[Show abstract][Hide abstract] ABSTRACT: MicroRNAs are 18-22 nucleotides long, non-coding RNAs that bind transcripts with complementary sequences leading to either mRNA degradation or translational suppression. However, the inherent differences in preferred mode of miRNA regulation among cells of different origin have not been examined. In our previous transcriptome profiling studies, we observed that post-transcriptional regulation can differ substantially depending on the cell in context. Here we examined mechanistic differences in the regulation of a let-7a targeted (wild type) or resistant (mutant) engineered renilla transcript across various mammalian cell lines of diverse origin. Dual luciferase assays show that compared to mutant (mut), the reporter gene containing wild type (wt) let-7a binding sites was efficiently suppressed upon transfection in various cell lines. Importantly, the strength of miRNA regulation varied across the cell lines. Total RNA analysis demonstrates that wt renilla mRNA was expressed to similar or higher levels compared to mut suggesting that translation repression is a predominant mode of miRNA regulation. Nonetheless, transcript degradation was observed in some cell lines. Ago-2 immunoprecipitation show that miRNA repressed renilla mRNA are associated with functional mi-RISC (miRNA-RNA induced silencing complex). Given the immense potential of miRNA as a therapeutic option, these findings highlight the necessity to thoroughly examine the mode of mRNA regulation in order to achieve the beneficial effects in targeting cells.
Full-text · Article · Jan 2016 · International Journal of Molecular Sciences
[Show abstract][Hide abstract] ABSTRACT: Objective:
MicroRNAs (miRNA) are ubiquitous regulators of human biology and immunity. Previously, we have demonstrated an inhibitory role for miR-24 in the phagocytosis of Escherichia coli and Staphylococcus aureus bioparticles and the induction of cytokine secretion in response to lipopolysaccharide (LPS) of the same origin; also, we have identified divergent and convergent miRNA responses to LPS from the periodontopathic pathogens Aggregatibacter actinomycetemcomitams (Aa) and Porphyromonas gingivalis (Pg), and revealed cigarette smoke extract as an environmental modifier of Pg LPS structure (Pg CSE) impacting macrophage miRNA responses. This study was designed to investigate the role of miR-24 on macrophage polarization and plasticity.
Primary human macrophages were differentiated from CD14(+) monocytes isolated from peripheral blood mononuclear cells (PBMCs) by MACS positive selection and transfected with miR-24 miRNA mimics, inhibitors, or negative control mimic; followed by stimulation with cytokines and/or LPS under various conditions representing key stages of macrophage activation. Macrophage activation and polarization was assessed using assays for cytokine production (ELISA) and protein expression (flow cytometry, immunoblot). MiR-24 expression was assessed by RT-PCR.
Stimulation of macrophages with LPSs of Aa, Pg, and Pg CSE origin resulted in dissimilar levels of cytokine expression and differential expression of miR-24. Overexpression of miR-24 inhibited cytokine secretion in response to LPS. Priming of macrophages with interferon gamma (IFN-γ) did not overcome this inhibitory effect, but classical activation of macrophages with IFN-γ plus TNF-α, TNF-β, or IL-17, modulated the pattern of miR-24 mediated suppression in a cytokine-specific fashion. Overexpression of miR-24 enhanced CD206 upregulation during alternative macrophage activation and inhibited its downregulation in macrophage transitioning from alternative to classical activation states. Overexpression of miR-24 resulted in reduced expression of the Class 1A PI 3-kinase subunit p110 delta (p110δ).
Pathogen- and environment-specific modifications in LPS alter the expression of cytokines and miR-24 in human macrophages. MiR-24 is a negative regulator of macrophage classical activation by LPS and promotes alternative activation under conditions of polarization and plasticity. MiR-24 mediated inhibition of LPS-induced cytokine secretion is dependent upon macrophage activation state at the point of stimulation, and this may be due to the degree to which p110δ is involved in the intracellular signaling pathway/s that transduce receptor ligation into cytokine induction. While important differences were observed in the effect of miR-24 on macrophages, these data indicate that overexpression of miR-24 would be predominantly anti-inflammatory.
[Show abstract][Hide abstract] ABSTRACT: Micro-RNAs (miRNAs) are small noncoding RNAs that regulate various biological pathways. As their role in phagocytosis remains poorly understood, we investigated their impact on phagocytosis in myeloid inflammatory cells. Seven miRNAs (miR-24, -30b, -101, 142-3p, -652-3p, -652-5p, and -1275) that were differentially expressed during monocyte to macrophage (Mφ) and monocyte to dendritic cell (DC) differentiation were screened for their potential role in phagocytosis. Among these, overexpression of miR-24, miR-30b, and miR-142-3p in human monocyte-derived Mφ, DC, monocytes, and PBMCs significantly attenuate phagocytosis of Escherichia coli and Staphylococcus aureus, as well as the secretion of inflammatory mediators, including TNF-α, IL-6, and IL-12p40. miRNA-mediated changes in cytokine profiles were observed at transcriptional and/or posttranscriptional levels and importantly exhibit miRNA-specific impact. To examine the underlying mechanism, we monitored the expression of phagocytosis pathway-associated genes and identified several genes that were altered in Mφ and DC transfected with miR-24, miR-30b, and miR-142-3p mimics. Some of these genes with altered expression also harbor putative miRNA binding sites. We show that miR-142-3p directly regulates protein kinase Ca (PKCa), a key gene involved in phagocytosis. Interestingly, miR-142-3p and PKCa exhibit antagonistic expression during Mφ and DC differentiation. Short interfering RNA-mediated knockdown of PKCa in Mφ leads to reduced bacterial uptake, further highlighting the role of the gene in phagocytosis. Overall, these results demonstrate that miR-24, miR-30b, and miR-142-3p regulate phagocytosis and associated cytokine production in myeloid inflammatory cells through modulation of various genes involved in the pathway.
Full-text · Article · Feb 2015 · The Journal of Immunology
[Show abstract][Hide abstract] ABSTRACT: Background: Treatment of intra bony defects is an important therapeutic goal of periodontal therapy. The goal of this consensus report was to critically appraise the evidence for the available approaches for promoting periodontal regeneration in intrabony defects. In addition to evaluating the effectiveness of new regenerative approaches for intrabony defects, recommendations for future research were defined for this area. Methods: A systematic review was conducted utilizing computerized search of PubMed and Cochrane databases, supplemented with screening of references in original reports, review articles and a hand search in selected journals. All searches were focused on regenerative approaches with histological evidence of periodontal regeneration (proof of principle), clinical trials and case reports. For purposes of analysis, change in intrabony defect fill was considered the primary outcome variable, with change in clinical attachment as a secondary outcome. The SORT grade was used to evaluate the quality and strength of the evidence. During the consensus meeting, the Group agreed on the outcomes of the systematic review, pertinent sources of evidence, clinical recommendations, and areas requiring future research. Results: The systematic review, which was conducted for the consensus conference, evaluated the effectiveness of the use of biologics for the treatment of intrabony defects. Enamel Matrix Derivative (EMD) and recombinant human Platelet Derived Growth Factor-BB (rhPDGF-BB) with β-tricalcium phosphate (β-TCP) were shown to be efficacious in regenerating intrabony defects. The level of evidence is supported by multiple studies documenting effectiveness. The clinical application of biologics support improvements in clinical parameters comparable to selected bone replacement grafts and guided tissue regeneration (GTR). Factors negatively affecting regeneration included smoking and excessive tooth mobility. Conclusion: Periodontal regeneration in intrabony defects is possible on previously diseased root surfaces, as evidenced by a gain in clinical attachment, decreased pocket probing depth, gain in radiographic bone height, and overall improvement in periodontal health. These clinical findings are consistent with available histologic evidence. Clinical improvements can be maintained over long periods (> 10 years). While bone replacement grafts have been the most commonly investigated modality, GTR, biologics, and combination therapies have also been shown to be effective. Future research should emphasize patient reported outcomes, individual response differences, and emerging technologies to enhance treatment results. Clinical Recommendations: Early management of intrabony defects with regeneration therapies offers the greatest potential for the successful periodontal regeneration. The clinical selection and application of a regenerative therapy or combination of therapies for periodontal regeneration should be based on the clinician's experiences and understanding of the regenerative biology and technology. This decision making process should take into consideration the potential adverse influence of factors such smoking, poor oral hygiene, tooth mobility, and defect morphology on regeneration. Management should be coupled with an effective maintenance program for long-term success.
No preview · Article · Oct 2014 · Journal of Periodontology
[Show abstract][Hide abstract] ABSTRACT: Abstract The objective of this study was to evaluate the impact of smoking on the early molecular events involved in peri-implant healing at either a micro-roughened or a micro-roughened with superimposed nanofeatures surface implants in humans. Twenty-one subjects, 10 smokers and 11 non-smokers received 4 mini-implants (2.2 x 5.0 mm; 2 of each surface). After 3 and 7 days, paired mini-implants were retrieved by reverse threading and RNA isolated from implant adherent cells. Whole genome microarrays were used interrogate the gene expression profiles. The study failed to identify differences in the gene expression profiles of implant adherent cells at this early stage of osseointegration (up to day 7) comparing smokers and non-smokers individuals.
[Show abstract][Hide abstract] ABSTRACT: Background:
Previous systematic reviews of periodontal regeneration with bone replacement grafts and guided tissue regeneration (GTR) were defined as state of the art for clinical periodontal regeneration as of 2002.
The purpose of this systematic review is to update those consensus reports by reviewing periodontal regeneration approaches developed for the correction of intrabony defects with the focus on patient-, tooth-, and site-centered factors, surgical approaches, surgical determinants, and biologics. This review adheres to the PRISMA (Preferred Reporting Items for Systematic Reviews and Meta-Analyses) guidelines for systematic reviews. A computerized search of the PubMed and Cochrane databases was performed to evaluate the clinically available regenerative approaches for intrabony defects. The search included screening of original reports, review articles, and reference lists of retrieved articles and hand searches of selected journals. All searches were focused on clinically available regenerative approaches with histologic evidence of periodontal regeneration in humans published in English. For topics in which the literature is lacking, non-randomized observational and experimental animal model studies were used. Therapeutic endpoints examined included changes in clinical attachment level, changes in bone level/fill, and probing depth. For purposes of analysis, change in bone fill was used as the primary outcome measure, except in cases in which this information was not available. The SORT (Strength of Recommendation Taxonomy) grading scale was used in evaluating the body of knowledge.
1) Fifty-eight studies provided data on patient, tooth, and surgical-site considerations in the treatment of intrabony defects. 2) Forty-five controlled studies provided outcome analysis on the use of biologics for the treatment of intrabony defects.
1) Biologics (enamel matrix derivative and recombinant human platelet-derived growth factor-BB plus β-tricalcium phosphate) are generally comparable with demineralized freeze-dried bone allograft and GTR and superior to open flap debridement procedures in improving clinical parameters in the treatment of intrabony defects. 2) Histologic evidence of regeneration has been demonstrated with laser therapy; however, data are limited on clinical predictability and effectiveness. 3) Clinical outcomes appear most appreciably influenced by patient behaviors and surgical approach rather than by tooth and defect characteristics. 4) Long-term studies indicate that improvements in clinical parameters are maintainable up to 10 years, even in severely compromised teeth, consistent with a favorable/good long-term prognosis.
Preview · Article · Sep 2014 · Journal of Periodontology
[Show abstract][Hide abstract] ABSTRACT: MicroRNAs (miRNAs) regulate the synthesis of cytokines in response to Toll-like receptor (TLR) activation. Our recent microarray study comparing normal and inflamed human dental pulps showed that miRNA-181 (miR-181) family is differentially expressed in the presence of inflammation. Prior studies have reported that the dental pulp, which is composed primarily of TLR4/2+ fibroblasts, expresses elevated levels of cytokines including interleukin-8 (IL-8) when inflamed. In this study, we employed an in-vitro model to determine the role of the miRNA-181 family in the TLR agonist-induced response in human fibroblasts. TLR4/2+ primary human dental pulp fibroblasts were stimulated with lipopolysaccharide from Porphyromonas gingivalis (Pg LPS), a known oral pathogen, and IL-8 and miR-181 expression measured. An inversely proportional relationship between IL-8 and miR-181a was observed. In-silico analysis identified a miR-181a-binding site on the 3' untranslated region (UTR) of IL-8, which was confirmed by dual-luciferase assays. MiR-181a directly binds to the 3'UTR of IL-8, an important inflammatory component of the immune response, and modulates its levels. This is the very first report demonstrating miR-181a regulation of IL-8.Genes and Immunity advance online publication, 22 May 2014; doi:10.1038/gene.2014.24.
Full-text · Article · May 2014 · Genes and Immunity
[Show abstract][Hide abstract] ABSTRACT: miRNAs are small noncoding RNAs which act to post-transcriptionally silence gene expression through translational inhibition and mRNA destabilization. It is clear that miRNA impact the function of undifferentiated stem cells. Also, several miRNAs are known to regulate NF-&kappaB signaling. One unexplored avenue is the effect of NF-&kappaB transactivation on miRNA expression.
Objective: The goal of this study was to define by a gene profiling approach the spectrum of miRNA that are expressed in human mesenchymal stem cells (hMSCs) that express constitutively active NF-&kappaB.
Method: hMSCs were transduced with adenoviral vectors encoding NF-&kappaB and GFP to induce transgene expression. Cell lysates were collected after 4, 12, 24, and 48 hours. RNA was isolated, quantified and RNA integrity was checked. Real Time PCR (RT-PCR) was done for a known NF-&kappaB miRNA target gene (miR-146a) and miR-891 was selected as a control miRNA, which is not regulated by NF-&kappaB. RNU6 was used as the housekeeping gene. Also the miRNA expression profiles were checked by microarray using LNA-based arrays (Exiqon). ANOVA and t-test were used for statistical analysis.
Result: miR-146a expression levels were significantly induced after 12, 24 and 48 hours, reaching more than 10-fold high for NF-&kappaB samples compared to GFP control. For the microarray analysis, Benjamini- Hochberg test between the NF-&kappaB and GFP samples showed no differences in miRNA profiles for time points 4 and 12 hours, whereas 17 and 180 differentially expressed miRNAs were identified for time points 24 and 48 hours, respectively. Amongst the differently expressed miRNAs, miR-155, miR-93, miR-30b, miR-214, miR-15b have targets like Osx and Runx2, which are transcription factors related to bone formation and regeneration.
Conclusion: NF-&kappaB predominantly induced miRNA expression in undifferentiated hMSCs.
[Show abstract][Hide abstract] ABSTRACT: Objective: Over a twelve day period of osteoclast differentiation from monocytes, differentially expressed microRNAs (miRNAs) were investigated and bioinformatics analysis performed on miRNAs predicted to target genes linked to osteoclastogenesis. We aim to gain a deeper understanding of osteoclastogenesis, which may have future therapeutic implications upon diseases where bone remodeling/resorption play a role in pathogenesis.
Method: Leukophoresed buffy coats were obtained from healthy human donors (N=4) and CD14+ monocytes isolated. Monocyte-to-osteoclast differentiation was induced by culturing cells with sRANKL and M-CSF. Differentiation was confirmed by microscopy, TRAP+ staining and osteolysis. Freshly isolated monocytes and monocytes cultured in the presence of M-CSF alone were utilized as controls. On days 0, 3, 6, 9 and 12, cells were lysed, total RNA isolated and miRNA levels interrogated using miRCURY LNA™ microRNA Arrays (N=36). Bioinformatic analysis for predicted miRNA-mRNA target interactions was performed on significant, differentially expressed miRNAs observed during the differentiation process using publically available algorithms.
Result: MiRNA profiling of osteoclast differentiation revealed conserved and differentially expressed miRNAs. These included miRNAs previously reported known to be involved in osteoclastogenesis, such as miR-29b and miR-223, and novel miRNAs, such as miR-378e with predicted target, ELK4, a member of the Ets family of transcription factors. Further, a number of differentially expressed miRNAs were found to be present in both monocyte- to -osteoclast, and monocyte -to -macrophage differentiation; however, the magnitude of the expression change varied between the two groups.
Conclusion: This study profiled miRNA expression during monocyte-to-osteoclast and monocyte-to-macrophage differentiation and identified novel miRNAs not previously described. Our in-silico analysis reveals potential mRNA targets which serve as candidates for future investigation. This study highlights the role of miRNA in osteoclastogenesis and has implications in physiologic and pathophysiology processes affecting humans, such as periodontal disease, peri-implant disease, osteoporosis and rheumatoid arthritis.
[Show abstract][Hide abstract] ABSTRACT: Antioxidants possess significant therapeutic potential for the treatment of inflammatory disorders. One such disorder is periodontitis characterised by an antimicrobial immune response, inflammation, and irreversible changes to the supporting structures of the teeth. Recognition of conserved pathogen-associated molecular patterns is a crucial component of innate immunity to Gram-negative bacteria such as Escherichia coli, as well as the periodontal pathogen Aggregatibacter actinomycetemcomitans. In this study, we investigated the antioxidants Phloretin, Silymarin, Hesperetin, and Resveratrol to ascertain whether they altered the production of inflammatory mediators by innately-activated leukocytes. Peripheral blood mononuclear cells were stimulated with lipopolysaccharide purified from Aggregatibacter actinomycetemcomitans, and the production of cytokines, chemokines, and differentiation factors was assayed by enzyme-linked immunosorbent assay, cytometric bead array, and RT-PCR. Significant inhibition of these factors was achieved upon treatment with Phloretin, Silymarin, Hesperetin, and Resveratrol. These data further characterise the potent anti-inflammatory properties of antioxidants. Their ability to inhibit the production of inflammatory cytokines, chemokines, and differentiation factors by a heterogeneous population of leukocytes has clear implications for their therapeutic potential in vivo.
Full-text · Article · Feb 2014 · Mediators of Inflammation
[Show abstract][Hide abstract] ABSTRACT: Apical periodontitis is an inflammatory disease of the periradicular tissues caused by the host's immune response to infection of the root canal system. MicroRNAs (miRNAs) have been shown to play an important role in the regulation of inflammation and the immune response; however, their role in the pathogenesis of endodontic periapical disease has not been explored. The purpose of this study was to examine the differential expression of miRNAs in diseased periapical tissues as compared with healthy controls.
We first compared miRNA profiles in diseased periapical tissues collected from patients undergoing endodontic surgery with those of healthy pulps by using microarray analyses. The target genes of the differentially expressed miRNAs were identified by using miRWalk and PubMed. Selected miRNAs linked to inflammation and the immune response were then confirmed in a separate cohort of diseased and healthy tissues by using quantitative reverse transcription-polymerase chain reaction. Healthy pulps and periodontal ligaments were used as controls. Data were normalized to the level of SNORD 44, which served as an endogenous control.
Of the 381 miRNAs identified by using microarray, 24 miRNAs were down-regulated in diseased periapical tissues compared with controls (n = 13) (P < .003). The down-regulation of 7 miRNAs was confirmed from 9 selected miRNAs by using quantitative real-time polymerase chain reaction (n = 19) (P < .05). Target genes of these miRNAs include key mediators in the immune and inflammatory response such as interleukin-6, matrix metalloproteinase-9, and transforming growth factor-β.
These findings offer new insight into the pathogenesis of endodontic disease and have the potential to impact the development of new methods for prevention, diagnosis, and treatment of apical periodontitis.
No preview · Article · Dec 2013 · Journal of endodontics
[Show abstract][Hide abstract] ABSTRACT: To determine the early temporal-wide genome transcription regulation by the surface topography at the bone-implant interface of implants bearing microroughened or superimposed nanosurface topology.
Four commercially pure titanium implants (2.2 × 5.0 mm) with either a moderately roughened surface (TiOblast) or super-imposed nanoscale topography (Osseospeed) were placed (n = 2/surface) in edentulous sites of eleven systemically healthy subjects and subsequently removed after 3 and 7 days. Total RNA was isolated from cells adherent to retrieved implants. A whole-genome microarray using the Affymetrix Human gene 1.1 ST Array was used to describe the gene expression profiles that were differentially regulated by the implant surfaces.
There were no significant differences when comparing the two implant surfaces at each time point. However, the microarray identified several genes that were differentially regulated at day 7 vs. day 3 for both implant surfaces. Functionally relevant categories related to the extracellular matrix (ECM), collagen fibril organization, and angiogenesis were upregulated at both surfaces (day7 vs. day3). Abundant upregulation of several differential markers of alternative activated macrophages was observed (e.g., MRC1, MSR1, MS4A4A, SLC38A6, and CCL18). The biological processes involved with the inflammatory/immune response gene expression were concomitantly downregulated.
Gene regulation implicating collagen fibrillogenesis and ECM organization as well as the inflammatory/immune responses involving the alternative activated pathway are observed in implant adherent cells at early (3-7 days) after implantation. These gene expression events may indicate a pivotal role of collagen fibrillogenesis as well as immunomodulation in altering bone accrual and biomechanical physical properties of the implant-bone interface.
No preview · Article · Sep 2013 · Clinical Oral Implants Research
[Show abstract][Hide abstract] ABSTRACT: MicroRNAs (miRNAs) are a class of small, noncoding RNAs that regulate post-transcriptional expression of their respective target genes and are responsive to various stimuli, including LPS. Here we examined the early (4 h) miRNA responses of THP1-differentiated macrophages challenged with LPS derived from the periodontal pathogens, Aggregatibacter actinomycetemcomitans, Porphyromonas gingivalis or environmentally-modified LPS obtained from P. gingivalis grown in cigarette smoke extract. Predicted miRNA-gene target interactions for LPS-responsive miR-29b and let-7f were confirmed using dual-luciferase assays and by transfection experiments using miRNA mimics and inhibitors. Convergent and divergent miRNA profiles were observed in treated samples where differences in miRNA levels related to the type, concentration and incubation times of LPS challenge. Dual-luciferase experiments revealed miR-29b targeting of interleukin-6 receptorα (IL-6Rα) and IFN-γ inducible protein 30 and let-7f targeting of suppressor of cytokine signaling 4 and thrombospondin-1. Transfection experiments confirmed miR-29b and let-7f modulation of IL-6Rα and SOCS4 protein expression levels, respectively. Thus, we have demonstrated convergent/divergent miRNA responses to wild type LPS and its environmentally-modified LPS, and demonstrate miRNA targeting of key genes linked to inflammation and immunity. Our data indicate that these LPS-responsive miRNAs may play a key role in fine-tuning the host response to periodontal pathogens.
[Show abstract][Hide abstract] ABSTRACT: PurposeThis pilot study evaluated the molecular, histologic, and radiographic healing of bone to instrumentation with piezoelectric or high speed rotary (R) devices over a 3-week healing period.Material and Methods
Fourteen Sprague-Dawley rats (Charles River Laboratories International, Inc., Wilmington, MA, USA) underwent bilateral tibial osteotomies prepared in a randomized split-leg design using Piezotome® (P1) (Satelec Acteon, Merignac, France), Piezotome 2® (P2) (Satelec Acteon), High-speed R instrumentation, or sham surgery (S). At 1 week, an osteogenesis array was used to evaluate differences in gene expression while quantitative analysis assessed percentage bone fill (PBF) and bone mineral density (BMD) in the defect, peripheral, and distant regions at 3 weeks. Qualitative histologic evaluation of healing osteotomies was also performed at 3 weeks.ResultsAt 1 week, expression of 11 and 18 genes involved in bone healing was significantly (p < .05) lower following P1 and P2 instrumentation, respectively, relative to S whereas 16 and 4 genes were lower relative to R. No differences in PBF or BMD were detected between groups within the osteotomy defect. However, significant differences in PBF (p = .020) and BMD (p = .008) were noted along the peripheral region between P2 and R groups, being R the group with the lowest values. Histologically, smooth osteotomy margins were present following instrumentation using P1 or P2 relative to R.Conclusions
Piezoelectric instrumentation favors preservation of bone adjacent to osteotomies while variations in gene expression suggest differences in healing rates due to surgical modality. Bone instrumented by piezoelectric surgery appears less detrimental to bone healing than high-speed R device.
No preview · Article · Jun 2013 · Clinical Implant Dentistry and Related Research
[Show abstract][Hide abstract] ABSTRACT: miRNA regulates immunity by modulating the expression of key genes involved in leukocyte development and function. Furthermore, immunity includes innate and adaptive systems comprising myeloid-derived cells and lymphocytes which mediate nonspecific and pathogen-specific responses. Innate immunity encompasses the generation of myeloid-derived cells and their ability to mount a rapid and appropriate response to pathogen-associated danger. Here we highlight common components of research investigating the role of miRNA in innate immunity. The identification of miRNAs involved in immunity generally begins with the profiling of samples generated ex vivo or in vitro which include the cell types and immunological conditions of interest. From here a core set of methods and technologies are utilised. Various technologies exist for quantifying miRNA expression, including qRT-PCR and microarray platforms capable of profiling all known human miRNAs as listed by miRBASE. miRNA array expression data may be confirmed by qRT-PCR before in silico analysis of predicted miRNA targets, and these miRNA-target interactions validated by dual luciferase assays. Finally, the biological significance of any miRNA is dependent upon the degree of modulation it exerts combined with the importance of it target within the relevant system. A widely used approach for ascertaining this is to perform experiments in which the miRNA is constitutively expressed or knocked-down and altered function or experimental outcome tested. To date these methods have revealed several important roles for miRNA in the functioning of the immune system. As the technologies and techniques described here continue to develop so will our understanding of the function
[Show abstract][Hide abstract] ABSTRACT: Inflammation of the pulp and periradicular tissues is estimated to affect approximately 22 million people in the United States. Pulpitis represents an immune response to bacteria in root canal systems and is the most common reason for patients seeking emergency dental care. MicroRNAs (miRNAs) play a pivotal role in the regulation of immune responses, including the acute inflammatory response triggered by activation of toll-like receptors (TLR); however, their role in pulpal immune response is still being explored. Our recent microarray study showed that the hsa-mir 181 family is differentially expressed in inflamed pulps and in macrophages stimulated with TLR agonists. At present, no study has characterized the expression of miR181 family in pulpal fibroblasts. Objectives: The objectives of this study are: 1) to characterize the expression of hsa-mir-181 family in primary human fibroblasts; and 2) to investigate if their expression level is influenced by TLR agonists. Methods: Primary cultures of human pulpal fibroblasts (HPF) were exposed at 1, 4 and 8 hours to 1μg, 100 ng and 10 ng per milliter of P. gingivalis LPS. Unchallenged HPF served as controls. Expression of the miR181 family was quantified by qRT-PCR using RNU-6b as the endogenous control. Relative quantification values were calculated together with the statistical significance using one-way ANOVA. P value was set at ‹0.05. Potential target messenger RNAs for the miR181 family were identified using the databases PUBMED and miRWalk. Results: Hsa-miR181 family is expressed in HPF. Our preliminary data suggest that TLR activation modulates expression of hsa-mir 181a and 181b. Bioinformatics search shows that miR181 family targets inflammatory mediators such as IL-2 and MMP-9. Conclusion: This is the very first report on the potential role of mir181 in regulation of pulpal immune response. Further functional studies are warranted to explore the role of miRNAs in pulpal response.