Identification and validation of m6A RNA regulatory network in pulpitis.

BACKGROUND: N6-methyladenosine (m6A) RNA modification regulators play an important role in many human diseases, and its abnormal expression can lead to the occurrence and development of diseases. However, their significance in pulpitis remains largely unknown. Here, we sought to identify and validate the m6A RNA regulatory network in pulpitis. METHODS: Gene expression data for m6A regulators in human pulpitis and normal pulp tissues from public GEO databases were analyzed. Bioinformatics analysis including Gene ontology (GO) functional, and Kyoto encyclopedia of genes and genomes (KEGG) pathway analyses were performed by R package, and Cytoscape software was used to study the role of m6A miRNA-mRNA regulatory network in pulpitis. Quantitative real-time PCR (qRT-PCR) was performed to validate the expression of key m6A regulators in collected human pulpitis specimens. RESULTS: Differential genes between pulpitis and normal groups were found from the GEO database, and further analysis found that there were significant differences in the m6A modification-related genes ALKBH5, METTL14, METTL3, METTL16, RBM15B and YTHDF1. And their interaction relationships and hub genes were determined. The hub m6A regulator targets were enriched in immune cells differentiation, glutamatergic synapse, ephrin receptor binding and osteoclast differentiation in pulpitis. Validation by qRT-PCR showed that the expression of methylases METTL14 and METTL3 was decreased, thus these two genes may play a key role in pulpitis. CONCLUSION: Our study identified and validated the m6A RNA regulatory network in pulpitis. These findings will provide valuable resource to guide the mechanistic and therapeutic analysis of the role of key m6A modulators in pulpitis.

Differentially Expressed Genes in Dental Pulp Tissues of Individuals With Symptomatic Irreversible Pulpitis With and Without History of COVID-19.

INTRODUCTION: Increased levels of proinflammatory markers have been reported in tissues of individuals with Coronavirus Disease 2019 (COVID-19). We hypothesize that inflamed dental pulp tissues of individuals with previous history of COVID-19 may present a differential inflammatory gene expression profile in comparison with individuals who never had COVID-19. MATERIALS AND METHODS: Dental pulp tissues were collected from 27 individuals referred for endodontic treatment due to symptomatic irreversible pulpitis. Of these, 16 individuals had a history of COVID-19 (6 months to 1 year post infection) and 11 individuals had no previous history of COVID-19 (controls). Total RNA from pulp tissue samples was extracted and subjected to RNA sequencing for comparison of differentially expressed genes (DEGs) among groups. DEGs showing log2(fold change) > 1 or < -1, and P < .05 were considered significantly dysregulated. RESULTS: RNA sequencing identified 1461 genes as differentially expressed among the groups. Of these, 311 were protein coding genes, 252 (81%) that were upregulated and 59 (19%) that were downregulated in the COVID group compared with controls. The top upregulated genes in the COVID group were HSFX1 (4.12-fold change) and LINGO3 (2.06-fold change); significantly downregulated genes were LYZ (-1.52-fold change), CCL15 and IL8 (-1.45-fold change). CONCLUSIONS: Differential gene expression in dental pulp tissues of COVID and non-COVID groups suggests potential contribution of COVID-19 on dysregulating inflammatory gene expression in the inflamed dental pulp.

Identification of the characteristics of infiltrating immune cells in pulpitis and its potential molecular regulation mechanism by bioinformatics method.

OBJECTIVE: The inflammation of dental pulp will also trigger an immune response. The purpose of this study is to demonstrate the immune cell's function and explore their regulatory molecules and signal pathways in pulpitis. METHOD: The CIBERSORTx method was used to quantitatively analyze 22 types of immune cells infiltrating in the GSE77459 dataset of dental pulp tissues. The immune-related differential genes (IR-DEGs) were further screened and enriched for the GO and KEGG pathways. Protein-protein interaction (PPI) networks were constructed and the hub IR-DEGs were screened. Finally, we constructed the regulatory network of hub genes. RESULTS: The GSE77459 dataset screened 166 IR-DEGs and was enriched for three signal pathways involved in pulpitis development: chemokine signaling, TNF signaling, and NF-κB signaling. Significant differences in immune cell infiltration were observed between normal and inflamed dental pulp. The proportions of M0 macrophages, neutrophils, and follicular helper T cells were significantly higher than that of the normal dental pulp, while the proportions of resting mast cells, resting dendritic cells, CD8 T cells, and monocytes were significantly lower. The random forest algorithm concluded that M0 macrophages and neutrophils were the two most important immune cells. We identified five immune-related hub genes IL-6, TNF-α, IL-1ß, CXCL8, and CCL2. In addition, IL-6, IL-1ß, and CXCL8 are highly correlated with M0 macrophages and neutrophils, and the five hub genes have many shared regulatory molecules: four miRNAs and two lncRNAs, three transcription factors. CONCLUSION: Immune cell infiltration plays an important role in pulpitis among which M0 macrophages and neutrophils are the most significant immune cells. IL-6, TNF-α, IL-1, CXCL8, and CCL2 may be essential molecule of the immune response regulation network in pulpitis. This will help us understand the immune regulatory network in pulpitis.

Association Between Single-nucleotide Polymorphisms in Candidate Genes and Success of Pulpal Anesthesia after Inferior Alveolar Nerve Block.

INTRODUCTION: The present study aimed to investigate the possible association between the single-nucleotide polymorphisms (SNPs) in the SCN9A, SCN10A, SCN11A, OPRM1, and COMT genes and the success rate of pulpal anesthesia after inferior alveolar nerve block (IANB). METHODS: A total of 70 patients (45 females and 25 males) presenting mandibular molar teeth with symptomatic irreversible pulpitis were included. Saliva samples were collected from the participants before the application of IANB. A standard IANB was performed with 1.8 mL 4% articaine with 1:100,000 epinephrine. Endodontic treatment was initiated 15 minutes after injection, and the patients were asked to report their pain level during the procedure on a 170-mm Heft-Parker visual analog scale. If the patient recorded a pain level of lower than 54 on the visual analog scale (no pain or mild pain), the anesthesia was considered successful. The DNA isolation and genotyping were performed, and the association between rs4286289, rs6746030, rs6795970, rs6801957, rs11709492, rs1799971, rs1799973, rs4680, rs6269, rs4633, and rs740603 SNPs and the success rate of anesthesia was investigated. RESULTS: The anesthesia success rate was significantly lower for the GG genotypes (45%) than the GA and AA genotypes (90%) for rs6795970 in the SCN10A gene. Additionally, the A allele for rs6795970 and the T allele for rs6801957 in the SCN10A gene were significantly associated with higher anesthesia success rates. CONCLUSIONS: SNPs in the SCN10A gene affect the success rate of pulpal anesthesia after IANB.

Overexpression of MicroRNA-155 aggravates pulpitis by targeting kinesin superfamily Proteins-5C based on illumina high-throughput sequencing.

AIM: To investigate the regulatory role of miR-155 and Kinesin Superfamily Proteins-5C (KIF-5C) in the progression of pulpitis based on bioinformatic analysis. METHODOLOGY: Normal pulp tissues and pulpitis pulp tissues were collected and subjected to high-throughput sequencing and the differentially expressed miRNAs were determined. An in vitro and in vivo pulpitis model was established. HE, IHC staining and histological evaluation were used to verify the inflammatory state of human and mouse pulp tissues. The mRNA expression of IL-1ß and TGF-ß1 were determined by RT-qPCR and protein expression of IL-1α, IL-4, IL-8, IL-13, IFN-γ, IL-6, IL-10 and MCP-1 were determined by protein chip. The target genes of miR-155 were predicted by miRanda database and verified by Dual-luciferase reporter assay, RT-qPCR and western blotting. MiR-155 lentivirus were used to upregulate or downregulate miR-155 and the siRNA of KIF-5C was used to downregulate KIF-5C. The expression of miR-155 or KIF-5C was determined by RT-qPCR. All statistics were analysed using GraphPad prism 8.2. RESULTS: The high-throughput sequencing results showed that 6 miRNAs (miR-155, miR-21, miR-142, miR-223, miR-486, miR-675) were significantly upregulated in diseased human pulp tissues, and miR-155 was significantly elevated among the six miRNAs. RT-qPCR results demonstrated that miR-155 expression was upregulated in human pulpitic tissue, mice pulpitic tissue and LPS-HDPCs. IL-1ß was increased while TGF-ß1 was decreased in lenti-miR-155 transfected LPS-HDPCs. Analysis of protein chip results indicated that lenti-miR-155 transfected LPS-HDPCs produced higher levels of IL-8, IL-6, MCP-1. The opposite results were obtained when miR-155 was inhibited. Through miRanda database screen and Dual-luciferase reporter assay, the target gene (KIF-5C) of miR-155 was identified. In lenti-miR-155 transfected LPS-HDPCs, the expression of KIF-5C was downregulated. However, when shRNA-miR-155 was transfected to LPS-HDPCs, the opposite result was obtained. Silent RNA was used to knock down KIF-5C, the results showed that when both KIF-5C and miR-155 were knocked down simultaneously, the downregulated expression of inflammatory factors observed in LPS-HDPCs following miR-155 knockdown was rescued. CONCLUSION: MiR-155 plays an important role in promoting pulpitis through targeting KIF-5C and may serve as a potential therapeutic target.

Identification of key module and hub genes in pulpitis using weighted gene co-expression network analysis.

BACKGROUND: Pulpitis is a common disease mainly caused by bacteria. Conventional approaches of diagnosing the state of dental pulp are mainly based on clinical symptoms, thereby harbor deficiencies. The accurate and rapid diagnosis of pulpitis is important for choosing the suitable therapy. The study aimed to identify pulpits related key genes by integrating micro-array data analysis and systems biology network-based methods such as weighted gene co-expression network analysis (WGCNA). METHODS: The micro-array data of 13 inflamed pulp and 11 normal pulp were acquired from Gene Expression Omnibus (GEO). WGCNA was utilized to establish a genetic network and categorize genes into diverse modules. Hub genes in the most associated module to pulpitis were screened out using high module group members (MM) methods. Pulpitis model in rat was constructed and iRoot BP plus was applied to cap pulp. Reverse transcription-quantitative polymerase chain reaction (RT-qPCR) was used for validation of hub genes. RESULTS: WGCNA was established and genes were categorized into 22 modules. The darkgrey module had the highest correlation with pulpitis among them. A total of 5 hub genes (HMOX1, LOX, ACTG1, STAT3, GNB5) were identified. RT-qPCR proved the differences in expression levels of HMOX1, LOX, ACTG1, STAT3, GNB5 in inflamed dental pulp. Pulp capping reversed the expression level of HMOX1, LOX, ACTG1. CONCLUSION: The study was the first to produce a holistic view of pulpitis, screen out and validate hub genes involved in pulpitis using WGCNA method. Pulp capping using iRoot BP plus could reverse partial hub genes.

MicroRNA and their implications in dental pulp inflammation: current trends and future perspectives.

MicroRNAs (miRNAs) are short, 19-23 nucleotide non-coding RNA molecules that regulate gene expression by silencing or degrading the target mRNA gene. Since their discovery in the nineties of the last century, they have emerged as key inflammatory regulators. Inflammation induces the synthesis of various miRNAs that modulate the expression of multiple molecules involved in orchestrating the inflammatory response. This review aims to provide an insight into the role of miRNAs as potential biomarkers, mediators, and potential therapeutic targets of dental pulp inflammation. A literature search was conducted using the keywords; biogenesis of microRNA, human dental pulp cells, pulpitis, and inflammation in PubMed and Scopus index databases for all the published articles dealing with the role of miRNAs in pulp inflammation in the last 10 years. According to the literature, there is a clear correlation between miRNAs and several physiological events, as well as their role as mediators of innate immune response and inflammation in dental pulp cells. Our narrative review stipulates that numerous miRNAs play a key role in modulating inflammation, delaying or enhancing cell repair, cell differentiation, and survival in dental pulp diseases. However, further studies are required for the validation of miRNAs as reliable biomarkers in dental pulp pathology and their targeted therapy.

Gene expression analysis of toll like receptor 2 and 4, Dectin-1, Osteopontin and inflammatory cytokines in human dental pulp ex-vivo.

BACKGROUND: Toll like receptors (TLR) 2 and 4 present on innate immune cells of the dental pulp detect cariogenic bacteria. Along with bacteria, C. albicans may also be present in dental caries. The presence of C. albicans can be detected by Dectin-1 a C type Lectin receptor. Expression of Dectin-1 in human pulpits has not been reported. Similarly, cytokines are released as a consequence of dental pulp inflammation caused by cariogenic bacteria. The T helper (Th) 1 inflammatory response leads to exacerbation of inflammation and its relationship with Osteopontin (OPN) is not known in pulp inflammation. OBJECTIVE: The aim of this study was to observe the expression of Dectin-1, TLR-2, OPN and pro-inflammatory cytokines in irreversibly inflamed human dental pulp and to observe relationship between Dectin-1/TLR-2 and OPN/Pro-inflammatory cytokines in the presence of appropriate controls. METHODS: A total of 28 subjects diagnosed with irreversible pulpitis were included in this ex-vivo study. Fifteen samples were subjected to standard hematoxylin and Eosin (H&E) and immunohistochemistry staining. Whereas, gene expression analysis was performed on 13 samples to observe mRNA expression of pro-inflammatory cytokines; tumor necrosis factor-alpha (TNF-α), interleukin (IL)-1 beta (ß), IL-6 Dectin-1, OPN, TLR-2 and TLR-4. SPSS version 21 was used for statistical analysis. One way analysis of variance (ANOVA), Pearson correlation and Chi-square test were used at p ≤ 0.05. RESULTS: Gene expressions of Dectin-1, TLR-2 and TLR-4 were observed in all samples. Dectin-1 and TLR-2 expressions were significantly correlated (r = 0.5587, p = 0.0002). Similarly, OPN and TNF-α expression showed a significant correlation (r = 0.5860, p = 0001). The agreement between histologic and clinical diagnosis was 69.2% in the cases of irreversible pulpitis. CONCLUSION: Dectin-1 was expressed by inflamed human dental pulp. Dectin-1 and TLR-2 expression pattern was suggestive of a collaborative receptor response in inflamed pulp environment. OPN and TNF-α expressions showed a positive correlation indicating a possible relationship.

MicroRNA-155 expression is associated with pulpitis progression by targeting SHIP1.

BACKGROUND: Pulpitis is a commonly seen oral inflammation condition in clinical practice, it can cause much pain for the patient and may induce infections in other systems. Much is still unknown for the pathogenic mechanism of pulpitis. In this work, we discovered that the expression of miR-155 was associated with dental pulpal inflammation both in vivo and in vitro. METHODS AND RESULTS: Our experiments of LPS stimulated odontoblast cell line MDPC-23 showed miR-155 could act as a positive regulator by increasing the production of pro-inflammatory cytokines IL-1ß and IL-6 during inflammatory responses, whereas knockdown of miR-155 can reverse the effects. Bioinformatics analysis demonstrated that SHIP1 is a direct target of miR-155 in odontoblasts, this result was further verified at both mRNA and protein level. Inhibition of miR-155 resulted in the downregulation of inflammation factors, while co-transfection of si-SHIP1 and miR-155 inhibitor promoted the inflammatory responses. Treatment with miR-155 mimic or si-SHIP1 up-regulated the protein level of p-PI3K and p-AKT. By contrast, miR-155 inhibitor exerted the opposite effects. miR-155 mimics could upregulate the gene expression of IL-1ß and IL-6. Co-transfection of LY294002 and miR-155 mimic attenuated the inflammatory responses. Consistent with in vitro results, miR-155-/- mice could alleviate inflammatory response, as well as decrease the activation of p-PI3K and p-AKT, whereas increase the activation of SHIP1. CONCLUSIONS: Our data revealed a novel role for miR-155 in regulation of dental pulpal inflammatory response by targeting SHIP1 through PI3K/AKT signaling pathway.

Identification of Immune-Related lncRNA Regulatory Network in Pulpitis.

Background: Long noncoding RNAs (lncRNAs) are emerging as critical regulators of various biological processes, including immune regulation. Methods: Due to the critical significance of immunological responses in the development and progression of pulpitis, we used an integrated algorithm to identify immune-related lncRNAs and then examined the lncRNA-immunity regulation network in pulpitis. Before identifying immune-related lncRNAs, the data from GEO datasets were precleaned. ConsensusClusterPlus was used to differentiate immune-related pulpitis subgroups. Enrichment analysis using GO and MSigDB databases was employed to determine the differences in molecular function, cellular component, and biological process between the two pulpitis subtypes. Results: An integrated algorithm was designed to filtrate immune-related lncRNAs accurately. 790 immune-related lncRNAs were found in 17 immunological categories, with 38 of them perturbated in pulpitis. The Cytoscape software was used to visualize the relationship between representative immune regulatory pathways and immune-related lncRNAs. Two immune-related pulpitis subtypes were discovered using differentially expressed immune-related lncRNAs. Subtype 2 has a stronger association with immune-related pathways than subtype 1 does. Conclusions: Our study identified many immune-related lncRNAs and investigated potential lncRNA regulation networks; meanwhile, the molecular subtypes of pulpitis were identified, all of which will be relevant for further research into inflammatory and immunological processes in pulpitis.

Expression Profiling of S100 Proteins in Healthy and Irreversibly Inflamed Human Dental Pulps.

INTRODUCTION: Several S100 proteins have been shown to play an important role in the innate immune response to infection and in regenerative processes. However, they have scarcely been investigated during inflammation of the dental pulp. Therefore, in this study, we performed gene expression profiling of S100 proteins in healthy and inflamed human dental pulps. METHODS: Tissue samples of human dental pulps were used, including 15 clinically diagnosed as symptomatic irreversible pulpitis (SIP), 7 as asymptomatic irreversible pulpitis (AIP), and 19 as healthy pulp (HP). S100 gene expression levels were quantitatively evaluated for S100A1, -A2, -A3, -A4, -A6, -A7, -A8, -A9, -A10, -A11, -A13, -A14, and -A16 by the quantitative polymerase chain reaction technique. In order to monitor the status of inflammation and degradation of pulp tissues, IL-8, COX-2, and HMGB-1 gene expression was also analyzed with GAPDH serving as the reference gene. Differential expression rates for each target gene between SIP, AIP, and HP were evaluated by analysis of variance followed by the Bonferroni post hoc test. RESULTS: Significantly reduced gene expression levels could be detected in SIP compared with HP for S100A1, -A2, -A3, -A4, -A6, -A10, and -A13 and for HMGB-1, whereas the gene expression of S100A8 and -A14 and IL-8 were significantly increased. In AIP, significantly increased expression levels compared with HP were only detected for S100A14 and -A16 and IL-8, with other genes of interest not being altered. CONCLUSIONS: The present study revealed significant differences in gene expression profiles of S100 proteins comparing samples from healthy and inflamed dental pulp. More pronounced differences were observed for symptomatic than for asymptomatic pulpitis.

Wnt signalling in oral and maxillofacial diseases.

Wnts include more than 19 types of secreted glycoproteins that are involved in a wide range of pathological processes in oral and maxillofacial diseases. The transmission of Wnt signalling from the extracellular matrix into the nucleus includes canonical pathways and noncanonical pathways, which play an important role in tooth development, alveolar bone regeneration, and related diseases. In recent years, with the in-depth study of Wnt signalling in oral and maxillofacial-related diseases, many new conclusions and perspectives have been reached, and there are also some controversies. This article aims to summarise the roles of Wnt signalling in various oral diseases, including periodontitis, dental pulp disease, jaw disease, cleft palate, and abnormal tooth development, to provide researchers with a better and more comprehensive understanding of Wnts in oral and maxillofacial diseases.

Identification of Pulpitis-Related Potential Biomarkers Using Bioinformatics Approach.

Inflammatory reaction of pulp tissue plays a role in the pathogen elimination and tissue repair. The evaluation of severity of pulpitis can serve an instructive function in therapeutic scheme. However, there are many limitations in the traditional evaluation methods for the severity of pulpitis. Based on the Gene Expression Omnibus (GEO) database, our study discovered 843 differentially expressed genes (DEGs) related to pulpitis. Afterwards, we constructed a protein-protein interaction (PPI) network of DEGs and used MCODE plugin to determine the key functional subset. Meanwhile, genes in the key functional subset were subjected to GO and KEGG enrichment analyses. The result showed that genes were mainly enriched in inflammatory reaction-related functions. Next, we screened out intersections of PPI network nodes and pulpitis-related genes. Then, 20 genes were obtained as seed genes. In the PPI network, 50 genes that had the highest correlation with seed genes were screened out using random walk with restart (RWR). Furthermore, 4 pulpitis-related hub genes were obtained from the intersection of the top 50 genes and genes in the key functional subset. Finally, GeneMANIA was utilized to predict genes coexpressed with hub genes, and expression levels of the 4 hub genes in normal and pulpitis groups were analyzed based on GEO data. The result demonstrated that the 4 hub genes were mainly coexpressed with chemokine-related genes and were remarkably upregulated in the pulpitis group. In short, we eventually determined 4 potential biomarkers of pulpitis.

Comprehensive Analysis of Differentially Expressed Genes in Clinically Diagnosed Irreversible Pulpitis by Multiplatform Data Integration Using a Robust Rank Aggregation Approach.

INTRODUCTION: Molecular diagnosis may overcome the limitations of clinical and histologic diagnosis in pulpitis, thereby benefiting many treatment techniques, such as vital pulp therapies. In this study, integrated microarray data on pulpitis were used to obtain a list of normalized differentially expressed (DE) genes for analyzing the molecular mechanisms underlying pulpitis and identifying potential diagnostic biomarkers. METHODS: A systematic search of public microarray and sequencing databases was performed to obtain expression data of pulpitis. Robust rank aggregation (RRA) was used to obtain DE gene lists (RRA_DEmRNAs and RRA_DElncRNAs) between inflamed pulp and normal samples. DE genes were evaluated by functional enrichment analyses, correlation analyses for inflammation-related RRA_DEmRNAs, and protein-protein interaction and competing endogenous RNA network construction. Quantitative real-time polymerase chain reaction validation was applied in snap-frozen pulp tissues. RESULTS: Using the GSE77459 and GSE92681 data sets, 280 RRA_DEmRNAs and 90 RRA_DElncRNAs were identified. RRA_DEmRNAs were significantly enriched in inflammation-related biological processes and osteoclast differentiation and tumor necrosis factor, chemokine, and B-cell receptor signaling pathways. The molecular complex detection and cytoHubba methods identified 2 clusters and 10 hub genes in the protein-protein interaction network. The competing endogenous RNA network was composed of 2 long noncoding RNAs (ADAMTS9-AS2 and LINC00290), 2 microRNAs (hsa-miR-30a-5p and hsa-miR-128-3p), and 3 messenger RNAs (ABCA1, FBLN5, and SOCS3). The expression between most top inflammation-related RRA_DEmRNAs in pulpitis showed positive correlations. Quantitative real-time polymerase chain reacation validated the expression trends of selected genes, including ITGAX, TREM1, CD86, FCGR2A, ADAMTS9-AS2, LINC00290, hsa-miR-30a-5p, hsa-miR-128-3p, RASGRP3, IL3RA, CCDC178, CRISPLD1, LINC01857, AC007991.2, ARHGEF26-AS1, and AL021408.1. CONCLUSIONS: The identified biomarkers provide insight into the pathology and may aid in the molecular diagnosis of pulpitis.

Clinical Value and Potential Target of miR-27a-3p in Pulpitis.

INTRODUCTION: This study investigated the clinical values of miR-27a-3p for pulpitis patients, and its association with TLR4. METHODS: Sixty-six patients with pulpitis and 34 cases without pulpitis were recruited; the pulp tissue and serum samples were collected from each participant. Real-time polymerase chain reaction was used for measurement of gene expression levels. The diagnosis values were assessed by the receiver operating characteristic curve. The target gene of miR-27a-3p was confirmed by the luciferase reporter assay. RESULTS: MiR-27a-3p was downregulated in both serum and pulp tissue of pulpitis patients. MiR-27a-3p could distinguish pulpitis patients from healthy controls and might be a predictor for the development of irreversible pulpitis. A high level of TLR4 was also detected in both peripheral blood monocytes and pulp tissues from pulpitis patients and showed a negative association with the miR-27a-3p level. TLR4 was a direct target gene of miR-27a-3p. DISCUSSION/CONCLUSION: MiR-27a-3p might be a promising biomarker for the diagnosis of pulpitis and predict the development of irreversible pulpitis. MiR-27a-3p might be involved in the pathogenesis of pulpitis via targeting TLR4.

NUTM2A-AS1 silencing alleviates LPS-induced apoptosis and inflammation in dental pulp cells through targeting let-7c-5p/HMGB1 axis.

BACKGROUND: Long non-coding RNA (lncRNA) NUTM2A antisense RNA 1 (NUTM2A-AS1) has been reported to be abnormally up-regulated in pulpitis tissues. However, the function of NUTM2A-AS1 in pulpitis remains unclear. The aim of this study was to investigate the role and working mechanism of NUTM2A-AS1 in pulpitis using lipopolysaccharide (LPS)-treated human dental pulp cells (HDPCs). METHODS: 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay, flow cytometry and lactate dehydrogenase (LDH) release detection assay were conducted to analyze the viability of HDPCs. Cell inflammatory response was analyzed through measuring the protein levels of interleukin-6 (IL-6) and IL-8. Western blot assay and quantitative real-time polymerase chain reaction (qRT-PCR) were applied to measure protein expression and RNA expression, respectively. Bioinformatic database StarBase was used to predict the possible targets of NUTM2A-AS1 and let-7c-5p, and dual-luciferase reporter assay was conducted to verify these intermolecular interactions. RESULTS: LPS stimulation restrained cell viability and induced cell apoptosis and inflammation of HDPCs. LPS exposure up-regulated the expression of NUTM2A-AS1 and High-Mobility Group Box 1 (HMGB1) and down-regulated the level of let-7c-5p. LPS-induced injury in HDPCs was partly attenuated by the silencing of NUTM2A-AS1 or HMGB1. Let-7c-5p was confirmed as a direct target of NUTM2A-AS1, and let-7c-5p bound to the 3' untranslated region (3'UTR) of HMGB1 messenger RNA (mRNA) in HDPCs. HMGB1 overexpression largely overturned NUTM2A-AS1 silencing-mediated effects in LPS-induced HDPCs. CONCLUSION: NUTM2A-AS1 knockdown attenuated LPS-induced damage in HDPCs partly through targeting let-7c-5p/HMGB1 axis.

The Genetic and Epigenetic Mechanisms Involved in Irreversible Pulp Neural Inflammation.

AIM: To identify the critical genetic and epigenetic biomarkers by constructing the long noncoding RNA- (lncRNA-) related competing endogenous RNA (ceRNA) network involved in irreversible pulp neural inflammation (pulpitis). MATERIALS AND METHODS: The public datasets regarding irreversible pulpitis were downloaded from the gene expression omnibus (GEO) database. The differential expression analysis was performed to identify the differentially expressed genes (DEGs) and DElncRNAs. Functional enrichment analysis was performed to explore the biological processes and signaling pathways enriched by DEGs. By performing a weighted gene coexpression network analysis (WGCNA), the significant gene modules in each dataset were identified. Most importantly, DElncRNA-DEmRNA regulatory network and DElncRNA-associated ceRNA network were constructed. A transcription factor- (TF-) DEmRNA network was built to identify the critical TFs involved in pulpitis. RESULT: Two datasets (GSE92681 and GSE77459) were selected for analysis. DEGs involved in pulpitis were significantly enriched in seven signaling pathways (i.e., NOD-like receptor (NLR), Toll-like receptor (TLR), NF-kappa B, tumor necrosis factor (TNF), cell adhesion molecules (CAMs), chemokine, and cytokine-cytokine receptor interaction pathways). The ceRNA regulatory relationships were established consisting of three genes (i.e., LCP1, EZH2, and NR4A1), five miRNAs (i.e., miR-340-5p, miR-4731-5p, miR-27a-3p, miR-34a-5p, and miR-766-5p), and three lncRNAs (i.e., XIST, MIR155HG, and LINC00630). Six transcription factors (i.e., GATA2, ETS1, FOXP3, STAT1, FOS, and JUN) were identified to play pivotal roles in pulpitis. CONCLUSION: This paper demonstrates the genetic and epigenetic mechanisms of irreversible pulpitis by revealing the ceRNA network. The biomarkers identified could provide research direction for the application of genetically modified stem cells in endodontic regeneration.

Long noncoding RNA MEG3 expressed in human dental pulp regulates LPS-Induced inflammation and odontogenic differentiation in pulpitis.

Pulpitis refers to inflammation of the inner pulp by invading microbes, and tissue repair occurs due to odontogenic differentiation of human dental pulp cells (hDPCs) with multidifferentiation potential. Long noncoding RNAs (lncRNAs) can modulate numerous pathological and biological processes; however, the role of lncRNAs in the inflammation and regeneration of the dentin-pulp complex in pulpitis is unclear. Here, we performed high-throughput sequencing to identify differentially expressed lncRNAs between human normal and inflamed pulp and concluded that lncMEG3 (lncRNA maternally expressed gene 3, MEG3) was significantly upregulated in both inflamed pulp and LPS-treated hDPCs. MEG3 expression in the pulp tissue was detected using the RNAscope® technique. RNA pulldown assays identified the MEG3-interacting proteins and the potential mechanisms. With MEG3 knockdown, we investigated the role of MEG3 in the secretion of inflammatory cytokines in LPS-treated hDPCs and odontogenic differentiation of hDPCs. MEG3 downregulation inhibited the secretion of TNF-α, IL-1ß and IL-6 in LPS-treated hDPCs, and the p38/MAPK signaling pathway may be related to this effect. MEG3 knockdown promoted odontogenic differentiation of hDPCs by regulating the Wnt/ß-catenin signaling pathway. Our study suggested that MEG3 has a negative effect on inflammation and regeneration of the dentin-pulp complex in pulpitis.

Diagnostic biomarker candidates for pulpitis revealed by bioinformatics analysis of merged microarray gene expression datasets.

BACKGROUND: Pulpitis is an inflammatory disease, the grade of which is classified according to the level of inflammation. Traditional methods of evaluating the status of dental pulp tissue in clinical practice have limitations. The rapid and accurate diagnosis of pulpitis is essential for determining the appropriate treatment. By integrating different datasets from the Gene Expression Omnibus (GEO) database, we analysed a merged expression matrix of pulpitis, aiming to identify biological pathways and diagnostic biomarkers of pulpitis. METHODS: By integrating two datasets (GSE77459 and GSE92681) in the GEO database using the sva and limma packages of R, differentially expressed genes (DEGs) of pulpitis were identified. Then, the DEGs were analysed to identify biological pathways of dental pulp inflammation with Gene Ontology (GO) analysis, Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analysis and Gene Set Enrichment Analysis (GSEA). Protein-protein interaction (PPI) networks and modules were constructed to identify hub genes with the Search Tool for the Retrieval of Interacting Genes/Proteins (STRING) and Cytoscape. RESULTS: A total of 470 DEGs comprising 394 upregulated and 76 downregulated genes were found in pulpitis tissue. GO analysis revealed that the DEGs were enriched in biological processes related to inflammation, and the enriched pathways in the KEGG pathway analysis were cytokine-cytokine receptor interaction, chemokine signalling pathway and NF-κB signalling pathway. The GSEA results provided further functional annotations, including complement system, IL6/JAK/STAT3 signalling pathway and inflammatory response pathways. According to the degrees of nodes in the PPI network, 10 hub genes were identified, and 8 diagnostic biomarker candidates were screened: PTPRC, CD86, CCL2, IL6, TLR8, MMP9, CXCL8 and ICAM1. CONCLUSIONS: With bioinformatics analysis of merged datasets, biomarker candidates of pulpitis were screened and the findings may be as reference to develop a new method of pulpitis diagnosis.

Long non­coding RNAs are novel players in oral inflammatory disorders, potentially premalignant oral epithelial lesions and oral squamous cell carcinoma (Review).

In recent years, a large number of studies have shown that the abnormal expression of long non­coding (lnc)RNAs can lead to a variety of different diseases, including inflammatory disorders, cardiovascular disease, nervous system diseases, and cancers. Recent research has demonstrated the biological characteristics of lncRNAs and the important functions of lncRNAs in oral inflammation, precancerous lesions and cancers. The present review aims to explore and discuss the potential roles of candidate lncRNAs in oral diseases by summarizing multiple lncRNA profiles in diseased and healthy oral tissues to determine the altered lncRNA signatures. In addition, to highlight the exact regulatory mechanism of lncRNAs in oral inflammatory disorders, potentially premalignant oral epithelial lesions and oral squamous cell carcinoma. The detection of lncRNAs in oral samples has the potential to be used as a diagnostic and an early detection tool for oral diseases. Furthermore, lncRNAs are promising future therapeutic targets in oral diseases, and research in this field may expand in the future.