SS is characterized by features of systemic autoimmunity and dysfunction and inflammation in the exocrine glands. The exact cause of exocrine dysfunction in SS has not been delineated but it is thought that significant contributions from both immunologically-mediated and non-immune mechanisms. The diagnosis is based on the combination of subjective symptoms and objective signs of dry mouth and/or dry eyes, the presence of autoantibodies, and an inflammatory infiltrate in the minor salivary glands. MicroRNAs (miRNAs) are a group of small RNAs involved in the regulation of a wide variety of cellular and physiologic processes. They exert their effects by two mechanisms: messenger RNA degradation and disruption of translation. A single mRNA is usually translated into a single protein;however, a single miRNA is capable of regulating the translation of a multitude of genes involved in a certain function. Changes in mRNA levels can be ultimately modulated or nullified by post transcriptional regulation, and thus may be less representative of the physiological status of the cell than miRNA. Based on my previously published work with miRNA arrays of SS minor salivary glands (Arthritis and Rheumatism, 2011 Feb;63(2):535-44), we have identified several interesting and novel target proteins that, when modulated by miRNAs, might play significant roles in the pathogenesis and dysfunction of SS. Based on these results, I have developed the following distinct projects to further investigate the role of miRNAs in the mechanistic regulation of SS. Ebv-mir-BART13 expression may be mediated by inflammation and induce salivary gland dysfunction. Loss of secretory function of salivary glands is one of the most important functional consequences of SS. The previously mentioned miRNA profiling studies identified that Epstein Barr Virus (ebv) miRNA (ebv-mir-BART13) was significantly over-expressed in minor salivary gland biopsies of SS patients independent of degree of inflammatory infiltrate, when compared to the expression in minor salivary glands from healthy volunteers. Using the RNA22 target prediction algorithm, we identified STIM1 as a major target of ebv-mir-BART13. STIM1 is a protein that has recently been identified as a critical molecular component of the store-operated channels, including Orai1-CRAC (calcium release-activated calcium channel) and TRPC1-SOC channels. After validating that ebv-mir-BART13 does indeed bind to the STIM1 transcript and subsequently downregulates its protein expression, the functional effects of STIM1 dowregulation in salivary epithelial cells was tested. While the intracellular calcium release was not affected, the calcium influx was substantially reduced in ebv-miR-BART13 transfected cells as compared to controls. Further, a decrease in calcium influx-dependent NFAT nuclear translocation due to decreased calcineurin activity in the presence of ebv-mir-BART13 was observed. Together, these functional measurements suggest that the presence of ebv-miR-BART13 in the salivary glands of SS patients might contribute to their salivary gland dysfunction by reducing the expression of STIM1, a critical component of the saliva secretion mechanism. Through in situ hybridization, we observed an increased expression of this miRNA in salivary ducts of SS minor salivary glands, especially those surrounded by inflammatory cells. To determine whether this was a potential mechanism whereby this viral miRNA could be introduced to epithelial cells from the B cell residents within the minor salivary glands, we used a Transwell system to co-culture human salivary gland cell line HSG and the X50-7B-cell line which is stably transfected with ebv. In addition, we also set up co-cultures of primary salivary epithelial cells derived by human minor salivary gland biopsies with the X50-7 B-cell line. After 7 days of co-culture, the expression of ebv-miR-BART13 was significantly increased in both co-cultured epithelial cells (by more than 4-fold). These findings have been detailed in a recently-prepared manuscript to be submitted for publication. However, a number of questions remain regarding the role of ebv-mir-BART13 and other viral miRNAs in SS salivary gland dysfunction. More specifically the upstream and downstream cellular events related to this overexpression need to be characterized, especially in the context of a previous viral infection causing or being strongly associated with SS. Moreover, since in vitro the effect of ebv-miR-BART13 is so remarkable, I am planning to perform animal studies to assess the potential of silencing of this microRNA as a potential therapeutic for SS. MiRNA-mediated EZRIN is differentially expressed in SS and correlates with secretory function. Besides ebv-mir-BART13, two other miRNAs from the initial experiments are examined for their role in salivary hypofunction. Hsa-mir-183 and hsa-mir-22 have been shown to bind to the EZRIN mRNA and downregulate its expression. In our arrays, both microRNAs were shown to be downregulated concurrent with upregulation of ezrin in SS. Ezrin is a cytoplasmic peripheral membrane protein (80 kDa) that regulates the microvilli organization and secretion of exocrine cells. My lab has been investigating if the overexpression of ezrin in the salivary glands of SS can be explained by the downregulation of hsa-mir-183 and/or hsa-miR-22. In SG biopsies of SS patients (n=17) and healthy volunteers (n=3), we analyzed the levels of those miRNAs by qRT-PCR. Additionally, we have tested the effect of decreased expression of hsa-mir-183 on ezrin levels in primary salivary gland epithelial cells, the HSG salivary gland cell line, 3D culture of primary and HSG cells, as well as in vivo in mice (with local delivery of Locked Nucleic Acid anti-microRNAs). LNA-antimir in vivo transfection in parotid glands of C57/BL6 mice produced an increase of protein levels of ezrin and significantly affected the granule secretion of acinar cells, as imaged with confocal imaging. This suggests that hsa-mir-183 expression may directly mediate secretion through modulation of ezrin expression and further indicates that miRNA expression may be a critical mechanism by which salivary gland function in SS patients is disrupted. Further experiments to test the effect of these miRNAs in hyposalivation will be explored.

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National Institute of Dental & Craniofacial Research
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Burbelo, Peter D; Ambatipudi, Kiran; Alevizos, Ilias (2014) Genome-wide association studies in Sjögren's syndrome: What do the genes tell us about disease pathogenesis? Autoimmun Rev 13:756-61
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