Saliva is mainly secreted from three major salivary glands namely the SMGs, parotid glands and sublingual glands. In humans, approximately 60% of resting saliva and 40% of stimulated saliva is secreted from the SMGs [29]. The SMGs are an important for maintaining saliva secretion and oral health [30]. Notably, the SMGs are more sensitive to changes of physiological and metabolic changes in the body. Therefore, damage to SMGs is related to a variety of diseases. For example, the destruction of SMGs acinar tissue leads to a decrease in saliva secretion in patients with Sjogren’s syndrome [31]. However, studies focusing on diabetes-induced damage to the SMG are very limited. Therefore, the present study collected SMG tissues of db/db mice and db/m mice for microarray analysis in order to investigate the underlying mechanisms of lncRNAs in diabetes-induced hyposalivation. Compared with db/m mice, 536 upregulated and 737 downregulated lncRNAs were identified; these differentially expressed lncRNAs may play an important role in diabetes-induced hyposalivation.
The present study performed RT-qPCR validation on 11 lncRNAs with the most significantly dysregulated expression levels. From this validation, the results of five lncRNAs were consistent with those of high-throughput sequencing. We previously determined the whole genome expression profile of SMG tissues in db/db mice and found that 1,146 mRNAs exhibited a significantly dysregulated expression; of these, 606 mRNAs were upregulated and 540 mRNAs were downregulated [32]. LncRNAs are regarded as the primary section of the CNC network, particularly the ceRNA network. However, the functional roles and regulatory mechanisms of the CNC and ceRNA networks in diabetes-induced hyposalivation remain unknown. Therefore, the present study used these five lncRNAs to perform CNC and ceRNA network analyses in combination with 1,146 dysregulated mRNAs.
GO analysis of mRNA obtained from the CNC network of five lncRNAs showed that “Calcium ion binding” is highly enriched in the molecular function. The mobilization of Ca2+ plays an important role in salivary secretion, the activation of muscarinic cholinergic receptors rapidly triggers the release of intracellular Ca2+ from the endoplasmic reticulum and subsequently the influx of Ca2+ from the extracellular medium, resulting in a sustained increase in intracellular Ca2+ [33, 34]. The increased intracellular Ca2+ induces the transport of aquaporin 5, leading to the formation of water pores and thus promoting a rapid increase in transcellular water permeability [35]. In addition, a study found that adiponectin can also induce salivary secretion of the db/db mouse by activating adenosine monophosphate-activated protein kinase and the Ca2+ signaling pathway played an important role in this process [36]. In the human and rabbit SMGs, the activation of muscarinic cholinergic receptors and transient receptor potential vanilloid subtype 1 increased salivary secretion via increased intracellular Ca2+ [37, 38]. In patients with epiphora following human transplanted epiphora SMG transplantation, t the elevated intracellular Ca2+ mobilization induced by muscarinic acetylcholine receptors activation contributed to hypersecretion [37]. In spontaneously hypertensive rats, the damaged Ca2+ response to carbachol was confirmed in acinar cells of spontaneously hypertensive rats, which may also be related to the reduced salivary secretion caused by hypertension [39]. These studies indicate that the increased intracellular Ca2+ derived from extracellular and intracellular Ca2+ plays an important role in the salivary secretion of SMGs. In the present study, the results from microarray analysis showed that significantly altered “calcium ion binding”, suggesting that the salivary secretion from the SMGs may be also affected by the Ca2+ signaling pathway during diabetes, and this process may be regulated by these five lncRNAs. However, the regulatory mechanisms of the related lncRNA-mRNA interaction require further research.
LncRNAs regulate mRNAs via various mechanisms, one of which is ceRNA-mediated changes in the expression of downstream molecules regulated by miRNAs. Therefore, the present study performed ceRNA network analysis on five significantly altered lncRNAs and 1,146 dysregulated mRNAs to identify the related pathways regulated by the miRNA pathway. In addition, the obtained mRNAs of the ceRNA network were analyzed by GO and KEGG analyses. KEGG analysis revealed that the “mTOR signaling pathway” was significantly enriched, suggesting that these five lncRNAs may affect the downstream “mTOR signaling pathway” through ceRNAs. The phosphatidylinositol 3-kinase (PI3K)/protein-serine-threonine kinase (Akt)/mTOR pathway is an intracellular signaling pathway that plays a key role in regulating cell cycle-mediated processes, including cellular quiescence and cell proliferation [40], as well as various disease such as epithelial ovarian cancer [41]. It has been demonstrated that the “mTOR signaling pathway” plays an important role in the pathophysiological process of diabetes. In MC3T3-E1 cells, high glucose levels have been shown to increase the production of reactive oxygen species and induced autophagy by inhibiting the Akt /mTOR pathway [42]. The mTOR/PI3K/Akt pathway is involved in the regulation of autophagy in diabetic kidney diseasde [43]. The PI3K/Akt/mTOR pathway has also been shown to be significantly downregulated in the brains of rats with streptozotocin-induced type 2 diabetes; this may explain the neurodegeneration commonly observed in diabetes [44]. In addition, the mTOR pathway is associated with the process of salivary secretion. Bleomycin has been shown to induce the epithelial-to-mesenchymal transformation of human SMG cells via the Akt/mTOR pathway [45]. In our previous studies, it was found that autophagy induced aquaporin 5 degradation through the PI3K/Akt/mTOR pathway signaling pathway, resulting in a decreased salivary secretion from the SMG in db/db mice [9]. These studies suggest that the “mTOR signaling pathway” may play an important role in SMGs injury caused by diabetes. The five lncRNAs we verified may involve in the regulation of the “mTOR signaling pathway” through the ceRNA mechanism via miRNA sponging, but further research is needed.
Currently, it is difficult to rapidly and efficiently study the association between lncRNAs and diseases by relying only on traditional biological experiments. With the development of high-throughput sequencing technology and bioinformatics, the application of digital techniques and large data in the medical field is becoming increasingly extensive. It is very effective for the diagnosis, treatment of diseases and the improvement of the quality of life of patients. Liu et al [46] found eight lncRNAs, including WDFY3-antisense (AS)2, cancer susceptibility 8 and UGDH-AS1, which were associated with the overall survival of the patients with esophageal cancer using RNA-sequencing and bioinformatics analysis; their study provided a new direction for the search for novel molecular therapeutic targets and prognostic markers for esophageal cancer. By constructing a lncRNA-miRNA-mRNA ceRNA network and analyzing the ceRNAs in the development of cervical squamous cell carcinoma, Song et al [47] found two novel lncRNAs, ADAMTS9-AS2 and MEG3, which may play important roles in the pathogenesis of cervical squamous cell carcinoma. lincRNA-p21 has been identified as a potential novel prognostic biomarker for the prognosis of diffuse large B cell lymphoma, and has been shown to regulate cell proliferation and the cell cycle in vitro [48]. The application of bioinformatics technology and large data may be effective for the identification of potential lncRNA functions and lncRNA-disease associations, hence decreasing the time and cost of biological experiments. In the present study, microarray analysis was performed using SMG tissues, followed by bioinformatics analysis. This may aid in the rapid and efficient identification of lncRNAs associated with hyposalivation in patients with diabetes.