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Qistina Pilson, Joan Ní Gabhann, Conor C Murphy; Profiling microRNA in conjunctival impression cytology samples in Primary Sjogren’s syndrome. Invest. Ophthalmol. Vis. Sci. 2015;56(7 ):4809.
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© ARVO (1962-2015); The Authors (2016-present)
In Sjogren’s syndrome (SS), a combination of reduced aqueous tear production from lacrimal gland destruction and increased tear osmolarity leads to inflammatory damage to the ocular surface. microRNAs (miR) are known to alter the expression of inflammatory cytokines, which plays an important role in the pathogenesis and progression of SS. The aim of this study was to isolate miRs and mRNA from conjunctival epithelial cells of patients with primary SS (pSS) and healthy controls to identify potential biomarkers that might aid diagnosis and future therapy in pSS.
pSS patients with confirmed dry eyes and satisfying the American-European Consensus Group classification criteria (AECG) were recruited to this study. Age and sex matched healthy controls were evaluated for comparison. The isolation of miRs using miRNeasy kit and mRNA using TRIzol from impression cytology conjunctival samples was optimized using Immobilon, Biopore and Hanging culture insert (HCI) membranes. mRNA was sent to Ocean Ridge Biosciences for miR and mRNA microarray. Bioinformatic analysis using online platforms including miRWalk and MiRDB was performed to identify predicted targets and comparison was made with the mRNA microarray data. Validation experiments were performed in Hela cells following transfection with selected miR mimics and predicted genes were detected using real time PCR.<!--EndFragment-->
Biopore membrane gave a significantly higher average yield for miRs (p<0.05) compared to the other membranes. miR microarray found 11 significantly increased and 21 significantly decreased novel miRs in pSS patients compared to healthy controls. mRNA microarray found 62 significantly increased and 74 significantly decreased genes. Novel miR-A was chosen for further analysis as it was significantly increased in pSS (p=0.0079) and bioinformatics suggested Pellino3 (PELI3), a negative regulator of inflammatory cytokines, as a predicted target. The mRNA microarray showed a decrease in PELI3 in pSS patients compared to healthy controls (p=0.07). Additionally, over expression of a miR-A mimic in Hela cells resulted in decreased expression of PELI3, suggesting that it is a direct target for miR-A.
We have identified PELI3, a known negative regulator of cytokines, a potential target of novel miR-A which is over expressed in pSS. PELI3 and novel miR-A, may have biomarker and therapeutic potential for pSS related dry eyes.
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