Loss of secretory function of salivary glands is one of the most important functional effects of Sjogren's syndrome (SS), a chronic systemic autoimmune disease. Previous studies have reported the presence of Epstein Barr Virus (EBV) DNA in salivary glands of SS patients. MicroRNA profiling studies in our laboratory have identified an EBV microRNA (ebv-mir-BART13) as significantly over-expressed in minor salivary gland biopsies of SS patients, when compared to the expression in minor salivary glands from healthy volunteers. Using the RNA22 target prediction algorithm, we identified STIM1 3'UTR and coding sequence as targets of ebv-mir-BART13. STIM1, is a protein that has recently been identified as a critical molecular component of the calcium release-activated calcium current (CRAC) channels. Store-operated calcium entry (SOCE) and CRAC are critical events for the replenishment of intracellular calcium stores and have indispensable roles in various cellular functions, including salivary secretion. SOCE is activated by the depletion of calcium in the endoplasmic reticulum (ER). STIM1, is suggested to be the ER-Ca2+ sensor protein regulating SOCE in a number of different cell types. Attenuation of SOCE current underlies salivary gland dysfunction in mice lacking transient receptor potential 1 (TRPC1). We hypothesized that ebv-miR-Bart-13 may contribute to the salivary gland dysfunction present in SS by downregulating STIM-1 and decreasing calcium influx in salivary gland cells. To test this hypothesis, HSG -human submandibular salivary gland cells- were transfected for 24 and 48hrs with ebv-miR-BART13 analog and the mRNA and protein levels of STIM1 were measured. The mRNA level was not affected by the presence of this viral miRNA. However, the protein level was dramatically decreased both 24 and 48hrs after transfection, suggesting that the ebv-mir-Bart13 exerts its effects on STIM1 not by degrading its transcript, but by repressing STIM1 translation. To examine if the effect on STIM1 protein dowregulation was mediated by binding on the 3UTR, we co-transfected HSG cells with the STIM1 3UTR cloned sequence within a luciferase plasmid along with EBV-mir-BART13 mimics, and antagonists. A 40% decrease in the luciferase expression was observed with the mimic and plasmid co-transfection, confirming that this viral microRNA binds to the 3UTR of STIM1. To examine if binding also occurs on the coding sequence of the STIM1 transcript, we co-transfected HSG cells with a YFP plasmid containing STIM1 coding sequence with ebv-mir-BART13 mimics, and antagonists. Through immunofluorescence we observed a distinct decrease in STIM1 fluorescence levels in the EBV-mir-BART13 mimic transfected cells compared to controls. Functional Ca2+ assays through Thapsigargin-mediated depletion of the endoplasmic reticulum calcium in the ebv-mir-BART13 transfected cells showed a significant delay in the influx of calcium in the cytosol compared to controls. Together, these functional measurements suggest that the presence of ebv-miR-BART13 in the salivary glands of Sjogren's syndrome patients might be, at least partially, responsible for the salivary gland dysfunction in those patients, by reducing the expression of STIM1, a critical member of the saliva secretion mechanism. Besides ebv-mir-BART13, we identified several more microRNAs that are differentially expressed in the minor salivary glands of SS patients. Through extensive bioinformatics analysis we have identified genes known to be involved in salivary gland function that might be differentially expressed in SS salivary glands and we are in the process of validating their altered expression.

Project Start
Project End
Budget Start
Budget End
Support Year
3
Fiscal Year
2012
Total Cost
$771,246
Indirect Cost
Name
National Institute of Dental & Craniofacial Research
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Gallo, Alessia; Jang, Shyh-Ing; Ong, Hwei Ling et al. (2016) Targeting the Ca(2+) Sensor STIM1 by Exosomal Transfer of Ebv-miR-BART13-3p is Associated with Sjögren's Syndrome. EBioMedicine 10:216-26
Burbelo, Peter D; Iadarola, Michael J; Alevizos, Ilias et al. (2016) Transcriptomic Segregation of Human Autoantigens Useful for the Diagnosis of Autoimmune Diseases. Mol Diagn Ther 20:415-27
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Gallo, Alessia; Alevizos, Ilias (2013) Isolation of circulating microRNA in saliva. Methods Mol Biol 1024:183-90
Gallo, Alessia; Tandon, Mayank; Alevizos, Ilias et al. (2012) The majority of microRNAs detectable in serum and saliva is concentrated in exosomes. PLoS One 7:e30679
Imrich, Richard; Nikolov, Nikolay P; Bebris, Lolita et al. (2012) Adrenomedullary response to glucagon in patients with primary Sjögren's syndrome. Cell Mol Neurobiol 32:903-6

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