Background/Purpose:

Recent studies have identified distinct changes in cellular miRNA (miR) expression associated with systemic lupus erythematous (SLE). We have previously shown that toll-like receptor TLR7 and TLR8 are significantly up-regulated in PBMCs of SLE patients. TLR7 and TLR8 bind to single-stranded RNA of viral origin to stimulate innate inflammatory responses. We have demonstrated that EV-encapsulated miR-21 in-duces both TLR8-mediated cytokine signaling and EV secretion in vitro. In another recent publication from our group, we also discovered that high mobility group box (HMGB)1 is a substrate for transglutaminase-2-mediated autoantigen complex formation and HMGB1 complexes are significantly up-regulated in both extracellular plasma and PBMCs of SLE patients. The objective of this study was to identify miR and other small RNA signatures that may be used as diagnostic bi-omarkers and potential therapeutic targets to limit either HMGB1 or TLR-induced inflammatory response in SLE.

Methods:

Our pilot cohort consisted of four active female SLE patients meeting the revised criteria of the American College of Rheumatology and three age/sex matched healthy controls. Active disease was defined as a SLEDAI > 4 at the time of sample collection. Plasma-derived EVs were isolat-ed by ultracentrifugation and small RNA libraries were prepared for RNA-sequencing (RNAseq). Global small RNA reads were analyzed and cross-referenced with a sequencing database (miR-Base) of known miRs. RNAseq reads with more or less than a 2-fold change when comparing healthy to SLE were characterized and considered statistically significant if was p < 0.05.

Results:

Analysis of our RNAseq data resulted in a large collection of statistically significant small RNA reads (< 25nt) that were up or down-regulated in SLE patients relative to healthy controls; 77% of these RNA reads were unique non-coding RNA sequences that did not align with a database of known miRs, which suggests that many of the SLE-associated small RNAs in EVs are long non-coding RNA (lnRNA), piwi interacting RNA (piRNA), or novel/undiscovered miRs. Con-versely, 23% of the reads correlated with miRBase-aligned sequences for known miRs. Since our database analysis found that there are 47 predicted miRs targeting the 5030nt long mRNA se-quence for HMGB1 and there are 121 known miRs predicted to target HMGB1, we cross-referenced our results to identify miRs that may inhibit its expression that were down-regulated in the plasma-derived EVs of SLE patients. Our analysis revealed that miR-142-3p, a previously characterized negative regulator of HMGB1, was down regulated over 15-fold in EVs from SLE patients when compared to healthy controls. In examining the dataset for miRs that may bind HMGB1 to facilitate EV trafficking, we found that let-7b-5p was up-regulated in SLE EVs more that 4-fold.

Conclusion:

Our data demonstrate unique EV-derived small RNA signatures that may be targeted therapeuti-cally or used as diagnostic biomarkers for SLE. Also, these results identify HMGB1-associated miRs that are dysregulated in SLE and a collection of miRs that may function as TLR7/8 ago-nists. Future studies will expand our cohort and validate miRs of interest in the context of HMGB1 and TLR7/8.

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ACR Meeting Abstracts - https://acrabstracts.org/abstract/unique-mirna-signatures-detected-in-extracellular-vesicles-from-patients-with-systemic-lupus-erythematous/