Background/Purpose

Sjögren’s syndrome (SS) is a common autoimmune disorder characterized by immune-mediated exocrine gland destruction and systemic inflammatory responses that contribute to clinical heterogeneity. Widespread dysregulation of transcribed RNAs in SS, including coding and non-coding elements, has been identified, but the complex regulatory mechanisms governing these responses are poorly understood. We performed an RNA-sequencing (RNA-seq) study that identified over 2,600 differentially expressed (DE) transcripts associated with SS, including 969 long non-coding RNAs (lncRNAs). This study sought to validate, replicate, and functionally characterize one upregulated lncRNA mapped to chromosome 2p25.1, SSINCR1, to better understand its role in SS pathogenesis.

Methods

Whole blood RNA from 27 healthy controls and 57 SS patients was sequenced, and 2,632 statistically significant DE transcripts were identified. Technical validation and replication of SSINCR1 upregulation was assessed by qRT-PCR in an independent set of 36 SS patients and 21 controls. Bioinformatic analyses using GAMMA-seq and the lncRNAtor database were performed to identify co-expression patterns of SSINCR1 with other coding and non-coding transcripts and to identify candidate protein binding partners. To determine cellular expression patterns of SSINCR1, we employed fluorescence-assisted cell sorting (FACS) staining for 10 distinct immune cell subsets in a healthy control followed by RNA isolation and assessed SSINCR1 expression by qRT-PCR. Statistical comparisons were made using t-tests and Pearson correlations.

Results

RNA-seq showed significant upregulation of SSINCR1 when comparing healthy controls and SS patients (P_adj=3.69×10^-5 ; Fold Change=2.4). Technical validation by qRT-PCR using the RNA-seq cDNA library confirmed this finding (P=0.0096), and correlation with RNA-seq results was observed (r=0.869). Transcript expression in an independent sample set replicated and confirmed SSINCR1 upregulation (P=0.0183). Co-expression patterns by GAMMA-seq showed T, NK, and dendritic cell activation, development, and proliferation, and assessment of SSINCR1 using lncRNAtor suggested protein binding with cyclin T1 and FIP1L1. FACS analysis showed that SSINCR1 expression levels were highest in the CD3⁺CD56⁺ compartment (containing NKT cells; relative units [RU]= 8.25), followed by CD8⁺ T cells (RU=3.34), CD56int NK cells (RU=2.08), CD56hi NK cells (RU=0.83), and CD4⁺ T cells (RU=0.81). Expression was not detected in CD141⁺ and CD1c⁺CD11c⁺ myeloid DCs, monocytes, B cells, or pDCs.

Conclusion

We have identified, technically validated, and independently replicated the upregulation of a novel SS lncRNA, SSINCR1.   We show that transcript expression is highly enriched in CD4+ and CD8+ T cells, NKT cells, and NK cells. Ongoing studies are assessing potential protein binding partners and SSINCR1 expression in refined T and NK subsets and in expanded groups of affected and healthy individuals to determine subset-specific DE. This study establishes SSINCR1 as the first lncRNA associated with SS and lays the groundwork for further functional characterization in the pathogenesis of this complex disorder.

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ACR Meeting Abstracts - https://acrabstracts.org/abstract/characterization-of-the-sjogrens-syndrome-intergenic-non-coding-rna-1-ssincr1/