Background Despite concerted efforts to characterize dysregulated transcriptional responses observed in Sjögren's syndrome and related autoimmune disorders (both in whole blood and target tissues), the functional roles of non-coding RNAs (ncRNAs), many of which have been identified as critical players in transcriptional regulation of disease, remain poorly defined.
Objectives In the present study, we describe ongoing efforts to functionally characterize the upregulated ncRNA identified by RNA-seq and in silico approaches, AC092580.4 (FC=2.54), which we hypothesize plays a role in T and NK cell responses.
Methods To study the immunomodulation of the ncRNA AC092580.4, we carried out a time-course experiment (0–36 hrs) using either PMA/I (500x dil) or universal Type I Interferon. Relative gene expression changes were determined using the Livak method by qPCR using optimized primers for GZMA and AC092580.4 normalized to GAPD. Healthy PBMCs were subjected to stimulation by PHA (1mg/mL; 3 days), PMA/I (500x dil; 3 days), or anti-CD3/CD28 (50uL/1x106cells; 1 day). An average 150-bp RNA-seq reads were generated for each sample; alignment was carried out using STAR (hg38) and comparisions of stimulated vs unstimulated cells were done using DEseq. Pearson's correlation (r) was calculated for all 3,748 differentially expressed (DE) transcripts to identify transcripts co-expressed with AC092580.4.
Results Of the transcripts showing DE in our SS RNA-seq study, we identified 8 as having significantly correlated expression with AC092580.4 in the SSRo- expression matrix (r>0.70 or <-0.65). To understand the possible effects of immunomodulation on relevant cells, we stimulated HSB-2 cells with PMA/I at various time points and assessed AC092580.4 expression by. We observed downregulation of AC092580.4 and the co-expressed transcript GZMA by PMA/I (trough: 12–16hrs; FC=0.09) followed by slow recovery at 36hrs (FC=0.59). To characterize these transcriptional changes further, we performed RNA-seq using healthy PBMCs exposed to various T cell stimulants. We observed marked upregulation of both AC092580.4 and GZMA at 24/36hrs by all stimulants (FC=4.89–5.98). Other transcripts showed variable responses. CAV2 is upregulated by PMA/I, but downregulated by CD3/CD28 and PHA. Stimulation by PHA leads to upregulation of CD3D (FC=1.56) and SNRPD1 (FC=3.28) with little change in RPL36A (FC=1.09). Stimulation by CD3/CD28 similarly leads to upregulation of CD3D (FC=2.85) and SNRPD1 (FC=10.04), but clear downregulation of RPL36A (FC=0.64). We assessed AC092580.4 expression in HSB-2 cells exposed to I IFN and observed initial upregulation (6hrs, FC=1.46) followed by gradual downregulation (36hrs, FC=0.18).
Conclusions In the present study, we have initiated stimulation studies with to understand the immune relevance of AC092580.4 and co-expressed targets. AC092580.4 shows transcriptional induction by potent inducers of T cell responses (PMA/I, PHA, CD3/CD28) but is downregulated by type I IFN. Transcripts showing co-expressed with AC092580.4 by whole-blood RNA-seq show divergent expression patterns according to the specific stimulus, suggesting a complex regulatory network governing dysregulated T and NK responses.
Disclosure of Interest None declared