Summary of miRNAs selected for validation and their expression profiles compared in NGS vs ddPCR in equine blood plasma.

Summary of miRNAs selected for validation and their expression profiles compared in NGS vs ddPCR in equine blood plasma.</p

Statistical data of differentially expressed miRNAs equine plasma of OA vs OCD with a 1.5-log2FC or greater change in expression (p < 0.05).

Statistical data of differentially expressed miRNAs equine plasma of OA vs OCD with a 1.5-log2FC or greater change in expression (p < 0.05).</p

ddPCR count data.

Osteoarthritis (OA) is a leading cause of lameness in horses with no effective disease-modifying treatment and challenging early diagnosis. OA is considered a disease of the joint involving the articular cartilage, subchondral bone, synovial membrane, and ligaments. Osteochondritis dissecans (OCD) is a joint disease consisting of focal defects in the osteochondral unit which may progress to OA later in life. MicroRNAs (miRNAs) have been recognized as small non-coding RNAs that regulate a variety of biological processes and have been detected in biological fluids. MiRNAs are currently investigated for their utility as biomarkers and druggable targets for a variety of diseases. The current study hypothesizes that miRNA profiles can be used to actively monitor joint health and differences in miRNA profiles will be found in healthy vs diseased joints and that differences will be detectable in blood plasma of tested horses. Five horses with OA, OCD, and 4 controls (C) had blood plasma and synovial fluid collected. Total RNA, including miRNA was isolated before generating miRNA libraries from the plasma of the horses. Libraries were sequenced at the Schroeder Arthritis Institute (Toronto). Differential expression analysis was done using DESeq2 and validated using ddPCR. KEGG pathway analysis was done using mirPath v.3 (Diana Tools). 57 differentially expressed miRNAs were identified in OA vs C plasma, 45 differentially expressed miRNAs in OC vs C plasma, and 21 differentially expressed miRNAs in OA vs OCD plasma. Notably, miR-140-5p expression was observed to be elevated in OA synovial fluid suggesting that miR-140-5p may serve as a protective marker early on to attenuate OA progression. KEGG pathway analysis of differentially expressed plasma miRNAs showed relationships with glycan degradation, glycosaminoglycan degradation, and hippo signaling pathway. Interestingly, ddPCR was unable to validate the NGS data suggesting that isomiRs may play an integral role in miRNA expression when assessed using NGS technologies.</div

NGS analysis of OA vs OCD equine plasma.

a) PCA plot of OA and OCD equine blood plasma groups; b) Heatmap of differentially expressed miRNAs in OA vs OCD plasma. Towards red indicates a fold increase; towards blue represents a fold decrease; c) Pathway analysis using miRPath v.3 and Tarbase v7.0 assessing derived interactions and indicated pathways to have predicted interactions with miRNAs identified to be differentially expressed.</p

Statistical data of differentially expressed miRNAs equine plasma of OA vs C with a 1.5-log2FC or greater change in expression (p < 0.05).

Statistical data of differentially expressed miRNAs equine plasma of OA vs C with a 1.5-log2FC or greater change in expression (p < 0.05).</p

Volcano Plot of OA vs C plasma; LFC cut off = 1.5 and p-value cut off = 0.05; adjusted p-value was used; miRNAs marked in red are those shown to meet both the LFC and p-value cut offs.

Volcano Plot of OA vs C plasma; LFC cut off = 1.5 and p-value cut off = 0.05; adjusted p-value was used; miRNAs marked in red are those shown to meet both the LFC and p-value cut offs.</p

RNA Seq count data.

Osteoarthritis (OA) is a leading cause of lameness in horses with no effective disease-modifying treatment and challenging early diagnosis. OA is considered a disease of the joint involving the articular cartilage, subchondral bone, synovial membrane, and ligaments. Osteochondritis dissecans (OCD) is a joint disease consisting of focal defects in the osteochondral unit which may progress to OA later in life. MicroRNAs (miRNAs) have been recognized as small non-coding RNAs that regulate a variety of biological processes and have been detected in biological fluids. MiRNAs are currently investigated for their utility as biomarkers and druggable targets for a variety of diseases. The current study hypothesizes that miRNA profiles can be used to actively monitor joint health and differences in miRNA profiles will be found in healthy vs diseased joints and that differences will be detectable in blood plasma of tested horses. Five horses with OA, OCD, and 4 controls (C) had blood plasma and synovial fluid collected. Total RNA, including miRNA was isolated before generating miRNA libraries from the plasma of the horses. Libraries were sequenced at the Schroeder Arthritis Institute (Toronto). Differential expression analysis was done using DESeq2 and validated using ddPCR. KEGG pathway analysis was done using mirPath v.3 (Diana Tools). 57 differentially expressed miRNAs were identified in OA vs C plasma, 45 differentially expressed miRNAs in OC vs C plasma, and 21 differentially expressed miRNAs in OA vs OCD plasma. Notably, miR-140-5p expression was observed to be elevated in OA synovial fluid suggesting that miR-140-5p may serve as a protective marker early on to attenuate OA progression. KEGG pathway analysis of differentially expressed plasma miRNAs showed relationships with glycan degradation, glycosaminoglycan degradation, and hippo signaling pathway. Interestingly, ddPCR was unable to validate the NGS data suggesting that isomiRs may play an integral role in miRNA expression when assessed using NGS technologies.</div

Statistical data of differentially expressed miRNAs equine plasma of OCD vs C with a 1.5-log2FC or greater change in expression (p < 0.05).

Statistical data of differentially expressed miRNAs equine plasma of OCD vs C with a 1.5-log2FC or greater change in expression (p < 0.05).</p

ddPCR analysis of miRNA expression in blood plasma and synovial fluid.

a) miR-20a; b) miR-181a; c) miR-196b; d) miR-486; and e) miR-140. (*p<0.05).</p

Volcano Plot of OA vs OCD plasma; LFC cut off = 1.5 and p-value cut off = 0.05; adjusted p-value was used; miRNAs marked in red are those shown to meet both the LFC and p-value cut offs.

Volcano Plot of OA vs OCD plasma; LFC cut off = 1.5 and p-value cut off = 0.05; adjusted p-value was used; miRNAs marked in red are those shown to meet both the LFC and p-value cut offs.</p