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 of miRNA profiling 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, we have developed the following distinct projects to further investigate the role of miRNAs in the mechanistic regulation of SS. 1) Loss of secretory function of salivary glands is mediated by ebv-miR-BART13 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. STIM1 was identified 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. 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). 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. 2) 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 plays a crucial role in organizing membrane domains, in particular microvilli whose organization requires close interactions between the plasma membrane, membrane-associated cytoplasmic proteins and the underlying cytoskeleton. Recently, we found that SS patients present with a mislocalization and overexpression ezrin in acinar SG cells. Nothing is yet known about the functions and the mechanisms controlling ezrin gene expression in SGs. We hypothesized that Ezrin may be overexpressed in SS due to a decrease in the expression of hsa-miR-183 and or has-miR-22. Previously published work has demonstrated that those microRNAs target ezrin in its 3UTR in other tissues. After validation of the array results in independent SGs, with Real Time PCR and extensive immunohistochemistry for ezrin on human SG biopsies, we looked into the role of hsa-miR-183 in expression of ezrin in SG acinar cells and their connection to altered secretory function. To further confirm that in salivary gland hsa-miR-183 regulates ezrin expression we first used a model of acinar cells using HSG cultured in 3D. HSG cells cultured in 3D present a polarized phenotype similar to the acini in vivo. Under these conditions transfection of hsa-miR-183 also modulated the expression of ezrin. To finally probe that this modulation can occur in vivo, we took advantage of the fact that ezrin mRNA including the microRNA target areas is highly conserved between mouse and humans. We transfected salivary glands cells in vivo using Locked Nuclei Acids (LNA) conjugated to Fluorescein isothiocyanate (LNAs-Fitc). LNAs are a class of bicyclic conformational analogues of RNA, which exhibit high binding affinity to complementary RNA target molecules and high stability in blood and tissues in vivo. These LNAs (LNA-anti-miR) act as competitive inhibitors of miRNAs, impeding their ability to interact and repress cellular target mRNAs. LNA-anti-miRs can be delivered in vivo without additional conjugation or formulation chemistries. Also they possess enhanced pharmacological activity and optimized pharmacokinetic properties. The use of LNA-anti-miRs is relatively new, they have never previously been delivered in animals through SG retroductal cannulation. Our data in mouse parotid glands indicated that a dose of 25nM of LNA-anti-miR-183 is sufficient to observe a signal for fluorescein in fresh tissue, 72hrs after intraductal delivery. In these experiments we also observed a decrease in the granule secretion in parotid acini transfected with LNA. This dose was used to transfect both glands with LNA-anti-miR-183 and LNA-scramble (negative control) and established if silencing of hsa-miR-183 changed the expression levels of ezrin and decreased the volume of saliva secreted in mice. We observed that 48hrs after delivery of the LNA the proteins levels of ezrin increased in comparison with the glands transfected with negative control. Finally, saliva secretion after stimulation with pilocarpine decreased in every animal when compared with their secretion prior to transfection.
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