Abstract
Purpose :
Sjögren’s Syndrome (SS) is an autoimmune disease exhibiting inflammation and loss of function of exocrine glands including lacrimal gland (LG). Diagnosis of SS is lengthy and cumbersome. The male NOD mouse exhibits many symptoms of the autoimmune-mediated dry eye seen in SS patients. To identify tear biomarkers that might aid in diagnosis of SS-associated dry eye, we investigated the miRNA composition of tears from male NOD mice. miRNAs are 19-26 nucleotide long, highly-conserved, RNAs that regulate mRNA transcription and are altered in disease. Relative to proteins, miRNAs have less sequence heterogeneity and inter-individual variance, and have shown high sensitivity as biomarkers.
Methods :
Tears were collected with topical stimulation of LG with carbachol from male NOD (disease model), BALB/c (healthy control) and female NOD mice (strain control lacking LG inflammation) aged 12-14 weeks. Tear RNA was isolated from pools of 5 mice per sample with 5 samples of male NOD and BALB/c mice and 3 samples of female NOD. RNA quality was determined using Tapestation, then a small-RNA library was prepared using the Illumina TruSeq Small RNA library prep kit and sequenced on an Illumina HiSeq system. Following bioinformatics data analysis, miRNA hits were identified and validated using qRT-PCR. Hits were considered significant if they were up or downregulated in NOD tears with a log2FC of ± 0.5 in the same direction when compared to both male BALB/c and female NOD, and had a p value < 0.05 (DESeq2).
Results :
In comparison to both male BALB/c and female NOD mice, 8 tear miRNAs (miR-107-3p, miR-181a-5p, miR-181b-5p, miR-3572-3p, miR-3572-5p, miR-3963, miR-3076-3p) were upregulated whereas another 8 (miR-322-3p, miR-322-5p, miR-146b-5p, miR-146a-5p, miR-147-3p, miR-421-3p, miR-542-3p, miR-503-5p) were downregulated in male NOD. miRNAs 146a/b-5p which was significantly downregulated in male NOD mouse tears, and miR-155-5p, which was slightly but not significantly upregulated in male NOD mouse tears, have been implicated in autoimmune inflammation. The remaining miRNAs represent novel hits with their functional relevance under investigation.
Conclusions :
We have identified a panel of differentially expressed miRNA in male NOD mouse tears that may serve as novel biomarkers for early detection of SS.
This is a 2021 ARVO Annual Meeting abstract.