Dysregulation of lncRNA TFAP2A‐AS1 is involved in the pathogenesis of pulpitis by the regulation of microRNA‐32‐5p

Abstract Objective This study was designed to evaluate TFAP2A‐AS1 expression in the dental pulp of teeth with or without pulpitis and to determine the function of TFAP2A‐AS1 in pulp cells. Methods GSE92681 was analyzed to filter out differentially expressed lncRNAs. Pulp samples from teeth with pulpitis and healthy teeth (control) were examined using real‐time (RT) quantitative polymerase chain reaction (qPCR). Human dental pulp stem cells (hDPSCs) were cultured in a specific medium for osteogenic induction, or treated with lipopolysaccharide (LPS) to simulate inflammation. The viability and apoptosis of human DPSCs (hDPSCs) were determined by XTT assay and apoptosis detection kit. Inflammation was induced by LPS and assessed by measuring the expression and release of inflammatory cytokines after TFAP2A‐AS1 knockdown. Osteogenic differentiation of hDPSCs was investigated by determining expression levels of osteogenic markers and alkaline phosphatase (ALP) activity after TFAP2A‐AS1 overexpression. The downstream microRNA (miRNA) was predicted. Dual‐luciferase reporter was used to confirm the binding between miR‐32‐5p and TFAP2A‐AS1. Results The expression of TFAP2A‐AS1 was evaluated in inflamed pulp using RT‐qPCR. TFAP2A‐AS1 had a discriminatory ability for healthy individuals and patients with pulpitis. The expression of TFAP2A‐AS1 decreased upon the osteogenic differentiation of hDPSCs, and increased upon the LPS induction. TFAP2A‐AS1 can reverse the osteogenic differentiation of hDPSCs, as evidenced by decreased levels of dentine sialophosphoprotein, dentin matrix protein−1, and ALP activity. TFAP2A‐AS1 knockdown can promote cell proliferation of hDPSCs and relieve LPS‐induced inflammation, as evidenced by decreased levels of TNF‐α, IL‐1β, and IL‐6. miR‐32‐5p was identified as a downstream miRNA of TFAP2A‐AS1. Conclusion This study demonstrated the expression and potential function of TFAP2A‐AS1 in the human dental pulp. TFAP2A‐AS1 can inhibit odontogenic differentiation but promote inflammation in pulp cells.

Teeth, although small, have a complex structure comprised of components like dental pulp, each with unique features and functions. 1Dental pulp is the soft tissue at the core of teeth, encased by the crown and root and is lined with a layer of odontoblasts. 2Teeth are prone to damage from intense mechanical and chemical stress, as well as dense microbiologic colonization.This damage usually occurs in the form of caries, periodontal disease and trauma. 3If the mineralized tissues of enamel and dentine are damaged, the dental pulp would becomes susceptible to microbial invasion. 4The toxins from bacteria can penetrate the dentinal tubules.The bacterial antigens and lipopolysaccharides (LPS) can cause immunological reactions and increase the levels of immunoglobulins, prostaglandins, and other pro-inflammatory mediators in the infected pulp.In response to this insult, the dental pulp initiates a complex inflammatory response, leading to infection and inflammation.As bacteria in contact with the pulp, additional cell types of the pulp, such as stem cells, contribute great with a series of inflammatory-defense mechanisms, which are critical for tissue homeostasis. 5,6Stem cells exhibit both mesenchymal and neural characteristics. 7The pulpal stem cells may play a central role in the immunological responses of the dental pulp to oral microorganisms. 8A thorough understanding of molecular mechanism in pulpitis may lead to the identification of new therapeutic targets for this disease and enhance diagnostics strategies.
Epigenetics plays important roles in pulpitis, influencing aspects such as the inflammatory process and endodontic regeneration. 9,10Epigenetic processes refer to mitotically and meiotically heritable changes, independent of the DNA sequence.These processes have a complex molecular basis that involves noncoding RNAs (ncRNAs). 11Since epigenetic modifications can be reversed, understanding their mechanisms could help identify new therapeutic targets for inflammatory diseases. 12Over recent decades, modulation of ncRNA, despite lacking protein-coding function, has emerged as a new layer of gene regulation. 11ncRNAs can be classified as into two main types on length (shorter or longer than 200 nt)-long noncoding RNA (lncRNA) and microRNA (miRNA). 13They play potential roles in dental pulp tissue, including odontogenic differentiation, immune response, and bone resorption. 14,15For example, MEG3, an upregulated lncRNA in inflamed pulp and LPStreated human dental pulp cells, has been found to negatively affect inflammation and regeneration of the dentin-pulp complex in pulpitis. 16It's worth noting that lncRNA can function as natural miRNA sponges, binding to miRNAs and competitively sequestering them from their target genes, a process referred to as competing endogenous RNAs (ceRNAs).For instance, upregulated lncRNA DUXAP8 is found in pulpitis and associated with miR-18b-5p sponging function, which exacerbates the pathogenetic condition. 17n this study, GSE92681 data set from GEO database was analyzed to screen the differentially expressed lncRNAs.Then, TFAP2A-AS1 expression was evaluated in the healthy or inflamed dental pulp.Additionally, the function of TFAP2A-AS1 in osteogenic differentiation and inflammation was examined.

| Collection of dental pulp tissue
This study was approved by the Institutional Review Boards of Shijiazhuang Fourth Hospital (approval no.20230019).Each patient signed a written informed consent for use of the samples.Healthy dental pulp (n = 18) was obtained from wisdom teeth or premolars for orthodontic reasons.Simultaneously, inflamed dental pulp samples were extirpated from carious teeth with nerve broach in 37 patients with irreversible pulpitis (the American Association of Endodontists guidelines).The two groups were matched in age and gender (Table 1).The removed tissue was immediately placed in RNALater™ RNA Stabilization Reagent for Animal Tissue (Beyotime) and incubated at 4°C for 24 h.Then, the regent was discarded, and tissues were transferred to a prefrozen liquid nitrogen tube with a rotating cap.After rapid freezing with liquid nitrogen, the tissues were stored at −80°C.

| RNA isolation and RT-qPCR
The frozen healthy and inflamed dental pulp tissues were thawed and homogenized.Total RNA was isolated using RNAeasy™ Animal RNA Isolation Kit with Spin Column (Beyotime).Total RNA (400 ng) was subjected to reverse transcription, using the QuantiTect Reverse Transcription kit (Qiagen).The resulting cDNA was diluted 1:20 for RT-qPCR using the SsoAdvanced Universal SYBR-Green Supermix Kit (Bio-Rad) and primers.After RT-qPCR, quantification data were normalized using the geometric mean of the selected stable reference genes (Glyceraldehyde 3-phosphate dehydrogenase for lncRNA and mRNA, and U6 for miR-32-5p), with the 2 −ΔΔCt method.

| Cell proliferation and apoptosis assays
hDPSCs proliferation was determined by Cell Proliferation kit II (XTT) (Roche, Mannheim, Germany) abiding by the manufacturer's instructions.Cell apoptosis assays were completed by flow cytometry (CytoFLEX analyzer; Beckman Coulter), within 1 h of using FITC Annexin V Apoptosis Detection Kit (BD Biosciences) based on the recommendations from the manufacturer.

| Bioinformatics analysis
The bioinformatics tool, lncRNASNP v2, was used to search the possible miRNA targets of TFAP2A-AS1.Among the predicted miRNAs, miR-32-5p, known to be involved in osteogenic and adipogenic differentiation of dental pulp stem cells, was selected for further interaction studies with TFAP2A-AS1.

| Dual-luciferase reporter assay
Based on the binding sites between TFAP2A-AS1 and miR-32-5p, the wild-type and mutant binding sequence regions of miR-32-5p on the fragment of TFAP2A-AS1 were entrusted to Hanbio for chemical synthesis and inserted into pmirGLO luciferase reporter vectors (LMAI Bio).hDPSCs were transfected with reporter plasmids (wild or mutant TFAP2A-AS1) and miR-32-5p mimics or mimic negative control (NC).After 24 h of transfection, the cells processed as per the instructions of Dual-Luciferase Reporter gene assay kit (Promega).The activities of renilla luciferase and firefly luciferase were then measured using a GloMax luminometer (Promega).All data were transferred to statistics software (Graph-Pad Prism 9) for analysis, and the Kolmogorov-Smirnov normality test was applied.Data that were not normally distributed required Mann-Whitney method for parametric analysis.Normally distributed data were compared using either one way ANOVA or t tests.The correlation analysis between TFAP2A-AS1 and miR-32-5p was analyzed using Pearson's correlation coefficient.
The predictive performance of TFAP2A-AS1 was assessed by receiver operating characteristic (ROC) curve analysis, represented as the area under the curve (AUC).When p < .05, the difference was statistically significant.

| TFAP2A-AS1 expression in healthy and inflamed dental pulp
TFAP2A-AS1 was identified as a differentially expressed lncRNA in GSE92681 (Figure 1A).Then, the expression level of TFAP2A-AS1 was assessed in the healthy and inflamed dental pulp.Results showed that pulpitis caused a sharp significant (p < .001)elevation in TFAP2A-AS1 expression level in patients with inflamed dental pulp when compared with the healthy ones (Figure 1B).The TFAP2A-AS1 level manifested as a well-distinguishing tool for pulpitis and health, with an AUC of 0.818 (Figure 1C).During osteogenesis of hDPSCs, the TFAP2A-AS1 level decreased (p < 0.05) upon the culture time (Figure 1D).LPS exposure dose-dependently increased the expression of TFAP2A-AS1 in hDPSCs (Figure 1E).

| DISCUSSION
Pulpitis is one of the most common oral diseases.Its main clinical manifestations include spontaneous and paroxysmal pain, cold-and hot-stimulating pain, and nocturnal pain, which seriously affects the quality of life of patients. 20Previous studies prove that lncRNA is involved in the inflammatory process of the immune system, and is related to pulpitis. 21In a study by HUANG et al., lncRNA expression in pulpitis and normal human dental pulp tissues was assessed.They found significant differences in lncRNA expression between normal dental pulp tissue and pulpitis-affected tissue, suggesting lncRNA could play a crucial role in the pathogenesis of pulpitis. 22In our study, we analyzed GSE92681 data set, and identified TFAP2A-AS1 as a differentially expressed lncRNA in pulpitis.TFAP2A-AS1 has been reported to be related to the immune response in breast cancer treatment and oral squamous cell carcinoma progress. 23,24Subsequently, we identified TFAP2A-AS1 expression profiles in pulpitis tissues and found it was upregulated in pulpitis tissues compared with the healthy pulp.After induction of odontoblastic differentiation, TFAP2A-AS1 expression level was decreased in hDPSCs.In contrast, if hDPSCs were treated with LPS, TFAP2A-AS1 expression level increased.These findings imply TFAP2A-AS1 may be involved in the progress of pulpitis.
Functional pulp tissue is a prerequisite for completing root formation during tooth development and eruption.The odontoblasts lined in pulp can polarize and secrete a collagenous matrix, to physiologically and continuously form dentine. 25 The composition of dentine is similar to that of bone.This study explored the regulatory mechanism of TFAP2A-AS1 in the odontogenic differentiation.TFAP2A-AS1 was overexpressed in hDPSCs, followed by differentiating into odontoblasts in vitro.The expression profiles of osteogenic markers in TFAP2A-AS1downregulated hDPSCs were then examined and compared to those in non-downregulated cells.The results showed that the upregulation of TFAP2A-AS1 inhibited the osteo/odontogenic differentiation potential of DPSCs.Similarly, lncRNA-Ankrd26 from dental pulp stem cells can promote dental pulp restoration through osteoblastic differentiation of mesenchymal stem cells. 268][29] However, other lncRNAs, such as DANCR and LINC01133, have inhibitory effects on bone differentiation of hDPSCs. 30,31his study provides a new target for the regenerative treatment of endodontic diseases and enhances understanding of hDPSCs functions.
The cellular response to pulpal infection involves several resident cells, such as odontoblasts and fibroblasts, as well as immune cells.The recruited immune cells at the site of infection release the effector molecules including cytokines, chemokines and other proinflammatory mediators. 32The severity of inflammation dictates the outcome of dental pulp infection, which could result in regeneration, repair, or necrosis.hDPSCs play an important role in the regenerative and inflammatory response of the pulp to trauma and injury.Increasingly studies have evaluated the role of lncRNAs in the cellular and immune mediators in diseased dental pulps. 11For example, an in-vitro study revealed an upregulation of PVT1 in the pulpitis cell model, which faciliates injury to human dental pulp cells caused by LPS. 33This in-vitro study demonstrated TFAP2A-AS1 knockdown can reverse the LPS-induced increase in inflammatory mediators.Moreover, lncRNAs may regulate the proliferation and apoptosis of hDPSCs.LncRNA H19 is known to enhance the proliferation capability of hDPSCs. 34In the current study, TFAP2A-AS1 showed an inhibitory effect on proliferation capability and a promoting effect on the apoptotic capability of hDPSCs.These data provide insight into the regulatory effects of TFAP2A-AS1 on inflammatory response and growth of hDPSCs.
Considering the ceRNA function of lncRNAs, we further investigated the potential downstream miRNA for TFAP2A-AS1.Ultimately, miR-32-5p was verified to be a miRNA of TFAP2A-AS1 in hDPSCs.In oxidized lowdensity lipoprotein-induced human umbilical vein endothelial cells, miR-32-5p can suppress the expression of inflammatory factors including IL-1β, IL-6, TNF-α, ICAM-1, and VCAM-1. 18This indicates the antiinflammatory function of miR-32-5p in cell inflammation.Notably, miR-32 can be activated by enamel matrix proteins and regulate osteogenic and adipogenic differentiation of human exfoliated deciduous teeth. 19This miRNA potentially plays an important role in hDPSCs differentiation.Therefore, it was speculated that TFAP2A-AS1 may function in diseased dental pulps via sponging miR-32-5p.
The use of TFAP2A-AS1 could serve as a promising epigenetic biomarker for diagnosing pulpitis.Moreover, its role in various biological processes of pulpitis may highlight its potential as an epigenetic therapy.However, this study, conducted in our single center, has some limitations, including a small sample size and in vitro experiments.Therefore, future research is needed to explore the roles of TFAP2A-AS1 in pulpitis in vivo and the feasibility of a TFAP2A-AS1-inhibiting therapy.

| CONCLUSION
This study demonstrates TFAP2A-AS1 expression and potential function in human dental pulp.The expression pattern of TFAP2A-AS1 in human inflamed pulp was higher than that in healthy pulp.TFAP2A-AS1 appears to hinder odontogenic differentiation, but it promotes inflammation in pulp cells.These findings suggest that TFAP2A-AS1 may contribute to the pathogenesis of pulpitis Further research is needed to elucidate mechanism of TFAP2A-AS1 in pulpal diseases.
T A B L E 1 Clinical data comparison for subjects.ItemsNormal pulp (n = 18) Inflamed pulp (n = 37)