Validating Differentially Expressed Micro-RNAs That Distinguish Psoriatic Arthritis from Osteoarthritis

Objectives Biomarkers may help differentiate psoriatic arthritis (PsA) from osteoarthritis (OA). MicroRNAs have potential to be robust biomarkers, we aimed to 1) Use miRNA sequencing to analyze miRNA expression profiles in the synovial fluid of patients with PsA compared to those with OA, 2) Validate differential expression of select miRNAs in an independent cohort of PsA and OA patients through quantitative real-time PCR (qRT-PCR), and 3) Investigate the potential for differentially expressed miRNAs between PsA and OA to serve as biomarkers for PsA to enhance PsA diagnostic accuracy. Methods We performed miRNA sequencing on synovial fluid (SF) aspirated from knees of 12 PsA and 12 OA patients using Illumina NextSeq 550. We used linear modeling with empirical Bayes moderation (using the Limma R package) for assessing differential expressions of miRNAs. For validation, we extracted miRNA from SF samples from an independent set of 35 PsA and 37 OA patients. We investigated the differential expression of miRNAs between the disease states of 12 selected miRNAs using qRT-PCR (99a-5p, 100-5p, 10b-5p, 7c-5p, 125b-5p, 125a-5p, 27b-3p, 26b-5p, 142-3p, 142-5p, 223-3p, and 150-5p). We calculated the differential expression of miRNAs between PsA and OA using fold change, and calculated area under the receiver operating characteristic curve (AUC) values for the select miRNAs. Results After miRNA sequencing, 17 miRNAs were found to be significantly differentially expressed between PsA and OA (FDR adjusted p-value less than 0.05) (Figure 1). From the 12 selected miRNAs analyzed further by qRT-PCR in an independent set of patients, statistically significant differences were identified in the expression of 11 out of 12 miRNAs between PsA and OA. PsA patients had lower expression of 99a-5p (FC=0.162, p<0.0001), 100-5p (FC=0.091, p<0.0001), 125b-5p (FC=0.087, p<0.0001), 27b-3p (FC=0.191, p<0.0001), 7c-5p (FC=0.330, p<0.001), 10b-5p (FC=0.355, p<0.01), and 125a-5p (FC=0.500, p<0.05) compared to OA patients. PsA patients had higher expressions of 150-5p (FC=9.183, p<0.0001), 223-3p (FC=7.112, p<0.0001), 142-3p (FC=2.432, p<0.01), and 142-5p (FC=2.627, p<0.01) compared to OA patients. ROC analyses revealed that 4 miRNAs (miR-223-3p, miR-99a-5p, miR-125b-5p, miR-150-5p) had an area under the curve value greater than 0.8. Fig. 1. Volcano plot of log2 fold change (FC) against −log10 FDR adjusted p-value of miRNAs identified through miRNA sequencing. Red dots indicate differentially expressed miRNAs (FDR adjusted p value <0.05 and a log2 FC>±l). miRNAs with FC≥±1 but FDR corrected p-value of ≥0.05 are shown in green. Black dots represent non-significant miRNAs with a log2 FC<±1. Conclusion The validated miRNAs, shown to be differentially expressed in PsA and OA patient synovial fluid across 2 independent cohorts, may serve as potential biomarkers for PsA, and improve our understanding of PsA’s inflammatory mechanisms.

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