Background/Purpose: Loss of secretory function of salivary glands is one of the most important functional effects of Sjögren’s syndrome (SS), a chronic systemic autoimmune disease. We have previously shown that an EBV microRNA (ebv-miR-BART13) is significantly over-expressed in minor salivary gland biopsies of SS patients, when compared to the healthy volunteers. We have also shown that the over-expression of ebv-miR-BART13 in human salivary gland cell line is responsible of the down regulation of STIM1, an ER-Ca2+ sensor protein regulating SOCE and to the consequent decrease in Calcium influx. We demonstrated that the decrease of Calcium influx, due to the presence of ebv-miR-BART13, is responsible of the loss of translocation of NFAT, a transcriptional factor that drives the transcription of several genes, but most interestingly AQP5.  The objective of this work is to establish if ebv-miR-BART13 leads to the decreased level of AQP5 not only trough the Calcium-NFAT pathway but also by directly binding the 3’UTR of the AQP5 mRNA. 

Methods: We checked the AQP5 messenger lever by Real-time PCR in a primary salivary epithelial cells line. To investigate if the viral microRNA is able to bind the 3’UTR the AQP5 messenger we transfected HSG cells and a primary salivary epithelial cells line derived by human minor salivary gland biopsy with a luciferase plasmid containing the  AQP5 3’UTR fused with the luciferase firefly coding sequence and ebv-mir-BART13.

Results: We first checked if the mRNA level of the endogenous AQP5 is affected by the presence of ebv-miR-BART13. For this, we used as system primary salivary epithelial cells because they maintain their acinar phenotype thus expressing AQP5 mRNA level, compared to established salivary cell lines such as HSG cells. After 48 hour of transfection with ebv-miR-BART13, the AQP5 mRNA was decreased (4 fold change) by the presence of this viral miRNA.

In order to examine if ebv-miR-BART13 also exerts its effect by binding directly the AQP5  mRNA, we used a plasmid containing the AQP5 3’UTR sequence fused with the luciferase firefly sequence. The luciferase plasmid was co-transfected with ebv-miR-BART13 analogues and antagonists in both HSG cells (human submandibular salivary gland cells) and primary salivary epithelial cells line derived from human minor salivary gland biopsies. After 48 hour of transfection, the cells were collected, proteins were extracted and the luciferase activity was measured. Luciferase expression decreased by 30 % in HSG cells and by 40 % in the primary cell line, confirming that ebv-miR-BART13 directly targets the 3’UTR of AQP5 mRNA.

Conclusion: We first checked if the mRNA level of the endogenous AQP5 is affected by the presence of ebv-miR-BART13. For this, we used as system primary salivary epithelial cells because they maintain their acinar phenotype thus expressing AQP5 mRNA level, compared to established salivary cell lines such as HSG cells. After 48 hour of transfection with ebv-miR-BART13, the AQP5 mRNA was decreased (4 fold change) by the presence of this viral miRNA.

---

« Back to 2012 ACR/ARHP Annual Meeting

ACR Meeting Abstracts - https://acrabstracts.org/abstract/ebv-mir-bart13-affects-the-expression-of-aqp5-in-human-salivary-gland-cell-lines-contributing-to-the-pathogenesis-of-sjogrens-syndrome/