Promotion effect of apical tooth germ cell-conditioned medium on osteoblastic differentiation of periodontal ligament stem cells through regulating miR-146a-5p.

BACKGROUND: MicroRNAs (miRNAs) play an important role in gene regulation that controls stem cells differentiation. Periodontal ligament stem cells (PDLSCs) could differentiate into osteo-/cementoblast-like cells that secretes cementum-like matrix both in vitro and in vivo. Whether miRNAs play key roles in osteoblastic differentiation of PDLSCs triggered by a special microenviroment remains elusive. In this study, we aimed to investigate potential miRNA expression changes in osteoblastic differentiation of PDLSCs by the induction of apical tooth germ cell-conditioned medium (APTG-CM). METHODS AND RESULTS: First, we analyzed the ability of APTG-CM to osteogenically differentiate PDLSCs. The results exhibited an enhanced mineralization ability, higher ALP activity and increased expression of osteogenic genes in APTG-CM-induced PDLSCs. Second, we used miRNA sequencing to analyze the miRNA expression profile of PDLSCs derived from three donors under 21-day induction or non-induction of APTG-CM. MiR-146a-5p was found to be up-regulated miRNA in induced PDLSCs and validated by RT-qPCR. Third, we used lentivirus-up/down system to verify the role of miR-146a-5p in the regulation of osteoblastic differentiation of PDLSCs. CONCLUSIONS: In conclusion, our results demonstrated that miR-146a-5p was involved in the promotion effect of APTG-CM on osteoblastic differentiation of PDLSCs, and suggested that miR-146a-5p might be a novel way in deciding the direction of PDLSCs differentiation.

The expression levels of MicroRNA-146a, RANKL and OPG after non-surgical periodontal treatment.

OBJECTIVE: MicroRNA-146a (miR-146a) is a regulator of inflammatory response. Periodontitis is a disease with immune pathophysiology of the periodontium in which the inflammation results in the destruction of the soft tissues and alveolar bone. Therefore, the aim of this study was to investigate the expressions of miR-146a, OPG, and RANKL in diseased and healthy periodontal tissues to understand whether miR-146a expression level may associate with OPG and RANKL mRNA levels and OPG/RANKL ratio after non-surgical periodontal treatment. METHODS: 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. RESULTS: The levels of miR-146a in two subgroups within periodontitis patients were significantly higher than healthy subjects (P < 0.0001). MiR-146a showed the increased level in group A of patients compared with group B (P < 0.05). Clinical parameters such as probing depth (PD) and clinical attachment loss (CAL) were significantly higher in patients than control group (P < 0.05). The levels of OPG and RANKL were increased in patients compared with healthy subjects, although the elevated levels were not statistically significant. MiR-146a was not associated with OPG and RANKL levels and OPG/RANKL ratio. CONCLUSIONS: The results of this study failed to show the associations of miR-146a level with OPG and RANKL levels and OPG/RANKL ratio in periodontitis after non-surgical periodontal treatment.

LncRNA MAFG-AS1 regulates human periodontal ligament stem cell proliferation and Toll-like receptor 4 expression.

LncRNA MAFG-AS1 is predicted to interact with miR-146a, which can target Toll-like receptor 4 (TLR4), a key player in periodontitis. This study aimed to investigate the roles of MAFG-AS1 in periodontitis. It was observed that MAFG-AS1 was downregulated in the human periodontal ligament stem cells (PDLSCs) derived from periodontitis-affected teeth. Dual-luciferase assay revealed that co-transfection of MAFG-AS1 expression vector and miR-146a mimic showed significantly lower relative luciferase activity comparing to co-transfection of MAFG-AS1 expression vector and negative control (NC) miRNA. However, MAFG-AS1 and miR-146a failed to affect each other. Interestingly, MAFG-AS1 overexpression led to the upregulated TLR4. In addition, MAFG-AS1 overexpression also led to the inhibited proliferation of PDLSCs. Therefore, MAFG-AS1 may regulate the proliferation of PDLSCs and the expression of TLR4 to participate in periodontitis.

APRPG-modified nanoliposome loaded with miR-146a-5p inhibitor suppressed choroidal neovascularization by targeting endothelial cells.

INTRODUCTION: To explore the effect and mechanism of APRPG-modified nanoliposomes loaded with miR-146a-5p inhibitor (ANL-miR-146a-5p inhibitor) on endothelial cell proliferation, migration, tube formation, and choroidal neovascularization (CNV) in mice. METHODS: ANL-miR-146a-5p inhibitors were generated by thin film hydration; in vitro, endothelial cell uptake experiment was used to detect the targeting effect of ANL-miR-146a-5p inhibitor; endothelial cells proliferation, migration, and tube formation were detected, respectively, by CCK8 assay, scratch assay, and Matrigel tube formation assay. In vivo, the mice CNV models were established by 810 nm laser photocoagulation. Mice choroidal flatmounts were performed to detect the volume of CNV after intravitreal injection of L-miR-146a-5p inhibitor, ANL-miR-146a-5p inhibitor, or normal saline; the vascular endothelial growth factor (VEGF) expression of mice choroidal tissue was detected by ELISA; HE section and electrophysiology (ERG) were performed to check the toxicity of ANL-miR-146a-5p inhibitor on mice retina. RESULTS: ANL are targeted to endothelial cells and are more targeted in inflammatory environment. At the same concentration, ANL-miR-146a-5p inhibitor's ability to inhibit endothelial cell proliferation, migration, tube formation, CNV formation, and VEGF expression is better than L-miR-146a-5p inhibitor (p < 0.05); ANL-miR-146a-5p inhibitor had no toxicity on the structure of mouse retina; ANL-miR-146a-5p inhibitor had no toxicity on the a-wave and b-wave amplitudes and b-wave implicit times (p > 0.05). CONCLUSIONS: ANL-miR-146a-5p inhibitor can more effectively down-regulate the expression level of VEGF through its targeting to endothelial cells, thereby more effectively inhibiting endothelial cell proliferation, migration, tube formation, and mice CNV formation.

microRNA-146a and Hey2 form a mutual negative feedback loop to regulate the inflammatory response in chronic apical periodontitis.

Chronic apical periodontitis (CAP) is defined as chronic inflammation of the dental pulp and root canal system. Porphyromonas endodontalis lipopolysaccharide ( P. endodontalis LPS) plays an important role in inducing an inflammatory response in CAP. microRNA-146a (miR-146a) is a key regulator of inflammation and is induced by LPS. Hairy and enhancer-of-split related with YRPW motif 2 (Hey2) has been confirmed to be induced by the Notch signaling pathway, which is involved in tooth development, pulp regeneration, and repair after injury. Our study aimed to investigate the functional role of miR-146a via the targeting of Hey2 in CAP as well as the underlying mechanism. Compared with 13 healthy controls, miR-146a and Hey2 expressions were significantly higher in 20 patients with CAP. In addition, miR-146a, Hey2, interleukin (IL)-6, IL-1ß, and tumor necrosis factor (TNF)-α expressions were significantly increased in MC3T3-E1 cells stimulated with different concentrations (0-20 µg/mL) of P. endodontalis LPS for different amounts of time (0-48 hours). Moreover, miR-146a, which acts as an anti-inflammatory mediator, negatively regulated the expression of IL-6, IL-1ß, and TNF-α, and Hey2 was confirmed as a target gene of miR-146a by a luciferase reporter assay. Hey2 also negatively regulated miR-146a, IL-6, IL-1ß, and TNF-α expressions, and P. endodontalis LPS strongly induced Hey2 recruitment to the IL-6 promoter (-400 ~ -200 bp). These findings suggest that miR-146a and Hey2 form a mutual negative feedback regulatory loop, demonstrating a novel mechanism that regulates inflammatory responses in CAP.

Involvement of miR-146a-5p/neurogenic locus notch homolog protein 1 in the proliferation and differentiation of STRO-1+ human dental pulp stem cells.

Dental pulp stem cells (DPSCs) and stem cells from the apical papilla (SCAPs) are oral mesenchymal stem cells capable of self-renewal and have a potential for multilineage differentiation. Increasing evidence shows that microRNAs (miRNAs) play important roles in stem cell biology. Here, we focused on exploring miR-146a-5p and its relationship to the undifferentiated status of STRO-1+ SCAPs and STRO-1+ DPSCs, as well as its role during STRO-1+ DPSC differentiation and proliferation. Our data indicated that baseline miR-146a-5p expression is significantly lower in STRO-1+ SCAPs than in STRO-1+ DPSCs and increased in the latter during osteogenic induction. Moreover, we identified miR-146a-5p as a key miRNA that promotes osteo/odontogenic differentiation of STRO-1+ DPSCs and attenuates cell proliferation. Additionally, it was observed that STRO-1+ DPSC mineralization results in the downregulation of notch receptor 1 (NOTCH1) and hes family bHLH transcription factor 1 (HES1). Interference with neurogenic locus notch homolog protein 1 (Notch 1) signaling was verified to enhance differentiation and suppress STRO-1+ DPSC proliferation. It was further observed that miR-146a-5p directly targets the 3'-untranslated region (3'-UTR) of NOTCH1 and inhibits expression of both NOTCH1 and HES1mRNAs and Notch 1 and transcription factor HES-1 (HES-1) proteins in STRO-1+ DPSCs. We conclude that miR-146a-5p exerts its regulatory effect on STRO-1+ DPSC differentiation and proliferation partially by suppressing Notch signaling.

miR-146a regulates inflammatory cytokine production in Porphyromonas gingivalis lipopolysaccharide-stimulated B cells by targeting IRAK1 but not TRAF6.

It has been suggested that microRNAs (miRs) are involved in the immune regulation of periodontitis. However, it is unclear whether and how miRs regulate the function of B cells in the context of periodontitis. This study is to explore the role of miR-146a on the inflammatory cytokine production of B cells challenged by Porphyromonas gingivalis (P. gingivalis) lipopolysaccharide (LPS). Primary B cells were harvested from mouse spleen. Quantitative real-time polymerase chain reaction (qPCR), enzyme-linked immunosorbent assay (ELISA) were used to detect the expression of inflammatory cytokines in B cells in the presence or absence of P. gingivalis LPS and/or miR-146a. Bioinformatics, luciferase reporter assay and overexpression assay were used to explore the binding target of miR-146a. Our results showed that miR-146a level in B cells was elevated by P. gingivalis LPS stimulation, and the mRNA expressions of interleukin (IL)-1ß, 6 and 10, and IL-1 receptor associated kinase-1 (IRAK1), but not TNF receptor associated factor 6 (TRAF6), were also upregulated. The expression levels of IL-1ß, 6, 10 and IRAK1 were reduced in the presence of miR-146a mimic, but were elevated by the addition of miR-146a inhibitor. MiR-146a could bind with IRAK1 3' untranslated region (UTR) but not TRAF6 3'-UTR. Overexpression of IRAK1 reversed the inhibitory effects of miR-146a on IL-1ß, 6 and 10. In summary, miR-146a inhibits inflammatory cytokine production in B cells through directly targeting IRAK1, suggesting a regulatory role of miR-146a in B cell-mediated periodontal inflammation.

MicroRNA-146a and microRNA-155 as novel crevicular fluid biomarkers for periodontitis in non-diabetic and type 2 diabetic patients.

AIM: Recent studies point at the crucial role of epigenetic mechanisms in the development of multifactorial diseases such as periodontitis and diabetes mellitus (DM) type 2. In addition, circulatory microRNAs (miRs) have emerged as novel biomarkers for various diseases. Aim of this study was to investigate the levels of miR-146a and miR-155 and superoxide dismutase (SOD) activity in gingival crevicular fluid (GCF) of periodontitis patients with (CPDM) and without (CP) DM type 2 as well as in periodontally healthy, control groups (PHDM and PH, respectively). MATERIAL AND METHODS: miR modulation was analysed using quantitative real-time PCR while SOD activity was measured spectrophotometrically. RESULTS: The upregulation of miR-146a and miR-155 was observed in CP and CPDM patients' baseline, while the levels decreased after 6 weeks of the non-surgical therapy to the levels comparable to PH and PHDM, respectively. Expression levels of miRs positively correlated with SOD activity. Levels of miR-146a were higher in PHDM compared to PH patients. Multivariate analysis revealed that levels of miR-146a and miR-155 were significantly associated with periodontitis when adjusting for age and gender. CONCLUSIONS: miR-146a and miR-155 may be considered as possible novel biomarkers for periodontitis in non-diabetic and type 2 diabetic patients.

Portland cement induces human periodontal ligament cells to differentiate by upregulating miR-146a.

BACKGROUND/PURPOSE: Bioaggregates such as Portland cement (PC) can be an economical alternative for mineral trioxide aggregate (MTA) with additional benefit of less discoloration. MTA has been known to induce differentiations of several dental cells. MicroRNAs are important regulators of biological processes, including differentiation, physiologic homeostasis, and disease progression. This study is to explore how PC enhances the differentiation of periodontal ligament (PDL) cells in microRNAs level. METHODS: PDL cells were cultured in a regular PC- or MTA-conditioned medium or an osteoinduction medium (OIM). Alizarin red staining was used to evaluate the extent of mineralization. Transfection of microRNA mimics induced exogenous miR-31 and miR-146a expression. The expression of microRNAs and differentiation markers was assayed using reverse-transcriptase polymerase chain reaction. RESULTS: PC enhanced the mineralization of PDL cells in a dose-dependent manner in the OIM. Exogenous miR-31 and miR-146a expression upregulated alkaline phosphatase (ALP), bone morphogenic protein (BMP), and dentin matrix protein 1 (DMP1) expression. However, miR-31 and miR-146a modulates cementum protein 1 (CEMP1) expression in different ways. PC also enhanced ALP and BMP but attenuated CEMP1 in the OIM. Although the OIM or PC treatment upregulated miR-21, miR-29b, and miR-146a, only miR-146a was able to be induced by PC in combination with OIM. CONCLUSION: This study demonstrated that PC enhances the differentiation of PDL cells, especially osteogenic through miR-146a upregulation. In order to control the ankylosis after regenerative endodontics with the usage of bioaggregates, further investigations to explore these differentiation mechanisms in the miRNA level may be needed.

miR-146a and miR-155 expression in PBMCs from patients with Sjögren's syndrome.

BACKGROUND: An increasing number of studies have revealed that microRNA (miRNA) contributes to the pathogenesis of autoimmune diseases. The objective of this study is to investigate the miR-146a and miR-155 levels in peripheral mononuclear blood cells from patients with primary Sjögren's syndrome (pSS) who were not receiving medications and to examine the correlations between these miRNA levels and the clinical features of the disease. METHOD: Using real-time polymerase chain reaction analysis of miRNAs, the miR-146a and miR-155 expression levels were assessed in peripheral mononuclear blood cells from 27 patients with pSS and 22 healthy controls, and the relationships between these miRNA levels and the visual analog scale (VAS) scores for dry mouth, dry eyes, and parotid gland swelling were investigated. RESULTS: Compared with the healthy controls, the miR-146a expression level was significantly increased in the patients with pSS (P = 0.0182) and was positively correlated with the VAS scores for parotid swelling (r = 0.4475, P = 0.0192) and dry eyes (r = 0.4051, P = 0.0361). Although the miR-155 expression level was significantly decreased in the patients with pSS (P = 0.0131), the miR-155 expression positively correlated with the VAS score for dry eyes (r = 0.4894, P = 0.0096). CONCLUSION: Our results demonstrated miR-146a overexpression and miR-155 underexpression in the peripheral mononuclear blood cells of the patients with pSS. Furthermore, the expression levels of these miRNAs correlated with the patients' clinical features. Our data suggest that miR-146a and miR-155 might play important roles in the pathogenesis of pSS and that their expression levels may be useful for diagnosing pSS and for predicting disease activity and therapeutic responses.

From Bone Remodeling to Wound Healing: An miR-146a-5p-Loaded Nanocarrier Targets Endothelial Cells to Promote Angiogenesis.

Wound healing is a complex challenge that demands urgent attention in the clinical realm. Efficient angiogenesis is a pivotal factor in promoting wound healing. microRNA-146a (miR-146a) inhibitor has angiogenic potential in the periodontal ligament. However, free microRNAs (miRNAs) are poorly delivered into cells due to their limited tissue specificity and low intracellular delivery efficiency. To address this hurdle, we developed a nanocarrier for targeted delivery of the miR-146a inhibitor into endothelial cells. It is composed of a polyethylenimine (PEI)-modified mesoporous silica nanoparticle (MSN) core and a pentapeptide (YIGSR) layer that recognizes endothelial cells. In vitro, we defined that the miR-146a inhibitor and adiponectin (ADP) can modulate angiogenesis and the remodeling of periodontal tissues by activating the ERK and Akt signaling pathways. Then, we confirm the specificity of YIGSR to endothelial cells, and importantly, the nanocarrier effectively delivers the miR-146a inhibitor into endothelial cells, promoting angiogenesis. In a C57 mouse skin wound model, the miR-146a inhibitor is successfully delivered into endothelial cells at the wound site using the nanocarrier, resulting in the formation of new blood vessels with strong CD31 expression. Additionally, no significant differences are found in the expression levels of inflammatory markers interleukin-6 and tumor necrosis factor-α. This outcome not only brings new strategies for angiogenesis but also exhibits broader implications for bone remodeling and wound healing. The breakthrough holds significance for future research and clinical interventions.

Enterovirus 71 induces pyroptosis of human neuroblastoma SH-SY5Y cells through miR-146a/ CXCR4 axis.

Enterovirus 71 (EV71) is a predominant causative pathogen of hand-foot-and-mouth disease (HFMD) in children. Compared with other HFMD-associated viruses, EV71 tends to induce more severe neurological complications and even death. However, the detailed mechanism of EV71 causes nervous system disorder is still unclear. In this study, we found that EV71 induced the GSDMD/NLRP3-mediated pyroptosis of SH-SY5Y cells through up-regulated miR-146a. Through bioinformatic analysis, we identified C-X-C chemokine receptor type 4 (CXCR4) as the potential target of miR-146a. We noticed that the expression of CXCR4 was regulated by miR-146a during EV71 infection. Moreover, our results show that over-expression of CXCR4 attenuated EV71-induced pyroptosis of SY-SY5Y cells. These results reveal a previously unrecognized mechanism in which EV71 induces nervous system cells damage through regulating miR-146a/CXCR4 mediated pyroptosis.

MiR-146a-5p Promotes Dental Stem Cells Osteo/odontogenic Differentiation through NF-Kappa B Signaling Pathway by Targeting TRAF6.

OBJECTIVE: To screen miRNAs that could simultaneously regulate osteo/odontogenic differentiation of multiple stem cells, including dental pulp stem cells (DPSCs), stem cells from the apical papilla (SCAPs) and periodontal ligament stem cells (PDLSCs). METHODS: Differentially expressed miRNAs analysis on three miRNA microarrays data of dental stem cells undergoing osteo/odontogenic differentiation (GSE138180, GSE154466 and GSE159508) was performed, and miR-146a-5p were identified by bioinformatic prediction, dual-luciferase reporter assay and quantitative real-time polymerase chain reaction (PCR). In addition, differentially expressed genes between miR-146a-5p overexpressed group and control group (GSE79341) were applied for KEGG pathways enrichment analysis. RESULTS: MiR-146a-5p expression increased in the osteo/odontogenic differentiation of DPSCs, SCAPs and PDLSCs. Tumour necrosis factor receptor (TNFR)-associated factor 6 (TRAF6) was identified as the target gene of miR-146a-5p. Furthermore, miR-146a-5p could influence the NF-Kappa B signalling pathway. CONCLUSION: This study suggests that miR-146a-5p could promote differentiation in multiple dental stem cells through the NF-Kappa B signalling pathway by targeting TRAF6.

Expression of miR-155 and miR-146a in the saliva of patients with periodontitis and its clinical value.

OBJECTIVE: To explore the significance of microRNA-155 (miR-155) and microRNA-46a (miR-46a) in the diagnosis of periodontitis (PD). METHODS: A total of 41 patients with PD admitted to our hospital between September 2017 and August 2019 were enrolled into an observation group (obs group), and 35 healthy individuals during the same period were enrolled into a control group (con group). A qRT-PCR assay was carried out to quantify miR-155 and miR-146a expression, and the relationship between the expression of miR-155 and miR-146a and severity and clinical indicators of PD was analyzed. RESULTS: The obs group showed significantly higher expression of saliva miR-155 and miR-146a than the con group (P<0.05), and the expression of them was positively correlated with the severity of their PD. It was also positively correlated with gingival index (GI), attachment loss (AL), plaque index (PLI), probing depth (PD), and bleeding on probing (BOP) of them. Additionally, according to the receiver operating characteristic (ROC) curves, the area under the curve (AUC), specificity, and sensitivity of miR-155 in diagnosing PD were 0.887, 78%, and 97.14%, respectively, and those of miR-146a in diagnosing PD were 0.745, 58.54%, and 88.57%, respectively. CONCLUSION: MiR-155 and miR-146a were both highly expressed in the saliva of patients with PD, and the expression of them was positively correlated with the severity and clinical indexes of PD, so miR-155 and miR-146a might be involved in the development and progression of PD.

The Role of microRNAs in Pulp Inflammation.

The dental pulp can be affected by thermal, physical, chemical, and bacterial phenomena that stimulate the inflammatory response. The pulp tissue produces an immunological, cellular, and vascular reaction in an attempt to defend itself and resolve the affected tissue. The expression of different microRNAs during pulp inflammation has been previously documented. MicroRNAs (miRNAs) are endogenous small molecules involved in the transcription of genes that regulate the immune system and the inflammatory response. They are present in cellular and physiological functions, as well as in the pathogenesis of human diseases, becoming potential biomarkers for diagnosis, prognosis, monitoring, and safety. Previous studies have evidenced the different roles played by miRNAs in proinflammatory, anti-inflammatory, and immunological phenomena in the dental pulp, highlighting specific key functions of pulp pathology. This systematized review aims to provide an understanding of the role of the different microRNAs detected in the pulp and their effects on the expression of the different target genes that are involved during pulp inflammation.

RANKL blockade alleviates peri-implant bone loss and is enhanced by anti-inflammatory microRNA-146a through TLR2/4 signaling.

BACKGROUND: The present study was to determine the effect of local anti-RANKL antibody administration in the presence or absence of microRNA-146a on ligature-induced peri-implant bone resorption, and the potential role of TLR2/4 signaling in such effect. RESULTS: Titanium implants were placed in the left maxilla alveolar bone 6 weeks after extraction of first and second molars in C57/BL6 wild-type (WT) and TLR2-/- TLR4-/- (TLR2/4 KO) mice. Silk ligatures were tied around the implants 4 weeks after implantation. Anti-RANKL antibody (500 µg/mL) with or without microRNA 146a (miR-146a) (100 nM) was injected into palatal gingiva around implant on days 3, 6, and 9 during 2 weeks of ligation period. Bone resorption around the implants was assessed by 2D imaging using area measurement and 3D imaging using micro-computed tomography (µCT). Real-time quantitative PCR (RT-qPCR) was used to determine the peri-implant gingival mRNA expression levels of pro-inflammatory cytokines (TNF-α) and osteoclastogenesis-related cytokines (RANKL). In both WT and TLR2/4 KO mice, the bone resorption around implants was significantly increased in the ligation only group when compared to the non-ligation group, but TLR2/4 KO mice showed significantly less bone loss compared to WT mice after ligation. As expected, gingival injection of anti-RANKL antibody significantly reduced bone loss compared with the ligation only group in both WT and TLR2/4 KO mice. Moreover, injection of miR-146a in addition to anti-RANKL antibody significantly enhanced the inhibition of bone loss in WT mice but not in TLR2/4 KO mice. Gingival mRNA expressions of RANKL were significantly reduced by anti-RANKL antibody treatment in both WT and TLR2/4 KO mice but were not affected by the additional miR-146a treatment. Gingival mRNA expression of TNF-α was significantly reduced by miR-146a treatment in WT mice but not in TLR2/4 KO mice. The number of gingival inflammatory cell infiltration and peri-implant TRAP-positive cell formation was significantly reduced by the additional miR-146a treatment in WT mice but not in TLR2/4 KO mice. CONCLUSIONS: This study suggests that anti-inflammatory miR-146a enhance anti-RANKL-induced inhibition of peri-implant bone resorption through the regulation of TLR2/4 signaling and inhibition of TNF-α expression.

Dysregulation of miR-146a by periodontal pathogens: A risk for acute coronary syndrome.

BACKGROUND: Periodontitis is a polymicrobial, chronic inflammatory disease leading to loss of tooth-supporting structures. The bacteremia, endotoxemia, and systemic low-grade inflammation associate periodontitis with systemic illnesses such as diabetes mellitus and coronary artery disease. Periodontal pathogens have been detected from atheromatous plaque by amplification of the genetic material by using specific oligonucleotide primers in polymerase chain reaction. Though the association between periodontitis and cardiovascular diseases has been ascertained by systematic reviews and meta-analyses, its pathophysiology is not lucid. MicroRNAs are currently implicated in the regulation of many cellular processes including inflammation and may play a vital role in our understanding of this disease association. In this case-control study, we explored the role of the inflammatory microRNA, miR-146a, in acute coronary syndrome (ACS) subjects with and without chronic periodontitis (CP) and its regulation of the innate immune host response to periodontal pathogens. METHODS: Three groups each comprising 66 patients each, namely group 1 (ACS patients without CP), group 2 (ACS patients with CP) and group 3 (CP only) formed the study population. Subgingival plaque samples and serum samples were subjected to quantitative Polymerase Chain Reaction (qPCR) for detection of Porphyromonas gingivalis, a keystone pathogen and to assess the levels of circulating miR-146a and associated proinflammatory cytokines. RESULTS: miR-146a associated significantly in group 2 subjects with an odds ratio 1.434, 95% confidence interval 1.013-2.030, P < 0.042, and a predictive percentage of 83.3% and group 1 with a predictive percentage of 76.0.% The associated cytokines interleukin-6 (IL-6), tumor necrosis factor-α, and IL-1ß also showed an upregulation with statistical significance (P < 0.05). CONCLUSION: microRNA-146a is a key molecule associating periodontitis with acute coronary syndrome.

MiR-146a-5p Expression in Peripheral CD14⁺ Monocytes from Patients with Psoriatic Arthritis Induces Osteoclast Activation, Bone Resorption, and Correlates with Clinical Response.

In psoriatic arthritis (PsA), progressive bone destruction is mediated by monocyte-derived osteoclasts. MicroRNAs (miRNAs) regulate many pathophysiological processes; however, their function in PsA patient monocytes has not been examined. This study aims to address whether specific miRNAs in CD14⁺ monocytes and monocyte-derived osteoclasts cause active osteoclastogenesis in PsA patients. Candidate miRNAs related to monocyte activation (miR-146a-5p, miR-146b-5p and miR-155-5p) were measured in circulatory CD14⁺ monocytes collected from 34 PsA patients, 17 psoriasis without arthritis (PsO) patients, and 34 normal controls (NCs). CD14⁺ monocytes were cultured with media containing TNF-α and RANKL to differentiate into osteoclasts. Osteoclast differentiation and bone resorption were measured by TRAP immunostaining and dentin slice resorption, respectively. The results showed that the miR-146a-5p expression was higher in PsA patient-derived CD14⁺ monocytes compared to PsO and NCs. Activation and bone resorption were selectively enhanced in osteoclasts from PsA patients, but both were abrogated by RNA interference against miR-146a-5p. More importantly, after clinical improvement using biologics, the increased miR-146a-5p expression in CD14⁺ monocytes from PsA patients was selectively abolished, and associated with blood CRP level. Our findings indicate that miR-146a-5p expression in CD14⁺ monocytes derived from PsA patients correlates with clinical efficacy, and induction of osteoclast activation and bone resorption.

The effect of DNA methylation on the miRNA expression pattern in lipopolysaccharide-induced inflammatory responses in human dental pulp cells.

Endodontic infection is a widespread oral problem. DNA methylation is a key epigenetic modification that plays important roles in various inflammatory responses, but its role in dental pulp inflammation is poorly understood. In this study, we assessed the expression of DNA methyltransferases (DNMTs) in human dental pulp cells (hDPCs) during lipopolysaccharide (LPS)-induced inflammation and found that DNMT3B mRNA expression was reduced and DNMT1 mRNA and protein levels decreased significantly. Pretreatment with the DNMT inhibitor 5-Aza-2'-deoxycytidine (5-Aza-CdR) significantly enhanced the expression of the inflammatory cytokines IL-6 and IL-8 in LPS-stimulated hDPCs, indicating that DNA methylation may play a role in hDPC inflammation. Studies have reported that some microRNAs (miRNAs) are involved in dental pulp infection. DNA methylation can modulate the inflammatory response by regulating miRNA expression, but this phenomenon has not yet been reported in pulp inflammation. The present study used next-generation sequencing to examine the effect of 5-Aza-CdR on the miRNA expression profile of LPS-treated hDPCs, and the results showed that 5-Aza-CdR pretreatment changed the miRNA expression pattern in hDPCs during inflammation. Among the changed miRNAs, miR-146a-5p, which is a pulp inflammation-related miRNA, demonstrated the most noticeably altered expression. miR-146a-5p could be induced by LPS in hDPCs, and 5-Aza-CdR preincubation or DNMT1 knockdown markedly increased its expression level. However, no significant difference was found in the methylation pattern of the MIR146A promoter with 5-Aza-CdR pretreatment or DNMT1 knockdown in LPS-stimulated hDPCs. These results indicate that DNA methylation may regulate the LPS-induced inflammatory response by changing the miRNA expression in hDPCs.

Polymeric nanoparticles for the delivery of miRNA to treat Chronic Obstructive Pulmonary Disease (COPD).

RNA interference (RNAi) based therapeutics are considered an endogenous mechanism for modulating gene expression. In addition, microRNAs (miRNAs) may be tractable targets for the treatment of Chronic Obstructive Pulmonary Disease (COPD). In this study miR146a was adsorbed onto poly (glycerol adipate-co-ω-pentadecalactone), PGA-co-PDL, nanoparticles (NPs) to reduce target gene IRAK1 expression. NPs were prepared using an oil-in-water single emulsion solvent evaporation method incorporating cationic lipid dioleoyltrimethylammoniumpropane (DOTAP). This resulted in NPs of 244.80 ±â€¯4.40 nm at 15% DOTAP concentration, zeta potential (ZP) of +14.8 ±â€¯0.26 mV and miR-146a (40 µg/ml) maximum adsorption onto 15% DOTAP NPs was 36.25 ±â€¯0.35 µg per 10 mg NP following 24 h incubation. Using the MTT assay, it was observed that over 75% at 0.312 mg/ml of A549 cells remained viable after 18 h exposure to cationic NPs at a concentration of 1.25 mg/ml. Furthermore, the in vitro release profile of miR-146a from loaded NPs showed a continuous release up to 77% after 24 h. Internalization of miR-146a loaded cationic NPs was observed in A549 cell lines using fluorescence and confocal microscopy. The miR146a delivered as miR-146a-NPs had a dose dependent effect of highest NPs concentrations 0.321 and 0.625 mg/ml and reduced target gene IRAK1 expression to 40%. In addition, IL-8 promoter reporter output (GFP) was dampened by miR-146a-NPs. In conclusion, miR-146a was successfully adsorbed onto PGA-co-PDL-DOTAP NPs and the miR-146a retained biological activity. Therefore, these results demonstrate the potential of PGA-co-PDL NPs as a delivery system for miR-146a to treat COPD.