In a recent miRNA study [9], serum samples of systemic sclerosis (SSc) patients (n = 26) were screened for 758 miRNAs, of which 30 miRNAs were identified as differentially upregulated miRNAs. Among these 30 miRNAs, miR-483-5p showed higher expression level in an independent SSc cohort, especially in early stages of SSc. The miRNA was also up-regulated in localized scleroderma, but not in systemic lupus erythematosus (SLE) or primary SjS (pSjS), indicating that this miRNA possibly contributes to the development of skin fibrosis. Furthermore, functional assays involving miR-483-5p overexpression in fibroblasts and endothelial cells revealed altered expression of fibrosis-related genes. This provides strong evidence that the serum miRNA miR-483-5p may function as a potential regulator of fibrosis in SSc.

Peripheral blood mononuclear cell (PBMC) miRNA profiles in SLE (n = 8) were compared with those of pSjS (n = 8) and healthy controls (HC) (n = 7) by the Illumina next-generation sequencing technology (NGS) [10]. A total of 135 miRNAs were identified as differentially expressed in SLE compared to only 26 miRNAs in pSjS. Of 26 miRNAs differentially-expressed in pSjS, 25 miRNAs were also commonly overexpressed in SLE as well. Downregulation of miR-150-5p was only detected in pSjS, which appears to be associated with differential ratios of B cells in comparison with SLE [11]. Several miRNA molecules were overexpressed in SLE, but not changed in pSjS, which include miR-148a-3p, miR-152, miR-155, miR-223, miR-224, miR-326, and miR-342. Interestingly, expression levels of miR-223-5p, miR-150-5p, miR-155-5p and miR-342-3p were associated with the B cell subsets, such as native B cells or switched memory B cells in the PBMC samples. As B cell hyperactivity is characteristic in both pSjS and SLE, the miRNA expression signature may serve to dissect the pathological B cell regulation between those two conditions.

MiRNAs in synovial fluids and plasma in rheumatoid arthritis (RA, n = 40), osteoarthritis (OA, n = 38), and HC (n = 30) were analyzed by real-time quantitative reverse transcription-PCR (qRT-PCR) [12]. Synovial fluid concentrations of miR-16, miR-132, miR-146a, and miR-223 were significantly lower than their plasma concentrations, and there were no correlations between plasma and synovial fluid miRNAs. Interestingly, synovial tissues, fibroblast-like synoviocytes, and mononuclear cells secreted miRNAs in distinct patterns. Plasma miR-132 was significantly higher in HC than in RA or OA with high reliability, while synovial fluid concentrations of miR-16, miR-146a, miR-155, and miR-223 were significantly higher in RA than in OA. Plasma miRNAs or the ratio of synovial fluid miRNAs to plasma miRNAs, including miR-16 and miR-146a, significantly correlated with disease activity. Therefore, the authors proposed that synovial fluid and plasma miRNAs may serve as diagnostic biomarkers for RA and OA.

Whole sera in early rheumatoid arthritis (ERA) patients (n = 34) were compared with established RA (n = 28) and HC (n = 16) in an analysis of miR-146a, miR-155, miR-223, miR-16, miR-203, miR-132 and miR-124a expression by TaqMan qRT-PCR [13]. The levels of circulating miR-146a, miR-155 and miR-16 were decreased in the sera of ERA patients in comparison with established RA. Levels of circulating miR-223 in treatment naive ERA patients correlated with disease activity. However, neither miR-16 nor miR-223 could distinguish ERA from HC. The authors concluded that circulating miR-146a, miR-155, and miR-16 in the sera of ERA could indicate an early stage of the disease.

A recent study explored the correlation between miR-200c and the severity of interstitial lung disease (ILD) in patients (n = 218) with connective tissue diseases (CTDs) [14]. PBMCs were acquired from SSc (n = 23), dermatomyositis/polymyositis (DM/PM, n = 29), pSjS (n = 30), RA (n = 47), and HC (n = 23) for qRT-PCR analyses. The results indicated that the miR-200c level in the SSc group was significantly higher than in the DM/PM, pSjS, and RA groups. The level of miR-200c in the CTDs with ILD group was significantly higher than in the CTDs without ILD group, and the level in the severe ILD group was significantly higher than in the mild ILD group. The authors concluded that the level of miR-200c was positively correlated with the severity of ILD, proposing miR-200c in PBMCs as a biomarker of the severity of ILD in CTDs.

Microarray analyses have proven to be valuable in determining differentially-expressed miRNAs in the field. One of the first studies by Alevizos et al. [15] utilizing miRNA microarray for miRNAs in SjS MSG revealed a correlation between relative expression of selected miRNAs and the MSG focus score (FS). Among those miRNAs, miR-768-3p showed a positive correlation whereas miR-574 showed a negative correlation with FS, which was validated as markers of inflammation by qRT-PCR in an independent cohort. In addition, comparing miRNAs from patients with preserved vs. low salivary flow identified nine of differentially expressed miRNA, seven of which were up-regulated in the group with decreased salivary gland function. This suggests that the predicted targets of upregulated miRNA, which was associated with salivary dysfunction, may have a protective effect on epithelial cells.

Another comparative microarray analysis of miRNA expression in the MSGs of SjS and HC revealed distinctive miRNA signatures in SjS patients, associated with glandular inflammation and dysfunction [16]. MiRNAs that were predicted to target Ro/SSA and La/SSB autoantigens were upregulated in the MSG tissues (miR-181a), salivary gland epithelial cells (SGEC) (miR-200b) and PBMC (miR-223) of SjS in comparison with HC. miR-181a and miR-16 overexpression in MSG tissues of SjS patients has been previously shown to be associated with salivary dysfunction [15]. Interestingly, down-regulated expression of let-7b miRNA was observed in SGECs of SjS patients who were positive for Ro/SSA and/or La/SSB compared to seronegative patients. The authors hypothesized that the increased expression of miR-181a, miR-200b and miR-223 miRNAs in SjS may represent a negative feedback loop for the control of the elevated Ro/SSA and La/SSB expression and/or their inefficiency to regulate target expression.

In a subsequent study in 2015 by the same group, the authors further investigated 29 SjS patients (20 of 29 positive for anti-Ro/SSA and anti-La/SSB) and 24 non-SjS sicca controls to study possible associations between autoantigen mRNA expression and disease features [20]. The upregulated levels of miR-16 in MSGs, miR-200b-3p in SGECs, and miR-223 and miR-483-5p in PBMCs of SjS patients compared to non-SjS sicca controls were identified. Interestingly, the levels of miR-16 in MSGs and miR-483-5p in PBMC of SjS were not significantly altered in their previous study [16]. This may be due to the heterogeneous clinical features of SjS, resulting in subtle differences in the subjects recruited for those two studies. Nonetheless, the MSG and PBMC levels of let-7b, miR-16, and miR-181a were correlated positively with Ro52/TRIM21-mRNA. Significantly lower miR-200b-5p levels were also detected in SjS patients with mucosa-associated lymphoid tissue lymphoma compared to those SjS without lymphoma. The exact roles of dysregulated miRNAs in their study in promoting autoantigen expression and autoantibody production would be interesting to follow-up.

Recent high-throughput approaches such as NGS have been applied to sequence entire genomes or transcriptomes within days. This technology has yielded exciting discovery of unknown genes or small miRNAs with a challenge of mining large amounts of data. A study utilizing NGS revealed several previously unidentified miRNAs in the MSG of pSjS patients which were then validated in other cell lines [21]. The authors found hsa-miR-4524b-3p and hsa-miR-4524b-5p and hsa-miR-5100 particularly interesting to further pursue. Although the results were based on a relatively small number of patient samples, potentially due to cost-intensive NGS, from whole MSG biopsy specimens, thus including a mix of cells, the study showed a promising feasibility of discovering novel miRNAs by NGS.

A number of important contributions to the field of miRNA research have been made by identifying the functions of differentially-expressed miRNAs. An innovative study focusing on Epstein Barr virus (EBV) miRNA, ebv-miR-BART13-3p, indicated the presence of the miRNA in both B cells and SGEC in pSjS salivary glands [22]. As EBV typically infects B cells, the authors further demonstrated that ebv-miRBART13-3p can be transferred from B cells to SGEC through exosomes and that it targets stromal interacting molecule 1 (STIM1) and aquaporin 5 (AQP5), which are important regulators of salivary secretion via the Ca²⁺-mediated signaling pathway. The data suggest a mechanism by which EBV can contribute to secretory dysfunction in SjS. This might also explain why the degree of immune cell infiltration and secretory dysfunction are not always correlated in SjS. Additionally, the role of EBV in SjS pathogenesis needs to be revisited since it has been underestimated due to inconsistency in the prevalence, localization, or expression levels of virus in patients [26–30].

A recent study by Martini et al. investigated miR-126 and miR-335-5p in MSG biopsies and their known target molecule, cystatin S [23]. The results indicated that salivary cystatin S was significantly decreased in pSjS (n=40) vs. HC (n = 20), especially in those with reduced secretory function. A positive correlation was observed between salivary cystatin S and unstimulated flow rate. Salivary cystatin S was also significantly reduced in patients with a submandibular gland ultrasonography score ≥2. Interestingly, the expression levels of miR-126 and miR-335-5P increased inversely with salivary flow rate. The authors proposed cystatin S as a promising biomarker for pSjS as it strongly correlated with glandular dysfunction. The cystatin S transcript did not change significantly, suggesting posttranscriptional regulation.

We have shown that both miR-146a and miR-155 were up-regulated in response to the adaptive immune response in PBMC derived from SjS when testing humans and mice [19]. In this study, miR-146a expression in SjS patients as well as in the SjS-prone C57BL/6.NOD-Aec1Aec2 mouse model was investigated to elucidate its involvement in SjS pathogenesis. Expression of miR-146a was significantly increased in SjS patients (n = 25) compared with HC (n = 10), and was upregulated in the salivary glands of the SjS-prone mouse at both 8 weeks (prior to disease onset) and 20 weeks (full-blown disease) of age, and in the PBMC at 20 weeks of age. More importantly, functional analysis revealed the roles for miR-146a in increasing phagocytic activity and suppressing inflammatory cytokine production (tumor necrosis factor α [TNF-α], interleukin [IL]-1β, macrophage inflammatory protein [MIP]-1α, and IL-6), whereas cell migration, nitric oxide production, and expression of antigen-presenting/costimulatory molecules were not affected in human monocytic THP-1 cells. Our data suggest that abnormal expression/regulation of miRNAs in innate immunity may contribute to, or be indicative of, the initiation and early stage of SjS [19].

MiR-146a is known to be activated by nuclear factor κB (NF-κB), which controls Toll-like receptor (TLR)/IL-1 receptor signaling pathway through TNF-associated factor 6 (TRAF6), IL-1 receptor-associated kinase (IRAK1), the signal transducer and activator of transcription 1(STAT1), and IRF5 [31]. It is important to mention that the expression, the roles, and the target gene regulation of miRNA146a/b need to be interpreted in the context of a specific disease entity as its regulatory mechanisms appear to be more complex than initially anticipated. For instance, in the PBMC of RA patients, the overexpression of miR-146a was found without any change in its target genes [31]. However, in SLE, the expression of miR-146a was decreased with increased IRAK1 expression and unchanged TRAF6 [32]. In a study by Zilahi et al. [33], the authors have found elevated miR-146a/b in the PBMC of SjS with down-regulated IRAK1 and upregulated TRAF6, detected by RT-PCR. Because of these findings, Jacob et al. [34] hypothesized that TRAF6 could be specific for SjS while IRAK1 could be regarded as a risk gene for SLE. They previously reported a multiple defect (decrease) in the expression of various protein kinase C isoenzymes (included PKC) in the monocytes and T cells of SLE patients, but not existing in SjS [10]. Therefore, the authors proposed that TRAF6 contributes to the activation of the NF-κB pathway by the involvement of PKC, which exists normally in the disease, and TRAF6 gene could be a new biomarker of SjS [33].

Another study discovered changes in miRNA expression profiles in the PBMC of patients with SLE (n = 8), pSjS (n = 8), and HC (n = 7) by the Illumina NGS technology [10], which was mentioned earlier for SLE in this review. This study by Chen et al. [10] is interesting in the sense that miR-146a was overexpressed in SLE patient PBMC, which is inconsistent with the finding by Tang et al. [32] that showed downregulated miR-146a in the SLE PBMC. Chen et al. [10] hypothesized that this inconsistency may be due to a difference in the ethnic backgrounds of participants in the two studies; Chen et al.’s study [10] involved Europeans and Tang et al.’s study [32] involved Chinese. The authors also indicated various external factors as a potential reason for the discrepancy, such as dietary habits, exposure to different infectious agents, and other environmental elements, which may have influenced miRNAs expression level in the PBMC of the patients [35, 36].

In a recent study by Peng et al. [24], several miRNAs with a viral origin including miR-181a were shown to be upregulated in PBMC of patients with SjS. MicroRNA profiling by microarrays in Chinese patients with pSjS revealed elevated miRNA 181a in the PBMC [24]. MiRNA expression was initially profiled in female patients (4 pSjS and 3 HC), followed by a large cohort of patients (n = 33) and HC (n = 10). Among the 202 miRNAs that were upregulated and 180 miRNAs that were downregulated in the patients with pSjS, the authors confirmed that miR-181a was the most profoundly differentially expressed in pSjS in comparison with HC. The authors speculate that elevated miR-181a levels in the PBMC of these patients compromise the maturation of B cells, thus enabling them to recognize and attack autoantigens and resulting in disease phenotypes [24]. Interestingly, many virus-derived miRNA were unexpectedly upregulated in the SjS patients, suggesting that viral infection of PBMC may play an important role in SjS.

Although the sources and compositions of the cells were different, the detection of viral miRNAs in PBMC by Peng et al. [24] was in line with the study investigating the salivary glands by Gallo et al. [22]. The former showing the presence of EBV miRNAs and the upregulation of miRNA-181a in B cells, where EBV establishes cell tropism, provides a convincing scenario that viral miRNAs may drive the expression of host miRNAs in a cell-specific manner. The latter study, mentioned earlier, demonstrated that ebv-miR-BART13-3p was present in both B cells and salivary epithelial cells in pSjS salivary glands. The authors further demonstrated the transfer of ebv-miR-BART13-3p from B cells to salivary epithelial cells through exosomes, suggesting a possibility of its role in compromising calcium signaling by targeting STIM1 and consequent secretory dysfunction. The establishment of the interplay between viral miRNA and host miRNA will provide the exact etiological and pathological link between viral infection and SjS.

Monocytes represent a relatively homogenous population of important precursor cells to peripheral macrophages and dendritic cells [18]. Monocytes have innate immune functions including immunomodulation, cytokine production, and phagocytosis [37]. In SjS, monocytes secrete pro-inflammatory cytokines such as IL-6 and B cell-activating factor (BAFF) upon stimulation [38], express type I interferon (IFN)-regulated genes [39, 40] with reduced expression of NFκB inhibitor (IκBα) [41], and show decreased phagocytosis of apoptotic cells [42]. Thus, monocytes reflect the inflammatory state in SjS patients and mature monocytes are proposed to contribute to salivary gland inflammation in SjS [43].

In our study published in 2016, monocytes purified from pSjS (n = 18) blood samples were compared by miRNA microarray with those purified from other disease controls of RA (n = 10), SLE (n = 10), and HC (n = 10) [18]. To validate selected miRNAs from the microarray analysis, the microarray cohort and a new cohort of monocyte RNA samples from 9 HC, 12 SjS, 8 SLE, and 9 RA patients were evaluated by qRT-PCR. We demonstrated that miR-34b-3p, miR-4701-5p, miR-609, miR-300, miR-3162-3p, and miR-877-3p were differentially expressed in 43% of pSjS patients compared to 5.8% and 5.6% for SLE and RA, respectively. Interestingly, the canonical transforming growth factor β (TGF-β) signaling pathway showed the greatest coverage of predicted targets, averaging 40.5% ± 23.1% pathway coverage for the six SjS-associated miRNAs. This suggests that these miRNAs preferentially target the canonical transforming growth factor β (TGFβ) signaling pathway as opposed to pro-inflammatory IL-12 and Toll-like receptor/NFkB pathways in SjS pathogenesis [18].

As our study was limited to RNA analyses, relationships between miRNAs and their post-transcriptional inhibition of target genes at the protein level need to be addressed as soon as sufficient human monocytes are collected. Nevertheless, SMAD4 gene expression exhibited repression in a subpopulation of pSjS patients by our SjS-associated miRNAs [18]. The SMAD4 protein of the family of TGFβ signaling molecules acts as an essential common mediator for receptor-regulated SMADs to enter the nucleus. Interestingly, miR-300 and miR-609 showed the strongest co-association by regression analyses and target prediction analyses, which indicates that SMAD4 may be targeted by SjS-associated miRNAs in a cooperative manner. To our knowledge, this is the only study where SjS monocytes were analyzed for miRNA profiling.

Dysregulated miRNA expression in purified T and B lymphocytes from patients with pSjS was reported this year [25]. The authors purified CD4＋ T cells and CD19＋ B cells from PBMC of two independent cohorts. Cohorts of pSjS and HC for discovery (n = 17 and n = 15, respectively) and another cohort for replication (27 for T cells and 25 for B cells for pSjS and 12 HC, respectively) were included in the study. The Exiqon system containing 372 miRNAs was utilized. In T lymphocytes, the study showed upregulation of haslet-7d-3p, hsa-miR-155-5p, hsa-miR-222-3p, hsa-miR-30c-5p, hsa-miR-146a-5p, hsa-miR-378a-3p and hsa-miR-28–5 p in both the discovery and the replication cohorts. In B lymphocytes, hsa-miR-378a-3p, hsa-miR-222-3p, hsa-miR-26a-5p, hsa-miR-30b-5p, and hsa-miR-19b-3p were significantly differentially expressed. Interestingly, expression of BAFF mRNA was inversely correlated with the down-regulated expression of hsa-miR-30b-5p in B cells from SjS patients. Furthermore, transfection of THP-1 cells with an antagomir (miRNA inhibitor) for hsa-miR-30b-5p led to a strong increase in BAFF expression, providing strong evidence that the loss of hsa-miR30b-5p could contribute to the increased expression of BAFF in patients with pSjS [25].