28), was directly correlated to clinical scores (CAL and PD) of periodontitis. Furthermore, it was observed that level of miR-146a had a direct association with the clinical scores of disease severity in aggressive periodontitis (10, 11), as another form of the disease according to the old classification of periodontitis (27). These findings suggest that miR-146a may be associated with the pathobiology of disease. In an effort to explore possible downstream targets by which miR-146a may contribute to the pathobiology of disease, the levels of the main pro-inflammatory cytokines such as TNF-α, IL-1β, and IL-6 were studied in patients with chronic and aggressive periodontitis (29, 30). With the expectation of IL-1β level in chronic periodontitis, the results showed significant reductions in the levels of pro-inflammatory cytokines in two subgroups within periodontitis patients (10, 11). However, these studies failed to indicate the associations of pro-inflammatory cytokines with the major clinical factors of disease severity. Thus, the key question was how miR-146a may participate in the development and outcome of the disease. In the present study, the levels of two molecular factors involved in the pathogenesis of periodontitis along with miR-146a level were investigated.
Our study showed that miR-146a had an increased level in two subgroups within periodontitis. Interestingly, group A of patients experienced a significant increase in miR-146a level compared with group B of patients. In agreement with these observations, our previous studies revealed that the expressions of IL-1β, IL-6, and TNF-α were higher in gingival tissue of patients with chronic periodontitis than subjects with aggressive periodontitis (29, 30). These findings suggest that inflammation reactions have higher severity in chronic periodontitis or early disease stages than aggressive form or late disease stages, which the disease progression occur in higher rate (31). Regarding the anti-inflammatory impacts of miR-146a, it is likely that the elevated level of this miRNA is a regulatory response to control destructive and inflammatory responses, leading to the reductions in the destructions of the connective tissue and alveolar bone in group A of patients compared with subjects in group B.
In an attempt to determine other downstream targets influenced by miR-146a, the levels of OPG and RANKL were assessed in two groups of patients. The elevated expressions of OPG and RANKL were observed in periodontitis patients upon preliminary phase of periodontal therapy, although these increases were not statistically significant, due perhaps to low sample size. Furthermore, the expression levels of RANKL and OPG were respectively decreased and increased upon the disease development. These findings were in contract with the results of some studies indicating the elevated level of RANKL accompanied by a significant reduction in OPG level during the disease progression (32–34). However, immunohistochemical studies on periodontal tissues have revealed a negative expression of RANKL and a positive expression of OPG in both oral and periodontal pocket epithelium (35). Moreover, several lines of evidence reveal that RANKL and OPG levels have the tendency to the reduction and elevation upon non-surgical periodontal treatment, respectively (36, 37).
As mentioned previous, miR-146a down-regulates IRAK-1 and TRAF 6, which are the key adaptors in signaling by toll like receptors (TLRs) and cytokine receptors (4). Down-regulation of these adaptor proteins result in the abolished activation of nuclear factor-kappa B, which is a central transcription factor in the transcription of pro-inflammatory genes (38). Other studies have demonstrated that pro-inflammatory cytokines stimulate the expression of RANKL and suppress the production of OPG (14–16). These studies suggest that the reduced level of RANKL and increased expression of OPG during the disease progression may relate to the elevated levels of miR-146a level in the early disease stage to exert a negative impact on pro-inflammatory cytokine productions and thereby reduce disease progression.
In the next step, to deremine the possible impact of miR-146a on the levels of RANKL and OPG, the correlations of miR-146a level with these molecular factors were evaluated. Our results showed that miR-146a level was not correlated to OPG level.