The secretory capacity of salivary gland (SG) is constantly challenged by physiological requirements as well as pathological perturbations to synthetize and fold membrane and soluble proteins of the secretory pathway. Labial SGs (LSG) from Sjögren's syndrome (SS) patients show a progressive disorganization that compromises the morphological and functional integrity of acini, ducts, and extracellular matrix [1]. Evidence suggests that altered epithelial cell homeostasis could be the initiating event leading to secretory dysfunction, autoimmunity, inflammation, and tissue damage [2]. The endoplasmic reticulum (ER) plays a pivotal role in the secretory pathway, modulating protein folding and quality control mechanisms [3]. This process is important because it allows a cell to successfully complete the synthesis of a protein. Under normal conditions, folding, quality control, and degradation mechanisms maintain protein homeostasis (proteostasis) [3]. Emerging studies on physiological and pathophysiological contexts claim that the ER also detects changes in other organelles through dynamic and transient membrane contact; orchestrating various cellular responses through signaling pathways between organelles [4]. When protein synthesis requirements exceed the ER folding capacity, many unfolded and misfolded proteins are generated, proteostasis is disturbed, and the ER activates an adaptive mechanism known as the unfolded protein response (UPR) to recover proteostasis. Alterations in metabolic and calcium homeostasis, inflammation, and oxidative stress can trigger it [5]. LSG from SS patients suffer chronic ER stress, evidenced by MUC1 accumulation in the ER [6], dilated ER cisterns [1] and altered levels of UPR components, such as activating transcription factor-6 (ATF6α) [7] and X box-binding protein 1 (XBP-1) [8].

ATF6α sensor is a type II transmembrane glycoprotein that dissociates from GRP78 chaperone during ER stress and traffics to the Golgi apparatus where it is processed by S1P and S2P proteases to release the N-terminal portion of ATF6α (ATF6f). ATF6f translocates into the nucleus as a transcription factor to induce expression of ER protein-folding chaperones and enzymes. Specifically, ATF6f binds to ER stress response (ERSE) element, which acts on the promoter of target genes such as ER chaperone target genes (GRP78, HSP90b1, and CALR), DDIT3, and XBP-1 [9], and proteins participating in ER-associated protein degradation (ERAD) [10]. The ERAD involves the recognition, retrotranslocation, ubiquitination, and degradation of substrates by the 26S proteasome, in which SEL1L (Suppressor Of Lin-12-Like Protein 1) and HERP (homocysteine-inducible ER protein with ubiquitin-like domain 1) participate, two direct targets of ATF6. SEL1L, is an adapter protein that participates in the retrotranslocation of ERAD substrates and HERP is an ER membrane scaffold protein essential for the recognition of ER glycoproteins. ERAD is essential to maintain optimal levels of proteins that regulate physiological and pathological processes [9]. Increased ATF6, SEL1L and HERP expression observed in LSG from SS patients were also observed in IFN-γ-stimulated 3D-acini [7], suggesting that IFN-γ regulates the expression of UPR and ERAD components. This is in line with accumulating evidence that shows the involvement of interferons (IFNs) in both the initiation and progression of SS [11,12]. In addition, IFN-γ induces increased MUC1 levels, a mucin that accumulates in the ER of acinar cells of SS patients [6], as IFN-γ activates STAT1, which interacts with STAT elements of the MUC1 promoter and induces its overexpression [13]. The increase in SEL1L and HERP is attributed to increased ATF6α signaling pathway activity, because both are target of ATF6f [14,15].

The IRE1α/XBP-1s signaling pathway is critical for specialized secretory cell functions that regulate the biogenesis of secretory organelles and lipid metabolism [16]. The processed XBP-1 mRNA encodes a potent transcriptional factor, XBP-1s, which translocates to the nucleus and induces the expression of a set of genes including ER chaperones (Dnajb9, Dnajb11, PDIA3, and Dnajc3), ERAD components (EDEM1, HERP, and HRD1), and foldases (PDIA6), among others as well as the ER translocon (SEC61a1) [17,18] to control various cellular functions, including lipid metabolism, biogenesis of important compartments of the secretory pathway, biosynthesis of glucose and immune responses [16]. XBP-1s and IRE1α transcript and protein levels were reduced in LSGs of SS-patients and IFN-γ-stimulated 3D-acini, partially explaining the secretory dysfunction of SS-patients as well as the role of IFN-γ in this process [8].

Differential expression of the UPR components could result from different regulatory mechanisms, such as hypomethylation of the ATF6 promoter and hypermethylation of the IRE1α and XBP-1s promoters [8,19]; other mechanisms may also be involved. However, it should keep in mind that DNA methylation can act together or separately with other mechanisms to regulate gene expression [20,21]. Therefore, this new approach to integrate microRNAs (miRNAs) and DNA methylation provides an integrative perspective that allows exploring the multiple mechanisms of regulation of gene expression [21]. microRNAs are evolutionarily conserved small non-coding RNA (sRNA) molecules that modulate gene expression by binding to target messenger RNAs (mRNAs), causing targeted mRNA decay or blocking translation [22]. Dysregulated miRNA expression may result from genetic and epigenetic variants and various environmental factors that play crucial roles in the pathogenesis of autoimmune diseases [23], including SS. Interestingly, there is an association between the expression of UPR pathway components with miRNAs [24], classifying them as proadaptive or proapoptotic miRNAs [25,26]. However, the contribution of miRNAs that target UPR molecules is unknown in LSG of SS patients. miRNA studies in SS patients mostly involve the use of peripheral blood mononuclear cells (PBMC) [[27], [28], [29], [30], [31], [32], [33], [34]], however, miRNAs in SG epithelial cells have been less studied in SS, despite the importance of these cells in the SS etiopathogenesis [[35], [36], [37], [38]]. Microarray or next generation sequencing (NGS) of miRNAs from SS-patients show downregulation of hsa-miR-424–5p and overexpression of hsa-miR-513c-3p [39,40], which both have UPR targets [41]. These miRNAs emerged as candidates that would regulate ATF6, SEL1L and XBP-1 and GRP78 levels. The present study focuses on the involvement of hsa-miR-424–5p and hsa-miR-513c-3p in regulating ATF6/SEL1L and XBP-1s/GRP78 levels, respectively, and their participation in mechanisms that ensure proteostasis in SS patients.