Micro RNA-146a effect on Periodontitis is Independent on OPG/RANKL Axis

Background MicroRNA-146a (miR-146a) is a regulator of inammatory response. Periodontitis is a disease with immune pathophysiology of the periodontium in which the inammation results in the destruction of the soft tissues and alveolar bone. Therefore, this study was investigated whether miR-146a may contribute to periodontitis through affecting the levels of OPG and RANKL. The levels of miR-146a, RANKL, and OPG in gingival tissues from patients with generalized periodontitis stages II and III and grades A and B (n = 15, group A), patients with generalized periodontitis stages III and IV and grade C (n = 15, group B), and healthy individuals (n = 10) were determined by real-time PCR. The associations of miR-146a expression with OPG and RANKL levels were evaluated. in two subgroups within patients were signicantly higher than healthy showed the increased level group A of patients compared (P Clinical parameters probing and clinical attachment (P The levels OPG and RANKL were increased in patients compared with although the elevated levels were not statistically signicant. not associated OPG and RANKL.

regions in messenger RNAs (mRNAs) resulting in their degradation and/or translation inhibition (1).
Periodontitis is a disease with immune pathophysiology of the periodontium which the in ammation damages the soft tissues and destroys the alveolar bone (5). The consequences are detachment of gingival tissues from the teeth, destruction of alveolar bone, and loss of teeth (5).
Alveolar bone destruction is stimulated by osteoclasts. The differentiation and function of osteoclast are induced by receptor activator of nuclear factor-kB ligand (RANKL) upon binding to its receptor, RANK (6). Osteoprotegrin (OPG) is a decoy receptor which binds to RANKL and inhibits its binding to RANK, and thereby preventing osteoclastogenesis (6). The in ammation affects bone homeostasis through enhancing the expression of RANKL and reducing the production of OPG (6).
Extensive data from the literature have indicated that patients with periodontitis had the increased level of miR-146a in comparison with healthy subjects (7)(8)(9). Previous studies revealed that the level of miR-146a was directly correlated to the clinical features of disease severity including probing depth (PD) and clinical attachment loss (CAL). It is suggested that miR-146a may contribute to the pathogenesis of the disease (10,11). Furthermore, animal studies have provided some evidence to show that miR-146a exerts anti-in ammatory impacts in periodontitis through inhibiting the expressions of the pro-in ammatory cytokines such as tumor necrosis factor-alpha (TNF-α), interleukin-1β (IL-1β), and interleukin-6 (IL-6) in periodontal tissue (7,12,13). On the other hand, several reports have demonstrated that pro-in ammatory cytokines stimulate the expression of RANKL and suppress the production of OPG (14)(15)(16). This study was therefore focused on investigating whether miR-146a exerts anti-in ammatory impacts on pathobiology of disease through RANKL/OPG axis.

Sample collection and clinical examination
Gingival tissue samples were obtained from 30 periodontitis patients and 15 healthy subjects. Patients were divided into two groups according to the new classi cation of periodontitis (17) including: 1) patients with generalized periodontitis stages II and III and grades A and B (n = 15, group A); 2) patients with generalized periodontitis stages III and IV and grade C (n = 15, group B). The diagnosis of periodontitis and its stage, extent, and grade was performed by a periodontist based on the new classi cation of periodontitis (17). Supra-and sub-gingival scaling, polishing, and oral hygiene instructions were performed at least 1 month before surgery as the preliminary phase of periodontal therapy. The average sizes of gingival tissue samples obtained from periodontits and healthy subjects were about 25 and 8.5 mm, respectively.
Gingival tissues used as control group were obtained from healthy individuals during crown-lengthening surgery with the inclusion criteria including PD < 3 mm, CAL < 3 mm and no evidence of the alveolar bone destruction based on the radiographic images.
The informed consent was collected from the participants. This study was approved by the Ethics Committee of Kashan University of Medical Sciences and performed according to the principles of Helsinki Declaration. Clinical parameters (PD and CAL) were measured by a calibrated periodontal probe (Hu Friedy, Chicago, Illinois, USA).
Measurements of the expression levels of miR-146a, OPG, and RANKL To determine the relative expression level of miR-146a, gingival tissue samples were homogenized using 1.0-mm silicon carbide beads (BioSpec products, Bartlesville, OK, USA). The researchers were blinded to sample information. Total RNA was isolated using a mirVana miRNA isolation kit (Ambion, Thermo Fisher Scienti c, Waltham, MA, USA) according to the manufacturer's protocol. The yield and purity of RNAs were quanti ed by nano drop (BioTek, Epoch, USA). Afterwards, RNA was reverse transcribed to complementary DNA (cDNA) using a TaqMan microRNA Reverse Transcription Kit (Applied Biosystems, Thermo Fisher Scienti c, USA) based on the manufacturer's instructions. The level of mature miR-146a was quanti ed by real-time polymerase chain reaction (Real-time PCR). This method was performed using a Rotor Gene 6000 (Qiagen, Hilden, Germany), TaqMan® Universal Master Mix II, no UNG, and hsa-miRNA146a kits (Applied Biosystems, USA). The machine was programmed as follows: 30 seconds incubation at 95˚C followed by 40 cycles consisted of a 95˚C denaturing temperature for 5 seconds and an annealing-extension temperature at 60˚C for 30 seconds. A non-template control containing 0.5 µL of Dnase/Rnase free water was used as the negative control. All analyses were performed in duplicate.
To measure the levels of OPG and RANKL, cDNAs were synthesized from total RNAs using a RevertAid First Strand cDNA Synthesis Kit (Thermo FisherScienti c, Wilmington, Delaware, USA). Afterwards, Realtime PCR was performed in a reaction mixture consisted of Master Mix (7.5 µL) (SYBR® Premix Ex Taq™ II; TaKaRa, Kyoto, Japan), 10 pM forward and reverse primers (0.5 µL), cDNA template (0.5 µL), and DNase-RNase free water (6 µL). The cycling parameters for OPG and RANKL were the same as those used to miR-146a. The melting curve was generated in 55˚C to 99˚C temperature range. Sequences of the primers are shown in Table. 1.
Glyceraldehyde 3 phosphate dehydrogenase (GAPDH) gene expression served as endogenous control to normalize the expression of genes (Table 1). With the exception of the annealing temperature (55˚C for 30 seconds), the mixture reaction and cycling parameters were similar to those for RANKL and OPG. Relative expressions of OPG and RANKL were calculated using 2 −∆∆ct method (18).  Data are representative of the mean ± SD. The scores of CAL and PD in periodontitis patients were signi cantly higher than healthy subjects. * P < 0.05.

Statistical analysis
Data analyses were carried out using the SPSS program (v. 20; SPSS, Chicago, USA). The results are presented as mean ± standard deviation (SD). Kolmogrov-Smirnov test was used to determine normal distribution of data. The comparisons of two groups in normal and non-normal distribution cases were performed by using student's t-test and Mann-Whitney U test, respectively. The correlation analyses were done using Pearson's test (for normal distributions) and Spearman's test (for non-normal distributions). P value < 0.05 was considered statistically signi cant.

Clinical ndings
To determine clinical features of patients and con rm periodontitis, the PD and CAL values of patients and healthy subjects were assessed. As shown in Table. 2, patients in two subgroups within periodontitis had statistically signi cant higher PD and CAL scores than control group (P < 0.05).
Expression level of miR-146a in gingival tissues of participants Regarding the fact that miR-146a can be considered as a negative feedback regulator of innate immunity and has possible role in the pathogenesis of some in ammatory diseases, the expression level of miR-146a in periodontitis was investigated. Our results revealed that both groups of patients with generalized periodontitis had an increased level of miR-146a compared with healthy individuals after one-month initial unsuccessful non-surgical treatment (P <0.001, Figure. 1). In addition, miR-146a level was signi cantly higher in group A of patients (subjects with generalized periodontitis stages II and III and grades A and B) than group B of patients (subjects with generalized periodontitis stages III and IV and grade C) (P <0.05, Figure. 1).
The expression levels of RANKL and OPG in gingival tissues of participants Having considered that RANKL induces the destruction of alveolar bone, its expression level was analyzed using real-time PCR assay. The expression level of RANKL in gingival tissue of patients in two groups of generalized periodontitis did not statistically differ from that of healthy individuals ( Figure. 2A).
In addition, the expression level of OPG, a main inhibitor of the bone destruction, in both groups of patients did not show signi cant changes compared with healthy subjects (Figure. 2B).

Associations of miR-146a level with RANKL and OPG levels in gingival tissues
To explore possible mechanism(s) suggesting miR-146a impact on the pathogenesis of periodontitis, correlations of miR-146a level with RANKL and OPG were analyzed. No signi cant association was observed between miR-146a and OPG and RANKL levels.
Thus, dysfunction and/ or down-regulation of miR-146a results in the pathogenesis of in ammatory diseases, especially periodontitis (8). In our knowledge, there is no report pointing to anti-in ammatory 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 led.
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 classi cation 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 classi cation of periodontitis (27). These ndings 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-in ammatory 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 signi cant reductions in the levels of pro-in ammatory cytokines in two subgroups within periodontitis patients (10,11). However, these studies failed to indicate the associations of pro-in ammatory 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 signi cant 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 ndings suggest that in ammation 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-in ammatory impacts of miR-146a, it is likely that the elevated level of this miRNA is a regulatory response to control destructive and in ammatory 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 in uenced 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 signi cant, due perhaps to low sample size. Furthermore, the expression levels of RANKL and OPG were respectively decreased and increased upon the disease development. These ndings were in contract with the results of some studies indicating the elevated level of RANKL accompanied by a signi cant reduction in OPG level during the disease progression (32)(33)(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-in ammatory genes (38). Other studies have demonstrated that proin ammatory 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-in ammatory 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.

Conclusion
Although it is thought that miR-146a has indirectly effects on RANKL/OPG axis through reducing proin ammatory cytokines. The results of this study provide evidence to indicate that anti-in ammatory impacts of miR-146a are independent on RANKL/OPG axis. Nevertheless, additional studies with larger sample sizes are required to con rm our ndings and explain the possible mechanism(s) involved in antiin ammatory effects of miR146a on disease development. It is worthy that future studies will be designed to clarify the impacts of miR-146a on other in ammatory mediators such as prostaglandins, leukotrienes, macrophage colony-stimulating factor, and broblast growth factor, which play indispensable roles in bone homeostasis and periodontal disease progression (39).

Declarations
Ethics approval and consent to participate This work was con rmed by the Ethics Committee of Kashan University of Medical Sciences. Written informed consent to participate in the study was obtained from all subjects before entering the study.

Consent for publication
Not applicable. No personal information was provided in this paper.

Availability of data and materials
All data generated or analyzed during this study are included in this published article. No sequencing data generated in this study to deposit in a suitable public repository such as the National Center for Biotechnology Information (NCBI) database. The raw data are available from the authors to any researcher who wishes to collaborate with us. Correspondence should be addressed to Hossein Motedayyen at the following email address. Email address: hmotedayyen@gmail.com. Speci c primers were designed using Allele ID 7.5 software (Premier Biosoft) and checked by Primer-BLAST (NCBI) which is an open access database.

Competing interests
The authors declare that there is no con ict of interests.

Funding
This study was nancially supported by Kashan University of Medical Sciences (KAUMS, grant no: 92081). KAUMS did not participate in the design of the study and collection, analysis, and interpretation of data and in writing the manuscript. The expression levels of miR-146a in patient and healthy subjects. The levels of miR-146a in gingival tissues from group A (n=15) and B (n=15) of patients and healthy subjects (n = 15) were measured by real-time PCR. Data are shown as mean ± SD of relative expression of miR-146a. * p<0.05, **** p<0.0001 Figure 2 The levels of RANKL and OPG in parient and healthy subjects. The relative expressions of RANKL and OPG in group A (n=15) and B (n=15) of patients and healthy subjects (n = 15) were measured by real-time PCR. The results are shown as mean ± SD.