Background
Paediatric dental caries is one of the most common chronic infectious diseases in childhood of great concern to parents and dentists, affecting up to 60% of schoolchildren in China, and remains a major problem in many countries [
1]. Cariogenic bacteria, cariogenic diets, susceptible host and affected time together contribute to the occurrence of dental caries. Caries causes lesions and cavities on tooth surfaces, leading to decay and even loss of tooth structure. The destruction can progress in a rapid speed if left untreated, resulting in pain and infection. Therefore, early diagnosis and prevention are of particular clinical significance [
2,
3]. Current studies seek to identify the risk factors for caries as well as to study oral defense functions in protecting against and preventing the development of this disease [
4]. The known factors influencing dental caries in children include: immature immune systems, cariogenic microorganisms, characteristics of saliva, and oral hygiene care in childhood [
5]. As one of the most important host-associated factors in the etiology of caries, saliva contacts closely with teeth, and the constituents of this biological fluid play an essential role in the occurrence and progression of dental caries [
6,
7].
Human whole saliva is primarily composed of water and originated mainly from three major salivary glands—parotid, submandibular and sublingual glands [
8], involving in maintenance of oral homeostasis [
9]. As the principal component of saliva, salivary proteins, although accounting for only a small proportion of saliva, play various important roles to keep the integrity of teeth depending on their ability to inhibit the growth of cariogenic bacteria or to modulate the demineralization/remineralization balance, such as lactoferrin, lysozyme, proline-rich proteins and statherin [
10]. Besides of these anti-cariogenic factors in saliva, there are many proteins taking part in the cariogenic progress through promoting the proliferation and tooth colonization of bacteria [
7]. For example, common salivary protein-1 was suggested to be able to enhance the binding of
Streptococcus mutans to the hydroxyapatite surface, indicating its potential influence on the initial colonization of pathogenic bacterium onto the tooth surface [
11]. In addition, elevated levels of salivary matrix metalloproteinase (MMP)-8 were found in patients with dental caries relative to healthy individuals, which was supposed to initiate the collagen degradation in caries process in dentin [
12]. Therefore, understanding the role of proteins in cariogenic saliva will be of great importance for both the assessment of caries susceptibility and caries prevention.
Mass spectrometry (MS)-based proteomics is a large-scale, high-throughput, systematic study, allowing for the comprehensive characterization of salivary proteins, even with a limited amount of samples [
13]. Easy and non-invasive collection of saliva made it interesting to be used for the assessment of a variety of oral diseases applying these techniques, such as Sjögren’s syndrome, oral squamous cell carcinoma and periodontitis [
14‐
16]. Also, salivary protein profiles of dental caries have been investigated in the last decade, but the results lacking of validation for candidate proteins still remain controversial. A high degree of similarity in the general composition of salivary proteins was shown between children with and without dental caries in a previous study [
17]. On the contrary, another study identified significant differences in salivary protein expression profiles between children with severe childhood caries and caries-free children [
18]. More recently, overexpression of salivary complement system and inflammatory markers were demonstrated in caries patients compared to healthy controls [
19]. Therefore, the relationship between dental caries and human salivary proteins is yet to be well defined, and the biomarker information remains unclear.
Isobaric tags for relative and absolute quantitation (iTRAQ) is one of the new techniques used in modern proteomics that couples stable isotopes labeling and tandem mass spectrometry to permit comparative quantification with good precision. Multiple reaction monitoring (MRM) is a powerful tool for targeted proteomics and is an emerging field of proteomics with high reproducibility across complex samples. To date, several studies have demonstrated the feasibility of using targeted MRM-MS for quantitative proteomic analyses, which could realize highly multiplex, precise, specific and standardized proteomic quantification [
20,
21]. Also, iTRAQ discovery combined with subsequent MRM conformation has recently been adopted for disease biomarker quantification studies [
22,
23], but it has rarely been used for the salivary proteomic analysis of childhood caries.
In the current study, for the first time, we applied iTRAQ as a discovery method, followed by a verification (MRM) to perform a comparative saliva proteomics analysis for identifying key candidate proteins with diagnostic or protective value for childhood caries. Our results will serve to better understand the roles of salivary proteins involved in the onset and progression of childhood caries as well as their potential impact on clinical practice for anti-caries.
Discussion
Paediatric dental caries is considered to be a major public health problem. As an endogenous factor, saliva and its components responsible for playing important roles in protecting oral structures, may partially determine why some children develop caries whereas others do not. The purpose of the present study was to explore significant salivary proteins involved in anti-cariogenicity or cariogenicity through comparing proteome profiling of saliva from children with dental caries to that of orally healthy individuals, as we hypothesized that the components of saliva might be associated with caries status. Proteomics and related techniques have advanced significantly over the last two decades, making this analysis possible. However, due to the lack of a coherent, demonstrably successful pipeline from the discovery to the verification stage, previous reports regarding to the proteome profiling of dental caries remained controversial. Herein, our study revealed the salivary proteome of the children with and without dental caries using iTRAQ-based MRM-MS for quantitative proteomic analysis.
In this study, 4369 unique peptides and 759 proteins were identified, which are much more than that in salivary peptidome and proteome profiling of childhood dental caries in recent studies [
18,
34]. The higher number of salivary protein identification in our study is probably due to the differences in sampling methods, grouping methods, saliva-based proteomic approaches and the subjects included in studies. Considering that stimulated saliva samples could be diluted the concentration of proteins, unstimulated saliva samples used in our study may be preferred for in-depth analysis of the salivary proteome. In order to avoid false identification of biomarkers due to nondisease related differences between children with and without dental caries, we controlled the inter-individual differences within groups and enrolled subjects with similar mean age and demographic characteristics to the diseased groups. Also as saliva is susceptible to many physiological and biochemical processes, all the saliva specimens were collected and processed consistently according to the strict method described above. Thus, the quality of samples and consequently the resulting data are reliable.
Since dental caries is a progressive disease, there is an important role for high-throughput methods to characterize the proteins involved in the disease process. Therefore, we divided the subjects into three groups according to the disease severity based on DMFT/dmft index. We postulated that some proteins in saliva of children with caries might be associated with the severity of disease. Consequently, we found 142 proteins expressed differentially in HDC vs LDC, of which 66 proteins were exclusive in this comparison from the results of iTRAQ. Besides, 40 of the 66 unique proteins were up-regulated in HDC, indicating their potential roles in the exacerbation of childhood dental caries. The up-regulated proteins involved in the response to stress and positive regulation of biological process in terms of GO annotation were enriched in HDC group compared with LDC group, and they were also categorized into 80 pathways, which were most represented by these proteins in metabolic pathways, focal adhesion as well as regulation of actin cytoskeleton. Among them, the proteins alpha actinin 1 and myosin regulatory light chain 12A both involved in the pathways for focal adhesion and regulation of actin cytoskeleton were associated with cell proliferation and cell moving [
35].
As for the differentially expressed proteins in HDC vs NDC, there were 62 up-regulated proteins in HDC groups from iTRAQ results, such as mucin 7, protein S100 A9, alpha-2-macroglobulin, zinc-alpha-2-glycoprotein, zymogen granule protein 16 homolog B and Ig kappa chain C region, which were also successfully validated in MRM analysis. As a result of MRM, protein S100 A9, protein S100 P, coronin 1A and alpha-2-macroglobulin were shared in HDC vs NDC and HDC vs LDC with higher expression levels in HDC group, indicating their potential diagnostic values in the childhood dental caries. In terms of the comparison between LDC and NDC, 97 salivary proteins were up-regulated in LDC, including mucin 7, mucin 5B, Ig kappa chain V-III region, which were also regarded as the special proteins of children with dental caries in a previous study [
34]. In addition, from the iTRAQ results, we found that complement C4-B was up-regulated in LDC when compared with NDC. Likewise, it has been reported that active components of the complement system in salivary metaproteome were associated with dental caries [
19], but their exact roles in the progression of dental caries still need further research. More importantly, of the unique proteins in LDC vs NDC in MRM analysis, basic salivary proline rich protein 2 was the only one down-regulated protein in LDC. The protective property of basic proline rich proteins (PRPs) in caries prevention has been reported previously, which was found to be able to neutralize acid produced by streptococci through attaching to a major adhesion antigen on the surface of
S. mutans and other oral streptococci [
36].
Both mucin 7 and mucin 5B are implicated in the prevention of dental cavity formation. The importance of mucin 7 in caries prevention has been demonstrated in elderly populations, who with lower mucin 7 concentrations were found to have higher
S. mutans titers in saliva [
37]. In fact, mucin 7 was able to bind to
S. mutans directly through the bacterium’s alpha-enolase surface protein, while mucin 5B could reduce the attachment and biofilm formation of
S. mutans, thereby accelerating the clearance of bacteria from the oral cavity [
38,
39]. As we know, salivary proteins seldom act alone, and they often bind together to perform their biological functions. It may thus be unrealistic to expect any single salivary factor to be adequate in protecting the integrity of teeth and counteracting the caries process. An important way mucin 7 and mucin 5B protect the oral cavity is by binding to select group of salivary proteins, through which they can influence the proteins’ localization in the oral cavity, increase their retention time, and then alter their biological activity [
40]. In this study, PPI network analysis was conducted to embed the differential salivary proteome with a biological framework, in which mucin 7, mucin 5B, histatin 1 and cystatin S interacted with each other. Histatin 1 was found to bind the N-terminal domain on the mucin 7 polypeptide backbone [
41], and mucin 5B also formed heterotypinc complexes with the same salivary proteins as mucin 7 [
42]. As these proteins all have antimicrobial properties, the complexes formation could increase their availability in saliva, protect proteins from proteolytic degradation and be beneficial to oral health. However, further studies are needed to better understand the effect of this complex on the biological activity of each component.
Among the differentially expressed proteins in different comparisons, the up-regulated proteins in NDC may also provide the source for the anti-cariogenic factors. Through iTRAQ analysis, statherin was found to be significantly up-regulated in NDC with the highest fold change in HDC vs NDC. In addition, among the unique proteins in comparison between HDC and NDC, cystatin S, cystatin SN and histatin 1 were down-regulated in HDC in MRM analysis. As for histatin 1, the value of MRM ratio for which was 0.71 in HDC compared with NDC, was involved in the biological process of biomineralization, antibacterial and antifungal response. Interestingly, these results were in line with previous findings. Vitorino et al. [
43] analyzed samples from caries-free and caries-susceptible subjects and revealed a strong correlation between the absence of dental caries and large amounts of histatin 1 and statherin, indicating the importance of these proteins in the maintenance of tooth integrity. Also, their subsequent study found significantly higher quantities of cystatin S and cystatin SN from caries-free group [
44]. These phosphorylated proteins, including histatin, statherin and cystatin, which were shown to maintain calcium saturation in saliva around teeth and then promoted the process of remineralization, may play important roles in the inhibition of caries process [
7,
45]. Although the differentially expressed salivary proteins in our study might be nonspecific to childhood caries, they seem to demonstrate an abnormal oral condition of those young children susceptible to dental caries. There are still more work to further investigate the mechanism of salivary biomarkers for dental caries in a larger sample size, and translate them from the laboratory level into the clinical practice.
Conclusions
The present study utilized iTRAQ/MRM methodology to characterize salivary components and their interactions, and constructed the comparative proteomics map for childhood dental caries, which increases knowledge about the salivary proteins functioned in this oral disease. Specifically, some key screened proteins, such as protein S100 A9, mucin 7, mucin 5B, statherin, histatin 1, cystatin S, cystatin SN and basic salivary proline rich protein 2, are worth studying for validation in a larger sample size in future studies. And these differentially expressed proteins in whole saliva with potential anti-cariogenic function in this study can be useful for drawing up caries-preventive agents for individualized preventive strategies in the future.
Authors’ contributions
KW collected the samples, supervised the overall experiment, and drafted the manuscript. YFW, XQW, QR and SLH helped collect samples and draft the manuscript. LJD and ZCL participated in the data processing and design of the study. KW, XDZ, WL and LLZ conceived of the overall study and participated in the study design, coordination and revision of the manuscript. All authors read and approved the final manuscript.