Identification and validation of m6A RNA regulatory network in pulpitis

Xu, Hui; Chen, Guang**; Zhou, Jiaying; Zhou, Xukang; Wang, Pengcheng; Chen, Chunhui; Xu, Zhi; Lv, Fengyuan; Li, **aofang

doi:10.1186/s12903-023-03578-8

Identification and validation of m⁶A RNA regulatory network in pulpitis

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Published: 17 November 2023

Volume 23, article number 878, (2023)
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Identification and validation of m⁶A RNA regulatory network in pulpitis

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Hui Xu¹^na1,
Guang** Chen^2,3,4^na1,
Jiaying Zhou^1,5,
Xukang Zhou^1,6,
Pengcheng Wang¹,
Chunhui Chen¹,
Zhi Xu^2,3,4,
Fengyuan Lv⁷^nAff8 &
…
**aofang Li¹

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Abstract

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.

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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” [Full size image

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.

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.

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.

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).

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.

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.

Data Availability

The datasets used and analyzed during the current study are available from the corresponding author on reasonable request.

Abbreviations

m⁶A:: N6-methyladenosine
GO:: Gene ontology
KEGG:: Kyoto encyclopedia of genes and genomes
qRT-PCR:: Quantitative real-time PCR
BP:: Biological processes
CC:: Cellular components
MF:: Molecular functions

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Acknowledgements

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Funding

The work was supported by the grants from the National Natural Science Foundation of China (No. 81901016 to Z.X.) and Wuhan Knowledge Innovation Program.

(No. 2022020801020469 to Z.X.).

Author information

Fengyuan Lv
Present address: Hubei Province Key Laboratory of Oral and Maxillofacial Development and Regeneration, Wuhan, 430022, China
Hui Xu and Guang** Chen contributed equally to this work.

Authors and Affiliations

Department of Stomatology, Affiliated **hua Hospital, Zhejiang University School of Medicine, **hua Municipal Central Hospital, **Hua, China
Hui Xu, Jiaying Zhou, Xukang Zhou, Pengcheng Wang, Chunhui Chen & **aofang Li
Department of Stomatology, Union Hospital, Tongji Medical College, Huazhong University of Science and Technology, Wuhan, 430022, China
Guang** Chen & Zhi Xu
School of Stomatology, Tongji Medical College, Huazhong University of Science and Technology, Wuhan, 430030, China
Guang** Chen & Zhi Xu
Hubei Province Key Laboratory of Oral and Maxillofacial Development and Regeneration, Wuhan, 430022, China
Guang** Chen & Zhi Xu
College of Medicine, Huzhou University, Huzhou, 313000, China
Jiaying Zhou
Department of Health Management Center, Affiliated **hua Hospital, Zhejiang University School of Medicine, **hua Municipal Central Hospital, **Hua, China
Xukang Zhou
Center of Stomatology, Tongji Hospital, Tongji Medical College, Huazhong University of Science and Technology, Wuhan, 430030, China
Fengyuan Lv

Authors

Hui Xu
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Guang** Chen
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Jiaying Zhou
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Xukang Zhou
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Pengcheng Wang
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Chunhui Chen
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Zhi Xu
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Fengyuan Lv
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**aofang Li
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Contributions

Hui Xu conducted the research and drafted the article. Guang** Chen analyzed the data and drafted the article. Pengcheng Wang, Zhi Xu and Chunhui Chen collected specimens. Jiaying Zhou and Xukang Zhou revised the article. Fengyuan Lv and **aofang Li designed the study and revised the article.

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Correspondence to Fengyuan Lv or **aofang Li.

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Ethical approval and consent

All methods were carried out in accordance with relevant guidelines and regulations. All experimental protocols was approved by the Ethics Committee of **hua Hospital Affiliated to Zhejiang University (No.S96; 16/05/2022). Written informed consent was obtained for all subjects after explaining the purpose of the study.

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Xu, H., Chen, G., Zhou, J. et al. Identification and validation of m⁶A RNA regulatory network in pulpitis. BMC Oral Health 23, 878 (2023). https://doi.org/10.1186/s12903-023-03578-8

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Received: 30 November 2022
Accepted: 25 October 2023
Published: 17 November 2023
DOI: https://doi.org/10.1186/s12903-023-03578-8

Identification and validation of m⁶A RNA regulatory network in pulpitis