Molecular mechanism and diagnostic marker investigation of endoplasmic reticulum stress on periodontitis

Periodontitis is a bacterial disease modified by multiple risk factors [1], which influence about 50% of the world's population [2]. Periodontitis usually shows few or only mild symptoms over many years, which are often not perceived or correctly classified by the patient [3]. The previous study has reported about the intricate molecular mechanism underlying this periodontitis [4]. However, the specific genes, cells and cellular mechanisms that may be involved in the pathogenesis of the disease are unknown [5], which further leads to the unsatisfactory clinical treatment and prognosis.

Changes at the cellular or molecular level are important factors in the development of diseases. Gaetano Isola et al. indicated that miRNAs such as miRNA 21-3p is closed associated with the risk of periodontitis [6]. The mRNAs such as transglutaminase genes change the response to chronic injury in the damaged gingival [7]. It is known to all that when under the action of various strong stimulators, cells will initiate a series of self-protection events including endoplasmic reticulum stress (ERS). A previous study shows that ERS triggered apoptosis that participates in the biological function of periodontitis on vascular calcification via the activation of the CHOP transcription pathway [8]. Periodontitis-related inflammation gave rise to the upregulated expression levels of ERS-related genes (ERSGs) including GRP78, PERK, ATF4, and CHOP, which further indicated that ERS might act as a promising therapeutic target against periodontitis [9]. Based on a bioinformatics analysis, Zhang et a. showed that three ERSGs including SERPINA1, ERLEC1 and VWF display the potential biomarkers of human disease, providing a target basis for diagnosis and therapy of periodontitis [10]. Actually, bacterial infection and immune-inflammatory process of the human body has played an indelible role during this process [11]. ERS can induce immune or inflammatory response in human disease process through cross talk with specific signaling pathways [12]. In addition, studies have proven that upregulated ERSGs including HSP60 in periodontitis participates in the immune response process [13]. Even though the ongoing research has further clarified the extensive relationship between periodontal disease and ERS, small parts of the puzzle remain a mystery and require further investigations.

In the present study, based on differentially expressed ERSGs (DE-ERSGs), the subtypes of periodontitis were explored, followed by periodontitis diagnostic markers investigation. Finally, a miRNA-gene interaction network was constructed. This study innovatively explored the functions of ERGs that may be related to diseases based on bioinformatics analysis. The overall design was clear and feasible. And we aimed to investigate the molecular mechanism of ERSGs in the progression of periodontitis, and provide potential ERS diagnostic markers for periodontitis.

The flow chart in current study was showed in Additional file 1: Fig. S1.

Although ERS has been revealed to be vital for the development of periodontitis, the detail molecular mechanism and valuable biomarkers based on ERS is still unclear. In this study, we explored 2 reliable periodontitis subtypes based on DE-ERGs between disease samples and normal samples. Then, totally 7 ERS diagnostic markers including ATP2A3, XBP1 and FCGR2B were further revealed from DE-ERGs by using two machine learning algorithms, followed by verification analysis on these markers. Finally, a miRNA-target gene interaction network was constructed to investigate the relation of ERGs and miRNA in the periodontitis. All these results suggested that ERGs might play vital role in the progression of periodontitis.

Protein processing, modification and folding in the ER are closely related regulatory processes that determine cell function, fate and survival [30]. In human diseases, different carcinogenic, transcriptional, and metabolic abnormalities combine to create an adverse microenvironment that disrupts ER homeostasis in malignant, stromal cells and infiltrating leukocytes [31]. These changes trigger a constant state of ERS characterized by the accumulation of misfolded or unfolded proteins [32]. A previous study shows that the differentially expression of ATP2A3 (ATPase Sarcoplasmic/Endoplasmic Reticulum Ca²⁺ Transporting 3, also called SERCA3) induces unfolded protein response in human lymphoma cells [33]. It has been proved that ATP2A3 overexpression can trigger ERS and changes of intracellular Ca²⁺ management to exert anti-cancer effects in various cancers [34, 35]. Since inflammation is associated with abnormal of ATP2A3 regulated Ca2⁺ levels, the host inflammatory and immune response ultimately leading to irreversible destruction of the periodontium [36, 37]. During the process of periodontitis, the periodontitis-related miRNA-gene regulatory network play an important role [38]. Actually, various miRNAs are found to be involved in the pathogenesis of periodontitis, leading to the tooth loss in adults [39]. A previous study proves that miR-671-5p take part in the inflammation and extracellular matrix degradation of chondrocytes [40]. Lien et al. indicated that the ability to suppress macrophage-mediated inflammation was controlled by miR-671-5p [41]. Via sponging miR-671-5p, circCDR1 is proved to promote autophagy and ERS in human tumor cells [42]. However, the detail effect of miR-671-5p and ATP2A3 in periodontitis is still unclear. In the current study, the ERSGs ATP2A3 was one of common genes related to periodontitis revealing by two machine learning algorithms. Meanwhile, the miRNA-target gene interaction network analysis showed that miR-671-5p was targeting with the up-regulated ERSGs ATP2A3. Thus, we speculated that the overexpression of miR-671-5p might take part in the progression of periodontitis via stimulating the expression of ERSGs ATP2A3.

ERS markers has been proved to play vital role in the progression the human disease [43]. And the inhibition of ERS-related genes presents therapeutic effects on periodontitis [44]. In this study, we revealed several DE-ERGs between Affected and Unaffected group, which were used to cluster all periodontitis samples into two reliable subtypes according to the results of immune infiltration and GSEA analysis. To further reveal the reason for different outcome of enrolled samples, totally 7 ERS diagnostic markers including XBP1 and FCGR2B were finally revealed.

XBP1 (X-Box Binding Protein 1) is a key effector molecule in UPR, which is necessary to save ERS and promote cell survival [45]. XBP1 is central requirements for plasma cell development, and high expression levels of XBP1 at diagnosis predict poor OS [46]. The role of XBP1 in the protective unfolded protein response to limit ERS and damage in human been revealed [47]. A previous study shows that XBP1 is up-regulated in treatment group than control of human periodontal ligament cells [48]. It has been proved that the ERS sensor XBP1 is activated and regulated transcription, and inhibiting XBP1 in CD8 + T cells effectively restore antitumor activity [49]. Due to the critical function for XBP1 in mammalian host defenses, XBP1 is considered as important biomarker in human disease [50]. Moreover, it has been proved that anti-inflammatory agent such as tacrolimus determined a more effective improvement when compared with anti-inflammatory mouthwash [51]. Some genes such as transglutaminases play important roles in the pathological mechanisms of autoimmune, inflammatory and degenerative diseases [7, 52].

FCGR2B (Fcγ receptor IIb), which can reduce the effectiveness of antibody immunotherapy, is an inhibitory molecule [53]. A previous study focus on inflammatory mediator polymorphisms associate with initial periodontitis in adolescents revealed the relation between FCGR2B variation and disease [54]. It is proved that higher FCGR2B expression was associated with significantly shorter progression-free survival in patients with diffuse large B-cell lymphoma, which indicating the diagnostic signature of FCGR2B [55]. In this study, AUC area under the diagnostic ROC curve of XBP1 and FCGR2B was more than 0.75, and the expression of all diagnostic genes were significantly different between Affected group and Unaffected group, indicating an ideal diagnostic effect for XBP1 and FCGR2B. Meanwhile, the correlation analysis between immune cell and diagnostic markers showed that both XBP1 and FCGR2B were significantly correlated with plasma cells, indicating the vital role of these ERS markers in periodontitis. Thus, we speculated that ERSGs including XBP1 and FCGR2B might be novel ERS diagnostic marker for periodontitis. However, there were some limitations in this study including small samples size and lack of clinical verification analysis. Thus, a further verification analysis based on a large sample size is needed.

The up-regulated miR-671-5p might take part in the progression of periodontitis via stimulating the expression of ERSGs ATP2A3. ERSGs including XBP1 and FCGR2B might be novel ERS diagnostic marker for periodontitis.