Background/Purpose: Sjögren’s syndrome (SS) is an autoimmune disease characterized by inflammatory destruction of the exocrine glands. Long non-coding RNAs (lncRNAs) are a functionally diverse class of non-protein coding RNA (ncRNA) with implications in immune cell regulation. The role of lncRNAs in SS pathogenesis is unknown.

Methods: Study received IRB approval. All SS cases met 2002 AECG criteria. RNA-seq was used on whole blood from SS patients (n=30 antibody negative (Ro^–); n=27 antibody positive (Ro⁺)) and healthy controls (HC, n=27) to identify differentially expressed (DE) lncRNAs (log₂ fold change (FC) ≥ 2 or  ≤ 0.5; p_adj < 0.05). Bioinformatic and pathway analyses were used to predict LINC01871 function.  CRISPR-targeted LINC01871 deletion in the T lymphoblastic cell line HSB-2 was performed by RNP transfection and assessed by using an enrichment method that increases the sequencing reads across the region using Oxford Nanopore Minion sequencing. Transcripts of interest were measured by qRT-PCR. Protein expression was analyzed using flow cytometry and multiplex bead-based ELISAs.

Results: We identified 1054 unique DE ncRNAs between Ro⁺, Ro^– and a combined analysis relative to HC. LINC01871 (SS^Ro-: FC=2.85; p_adj=1.1×10^-4) was a previously uncharacterized lincRNA that was co-expressed in the SS^Ro- transcriptome with several protein coding RNAs involved in immune regulation (TBX21, IL10RA, IL2RB, etc.).  DE of LINC01871 was confirmed in an independent set of 35 SS cases and 21 HCs using qPCR.  Bioinformatics analyses identified shared immune-related pathways including cytotoxic cell, cell migration, and T cell regulation. The Expression Atlas database showed DE of LINC01871 in several published RNA-seq studies in cancer, autoimmune diseases (such as discoid lupus), and T cell states.  CRISPR-targeting in HSB2 generated a single cell LINC01871^-/- clone (one of ~100) with no RNA expression by qPCR.  Using Nanopore Minion sequencing, the LINC01871^-/- cell line was found to have a homozygous deletion encompassing the entire coding interval for LINC01871 that was confirmed using PCR. RNA-seq analysis of LINC01871^-/- compared to unmodified HSB-2 cells identified 1166 DE transcripts. Pathway analyses clustered the DE transcripts into SS^Ro--similar immune regulatory, cytotoxic, and T cell pathways, with the addition of cancer/cellular growth and cell signaling. The LINC01871^-/- clone displayed impaired growth at 4 days (p< 0.05) and exhibited significant modulation of many T cell regulatory transcripts (CD8a, TBX21, IKZF3, CXCR3, PDCD1, and TIGIT) and secreted growth factors (IGFBP4, CSF1, CSF2, FGFPB2). Changes were confirmed by protein analyses using flow cytometry and multiplex ELISA assays (p< 0.05), indicating loss of LINC01871 resulted in widespread cellular changes affecting growth and immune regulatory pathways.

Conclusion: RNA-seq, bioinformatic data, and CRISPR technology identified and functionally characterized LINC01871 as a potential mediator of the dysregulated T cell inflammatory response pathways implicated in SS pathogenesis.

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ACR Meeting Abstracts - https://acrabstracts.org/abstract/dysregulated-expression-of-the-long-non-coding-rna-linc01871-implicated-in-sjogrens-syndrome-pathogenesis/