1. Identification of synovial fluid microRNA signature in knee osteoarthritis: differentiating early- and late-stage knee osteoarthritis 
Y.-H. Li, G. Tavallaee, T. Tokar, A. Nakamura, K. Sundararajan, A. Weston, A. Sharma, N.N. Mahomed, R. Gandhi, I. Jurisica, M. Kapoor 
Osteoarthritis and Cartilage 
Volume 24, Issue 9, Pages (September 2016)
DOI: /j.joca
Copyright © 2016 Osteoarthritis Research Society International Terms and Conditions

2. Fig. 1
a. Outline of the research design. b. Heat map of top 30 hits identified from a miRNA PCR array screening of 752 miRNAs in SF from four patients with early-stage OA and four patients with late-stage OA. Each row represents one miRNA, and each column represents one sample. The color scale shown on the top illustrates the relative expression level of a miRNA across all samples. c. Significant candidate miRNAs identified to be differentially expressed between late- and early-stage OA. Six miRNAs were up-regulated and five miRNAs were downregulated in late-stage OA SF compared to early-stage OA SF. Three miRNAs were only detected in late-stage OA SF. Results are represented as geometric mean fold-changes ±95% Confidence Interval.
Osteoarthritis and Cartilage  , DOI: ( /j.joca )
Copyright © 2016 Osteoarthritis Research Society International Terms and Conditions

3. Fig. 2
a. Validation study confirmed seven miRNAs (miR-23a-3p, miR-24-3p, miR-27a-3p, miR-27b-3p, miR-29c-3p, miR-34a-5p and miR-186-5p) were significantly increased in the SF of late stage OA (n = 26) compared to early-stage OA (n = 22). A numerical decrease in the expression of miR-27a-5p was also observed in SF of late-stage OA compared to early-stage OA. The expression of miRNAs was determined by quantitative PCR and normalized to miR-100-5p. Results are presented as the fold change using geometric mean ± 95% Confidence Interval (*P < 0.001). b. Comparative chart showing identification of differentially expressed miRNAs in screening and validation stages in late-stage compared to early-stage OA SF. c. Expression of miR-378a-5p was detected in majority of late-stage OA SF (23 out of 26, 88% detection) while largely undetectable in early-stage SF (15 out of 22, 32% detection) using Cq value of 37 as a cut-off. Cq values in undetectable samples were recorded as 40. KL grade I–IV.
Osteoarthritis and Cartilage  , DOI: ( /j.joca )
Copyright © 2016 Osteoarthritis Research Society International Terms and Conditions

4. Fig. 3
The source and release of validated miRNAs: The expression of miRNAs in the explant culture of cartilage, synovial tissue and synovial culture medium supernatant in response to (10 ng/ml) IL-1β treatment. a. Fold change in the expression of individual miRNAs in the cartilage explant tissue (n = 8/group) was determined by quantitative PCR and normalized to U6 with or without IL-1β stimulation. Results are presented as geometric mean ± 95% Confidence Interval. b. Fold change in the expression of individual miRNAs in the OA synovial explant tissue (n = 5/group) analyzed by quantitative PCR and normalized to U6 with or without IL-1β stimulation. Results are presented as geometric mean ± 95% Confidence Interval (*P < 0.001). c. Fold change in the expression of miRNAs in the culture medium of synovial tissue analyzed by quantitative PCR and normalized to miR-100-5p in response to IL-1β stimulation (n = 5/group). Results are presented as geometric mean ± 95% Confidence Interval. d. Schematic diagram of miRNA dysregulation in the synovium and cartilage, and release of miRNAs (miR-23a-3p and 27b-3p) into the SF from synovium in response to IL-1β-stimulation.
Osteoarthritis and Cartilage  , DOI: ( /j.joca )
Copyright © 2016 Osteoarthritis Research Society International Terms and Conditions

5. Fig. 4
a. Network of the SF-circulating miRNAs and their target genes. miRNAs are represented by white squares, their size corresponds to the number of gene targets. Target genes are represented by circles, whose color indicates the biological process given gene is primarily involved in. Red color highlights significantly targeted genes, i.e., genes targeted by more circulating miRNAs than expected by chance with respect to the total number of miRNAs targeting given gene (P < 0.05, hypergeometric test). The most targeted genes RC3H1, QKI are targeted by six out of eight circulating miRNAs and remaining 14 genes that are listed, are targeted by five miRNAs. Edges of the network were colored according to the product of the ranks of the confidence score that has been obtained across multiple resources, lower values indicate higher plausibility of the given miRNA-target couple. b. Heat map of the overlap between miRNAs targets as measured by JI. As shown, miR-27a-3p and 27b-3p share substantial number of their gene targets, while targets of the mir-378a-5p overlap poorly with targets of the other miRNAs. c. Barplot depicting the pathways that are significantly enriched by the significantly targeted genes (P < 0.05 and at least two genes). Bar height indicated P-value of the enrichment, color indicates the database source, and the numbers above indicate number of the miRNA targeted genes in the given pathway.
Osteoarthritis and Cartilage  , DOI: ( /j.joca )
Copyright © 2016 Osteoarthritis Research Society International Terms and Conditions

6. Fig. 5
a. Synovium: Treatment of OA synovial explants with IL-1β resulted in a significant reduction in the expression of RC3H1 and QKI (n = 8/group). Treatment of synovial explants with miR-27b-3p mimic resulted in a significant increase in miR-27b-3p expression and significant decrease in the expression of RC3H1 and QKI (n = 7). b. Cartilage: Treatment of cartilage explants with IL-1β resulted in a significant reduction in the expression of QKI with no change in RC3H1 expression (n = 6/group). Treatment of OA cartilage explants with miR-27b-3p mimic resulted in a significant increase in miR-27b-3p expression and significant decrease in the expression of QKI with no change in the expression of RC3H1 (n = 6). Results are presented as geometric mean ± 95% Confidence Interval.
Osteoarthritis and Cartilage  , DOI: ( /j.joca )
Copyright © 2016 Osteoarthritis Research Society International Terms and Conditions