THU0203 PROMOTER DNA METHYLATION AND HSA-MIR-424-5P REGULATE ATF6 ALPHA EXPRESSION IN SALIVARY GLANDS OF PATIENTS WITH SJÖGREN’S SYNDROME
Authors: Patricia Carvajal, Sergio Aguilera, María-José Barrera, Carolina Lagos, Isabel Castro, Sergio González, Daniela Jara, Claudio Molina, María-Julieta González
Journal: Annals of the Rheumatic Diseases (2019)

<h3>Background:</h3> Salivary glands (SG) from Sjögren's syndrome (SS)-patients show chronic inflammation and endoplasmic reticulum (ER) stress. To counteract ER stress, eukaryotic cells activate adaptive mechanisms referred to as the unfolded protein response via sensors present in the ER membrane, such as ATF6α<sup>1,2</sup>. In response to ER stress, ATF6α is translocated to the Golgi apparatus where is processed releasing ATF6f, which translocates into the nucleus as a transcription factor to activate ER-associated degradation and protein folding-related gene transcription<sup>3</sup>. The overexpression of ATF6α observed in Labial SG (LSG) from SS-patients<sup>1</sup> could be modulated by epigenetic mechanisms. <h3>Objectives:</h3> To evaluate the involvement of epigenetic mechanisms such as promoter-specific DNA methylation and microRNA (miRNA) regulation on ATF6α expression in LSG from SS-patients and controls. The effect of IFN-γ on both mechanisms was also evaluated in a 3D-acini model. <h3>Methods:</h3> SG biopsies from 12 SS-patients and 10 controls were analyzed. For in vitro assays 3D-acini incubated with 1 or 10 ng/mL IFN-γ for 24 h were employed. Specific DNA methylation of ATF6α gene promoter was evaluated by methylation sensitive-high resolution melting PCR. The profile of miRNAs differentially expressed was determined by miRNA sequencing (seq), of which hsa-miR-424-5p was selected because the <i>in silico</i> analysis showed that ATF6α mRNA is a target of this miRNA. The levels of hsa-miR-424-5p were determined by Taqman miRNA assays. Functional assays using miRNA mimic and miRNA inhibitors were also performed. <h3>Results:</h3> LSG from SS-patients showed a significant decrease of ATF6α promoter DNA methylation percentage (p=0.04), which was inversely correlated with high ATF6α mRNA levels (p= 0.02). The hsa-miR-424-5p was found downregulated by miRNA-seq in LSG from SS-patients. Taqman miRNA assays validated the significant decrease of hsa-miR-424-5p levels found in LSG from SS-patients (p=0.02), which were inversely correlated with ATF6α mRNA levels (p=0.02). Functional assays suggest that hsa-miR-424-5p regulates the mRNA levels of ATF6α. Finally, stimulation of 3D-acini with IFN-γ induced a significant increase of ATF6α mRNA levels together with a decrease in ATF6α promoter DNA methylation percentage and hsa-miR-424-5p levels. <h3>Conclusion:</h3> The results suggest that the ATF6α overexpression is regulated by two complementary epigenetic mechanisms: the DNA hypermethylation of its promoter and the silencing of its mRNA by hsa-miR-424-5p in LSG from SS-patients. IFN-γ affects the expression of ATF6α by modulation of epigenetic mechanisms, regulating the ability of epithelial cells to handle the chronic ER stress. Interestingly, these changes carry out under a global DNA hypomethylation condition<sup>4</sup>. <h3>References:</h3> [1] J Autoimmun2016;75:68-81 [2] Rheumatology2018;57:1021-1032 [3] Autoimmun Rev2018;17:796-808 [4] Clin Immunol2018;196:85-96 <h3>Acknowledgement:</h3> Fondecyt-1160015, Fondecyt-Postdoctorado-3170023, Fondecyt-Iniciación-11170049. <h3>Disclosure of Interests:</h3> None declared