Introduction

Pulpitis is a common disease in the oral cavity with a high incidence. Pulpitis is an inflammatory disease of the dental pulp which is caused by a progressive inflammatory reaction of the pulp-dentin complex caused by deep caries, trauma, etc. [1]. There is still no new breakthrough in the treatment of pulpitis at present [2]. The treatment for pulpitis is still root canal therapy to remove the pulp tissue. However, after the pulp tissue is removed from the affected tooth, there is no nutritional supply, and the brittleness increases, resulting in a decrease in the service life of the tooth. Pulpitis, as an inflammatory disease, is closely related to epigenetic modifications [3]. Research has shown that DNA methylation and RNA modification are involved in the occurrence and development of this disease [4, 5]. However, the mechanisms involved have not yet been fully elucidated, and the urgent search for the pathogenesis of pulpitis and new therapeutic targets is expected to bring good news to patients with pulpitis.

RNA in organisms can be post transcriptionally modified by more than 100 different chemical modifications, among which N6-methyladenosine (m6A) has been identified as the most abundant internal modification in eukaryotic mRNA [6]. m6 A is associated with many diseases such as cancer [7], and viral infection and inflammation are also diseases affected by m6 A [8, 9]. m6A modification is reversible and has become one of the current research hotspots. It is catalyzed by “writers” (METTL3, METTL14 and WTAP) to form a methyltransferase complex, which adds a methyl group to the N6 site of adenine in the RRACU sequence motif (R means G or A), while demethylation the enzymes ALKBH5 and FTO reverse this process, acting as an “eraser” [10]. When modified with m6A, transcripts can be recognized by “readers” (such as YTHDF1-3, YTHDC1 and IGF2BPs) to perform corresponding regulatory functions [11]. However, the role of m6A modification in the development of pulpitis disease remains unknown.

Bioinformatics studies on data from microarrays have emerged as promising and efficient tools for screening disease-related genes [12, 13]. This study used public datasets and bioinformatics analysis to identify the m6A RNA regulatory network in pulpitis. And the corresponding tissue samples were collected to further verify the expression of key related m6A regulators in pulpitis. This study can provide clues for further research on the occurrence and development mechanism of clinical pulpitis disease, which is crucial for identifying new targets for early detection and treatment of pulpitis.

Methods

Human samples

Human pulp tissue samples were obtained from patient dental specimens aged 18–35 years, all from completely extracted third molars (12 women and 8 men; age 25.65 ± 4.9 years). Pulpitis samples were obtained from 10 patients (6 women and 4 men; age 26.5 ± 5.2 years) with teeth diagnosed as irreversible pulpitis with a previous history of spontaneous pain and caused by cold and/or heat testing of persistent pain. These teeth had clinical and radiographic evidence of deep carious lesions extending into the pulp cavity. Control samples were obtained from 10 patients (6 women and 4 men; age 24.8 ± 4.8 years) with fully erupted teeth, intact crowns, and surrounded by healthy periodontal tissue, from orthodontic indications extracted third molar. The patients underwent tooth extraction after obtaining informed consent. This study is compliant with the Declaration of Helsinki. This study protocol was approved by the Ethics Committee of Jinhua Hospital Affiliated to Zhejiang University (No.S96; 16/05/2022).

Data collection and differentially analysis

The gene expression profile of human pulpitis was downloaded from GSE77459 through Gene Expression Omnibus (GEO) database (https://www.ncbi.nlm.nih.gov/geo/), which includes 6 normal dental pulp tissues and 6 inflammed pulp tissues. “Limma” R package were used to screen the differentially expressed genes (DEGs) and LogFC > 0.7 and P < 0.05 were set as the criterion. The volcano plot was visualized by “ggplot2” R package. The m6A regulators including “writers”: METTL3, METTL14, WTAP, ZC3H13, METTL16, ZCCHC4, RBM15 and RBM15B, “erasers”: FTO and ALKBH5, and “readers”: HNRNPA2B1, HNRNPC, IGF2BP1, IGF2BP2, IGF2BP3, YTHDC1, YTHDC2, YTHDF1, YTHDF2 and YTHDF3. The targets of differentially expressed m6A genes were acquired form m6A2Target database (http://m6a2target.canceromics.org).

Functional enrichment analysis

We further performed the Kyoto Encyclopedia of Genes and Genomes (KEGG) [14,15,16] and Gene Ontology (GO) pathway enrichment analyses including biological processes (BP), cellular components (CC) and molecular functions (MF) analysis of the targets of differential m6A genes by “clusterProfiler”, “org.Hs.eg.db” and “GOplot” R package.

Construction of miRNA-mRNA network of pulpitis-related m6A regulators

Upon the pulitis-related m6A regulators identified, we also predicted the potential interacted miRNA through miRwalk database (http://mirwalk.umm.uni-heidelberg.de/). Then we constructed and visualized the miRNA-mRNA network which showed the miRNAs interacting with the key m6A regulators in human pulpitis by Cytoscape software.

RNA isolation and real-time PCR analysis

Total RNA from dental pulp tissue was extracted using TRIzol (Vazyme, Nanjing, China). Reverse transcription reagents (Vazyme, Nanjing, China) were used for cDNA synthesis. Real-time PCR was performed on an ABI 7300 Real-Time PCR System (Applied Biosystems, Carlsbad, CA, USA) using the SYBR Green PCR protocol. Relative mRNA expression of target genes was normalized to GAPDH using the 2−ΔΔCT method and expressed as the mean ± SD of the number of replicates. The primers and primer sequences used in this experiment are listed in Table 1.

Table 1 Primers for PCR analysis
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Statistical analysis

The statistical analyses were carried out using Graphpad software. The data were manifested as means ± SD. The independent-sample t-test was applied for the comparisons between the normal and pulpitis group. P value < 0.05 was set as statistically significant.

Results

Identification of pulpitis-associated m6A regulators

To explore the functional m6A regulators in pulpitis, we preformed the differential analysis of GSE77459 dataset of pulpitis. Firstly, PCA analysis was performed and we found that the differential genes of the normal group and the pulpitis group were significantly grouped (Fig. 1A). Further differential expression analysis of genes between pulpitis tissue and normal pulp tissue was performed, as shown in the volcano plot (Fig. 1B). Then, the m6A-related genes were selected from these genes, including “writers”, “erasers” and “readers”, and further analysis was performed, showing the changes in m6A regulator gene expression between the pulpitis group and the normal group in Fig. 1C. Finally, the correlation analysis of these genes was carried out (Fig. 1D), and it was found that these genes were basically significantly correlated in pulpitis.

Fig. 1
figure 1

Identification of pulpitis-associated m6A regulators. (A) PCA plot: Differential gene significant grouping between normal group and pulpitis group. (B) Volcano plot: Differentially expressed genes between pulpitis group and normal group. Red indicates up-regulated mRNAs in pulpitis, blue indicates down-regulated mRNAs in pulpitis, and black indicates no differentially expressed mRNAs. (C) Heatmap of gene expression in pulpitis and normal groups. (D) Correlation analysis among m6A-related genes in pulpitis

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We further compared the expression of m6A-related genes in the normal group and the pulpitis group and made corresponding statistical analysis (Fig. 2). The results showed that there were significant differences in the genes ALKBH5, METTL14, METTL3, METTL16, RBM15B and YTHDF1 between normal group and the pulpitis group. It indicates that these genes may play specific roles in pulpitis.

Fig. 2
figure 2

Box plots for m6A-related genes expression between normal group and pulpitis group. *P < 0.05, **P < 0.01, ***P < 0.001

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m6A-related genes molecular interaction network and core genes interaction network

To explore the hub m6A regulators in pulpitis, we further constructed the molecular interaction network of m6A-related genes, and Cytoscape was used to extract the interaction network of core genes (Fig. 3), which further suggested that these genes RBM15B, METTL16, METTL14, YTHDF3 and ALKBH5 may be the main regulators in pulpitis m6A-modified genes.

Fig. 3
figure 3

m6A-related genes molecular interaction network and core genes interaction network

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Potential functions of genes modified by m6A regulators associated with pulpitis

To explore the role of m6A regulators in pulpitis, we further analyzed the downstream target molecules of core genes with statistical significance and intersected these molecules with differential genes in GEO (Fig. 4A), suggesting that these intersected molecules are m6A regulated molecules in pulpitis. And the number of intersected gene between GSE77459 and RBM15B targets, GSE77459 and YTHDF1 targets and GSE77459 and YTHDF3 is 110, 418, 70, respectively. We performed GO enrichment analysis and KEGG enrichment analysis at the same time (Fig. 4B-C), and the results showed that these further screened downstream genes were mainly enriched in biological processes such as cell adhesion, T cell activation, glutamatergic synapse, ephrin receptor binding, cytokine receptors, and osteoclasts differentiation. These results suggested that m6A regulators in pulpitis are mainly associated the biological processes of immune cell activation, glutamatergic synapse, cytokine activity and osteoclast differentiation, which are important for pulpitis progression.

Fig. 4
figure 4

(A) The intersection of the downstream target molecules of the above core genes and the differential genes in GEO. (B-C) GO enrichment analysis and KEGG analysis

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Molecular interaction network of upstream miRNAs regulating the core m6A genes

Pulpitis is a ubiquitous chronic inflammation in the dental pulp, and miRNAs have been found to act as promoters or inhibitors of oral inflammation [17]. To explore the potential upstream regulators of hub m6A genes in pulpitis, we used the miRwalkdatabase to predict the upstream miRNAs of the core m6A genes, including has-miR-4709-3p, has-miR-5010-3p, hsa-miR-4257, hsa-miR-550b-2-5p, has-miR-7977, has-miR-29b-2-5p, has-miR-149-3p, has-miR-363-5p, has-miR-329-3p, hsa-miR-329-3p, and made a molecular interaction network (Fig. 5).

Fig. 5
figure 5

Molecular interaction network of upstream miRNAs regulating the core m6A genes

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Validation of key m6A regulators in pulpitis

To further validate the key m6A regulators in pulpitis, the expression of ALKBH5, METTL3, METTL14, METTL16, RBM15B, YTHDF1, YTHDF3 and ZCCHC4 genes was detected by PCR on collected clinical tissue samples. METTL14 mRNA was confirmed to be significantly downregulated in pulpitis samples compared with controls (Fig. 6). However, YTHDF1, YTHDF3, METTL16 and METTL3 gene expression were not statistically different between the pulpitis group and the control group, and RBM15B, ZCCHC4 and ALKBH5 were up-regulated (Fig. 6). These results suggest that METTL14 may be a key regulator in pulpitis, further validating the differential expression of m6A-related genes between pulpitis and normal groups.

Fig. 6
figure 6

The gene expression of ALKBH5, METTL3, METTL14, METTL16, RBM15B, YTHDF1, YTHDF3 and ZCCHC4 in normal group and pulpitis tissue samples was analyzed by quantitative real-time PCR. *P < 0.05 was considered statistically significant

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Discussion

Pulpitis is a chronic progressive inflammatory and destructive disease that exists and occurs globally, affecting people’s quality of life to a certain extent [18]. The specific pathogenesis of the disease has not yet been elucidated [19], and it is believed to be related to a variety of factors.

In recent years, m6A regulators have been found to play an important role in the occurrence, development and prognosis of various human diseases [20, 21]. Studies have shown that m6A modification plays a role in inflammatory diseases [22], but the role of m6A modification in pulpitis has not been elucidated. This is the first systematic bioinformatics analysis and validation of m6A regulators in pulpitis disease, which can provide directions for the development mechanism of pulpitis and the targeted therapy of pulpitis.

We were the first to analyze and find the differences of m6A-related genes between pulpitis and normal groups, among which ALKBH5, METTL14, METTL3, METTL16, RBM15B and YTHDF1 were significantly different, suggesting that these genes may be involved in the occurrence of pulpitis important role in development. These hub genes have been reported in other inflammatory diseases such as rheumatoid arthritis [23], systemic lupus erythematosus [24] and scleroderma [25], further illustrating the importance and necessity of our study of the role of m6A in pulpitis. We analyzed functional enrichment analysis of key m6A target genes and found that these genes were mainly enriched in biological processes such as cell adhesion, T cell activation, glutamatergic synapse, ephrin receptor binding, cytokine receptors, and osteoclasts differentiation. These biological processes play an important role in the occurrence and development of pulpitis. Studies have shown that cell adhesion is related to the inflammatory progression of pulpitis and affects pulp regeneration [2]. Cytokine receptors can recruit stem cells and promote the differentiation of stem cells, which can promote the regeneration of dental pulp [26]. Invasion of caries-associated bacteria into dentin tissue resulted in enhanced osteoclastogenesis and inhibition of odontoblastogenesis [27], suggesting that osteoclast differentiation also plays an important role in the progression of pulpitis. These indicate that m6A is also very important for the progression of pulpitis. At the same time, we have made miRNAs that may regulate m6A, indicating that these miRNAs may also be important non-coding RNAs in the regulation of pulpitis.

In addition, the gene expression levels of ALKBH5, METTL3, METTL14, METTL16, RBM15B, YTHDF1, YTHDF3 and ZCCHC4 were detected in the collected human dental pulp tissue samples, and it was found that the expression of METTL14 in pulpitis was lower than that in the control group. The result indicates that METTL14 may be particularly important in pulpitis, and can be used as a biomarker for the progress of pulpitis, which provides a new idea for future therapeutic targets and drug development of pulpitis.