Extracellular vesicle-derived microRNA crosstalk between equine chondrocytes and synoviocytes – an in vitro approach

We describe a novel technique to analyze the extracellular vesicle (EV)-derived microRNA (miRNA) crosstalk between equine chondrocytes and synoviocytes. Donor cells (chondrocytes, n=8; synoviocytes, n=9) were labelled with 5-ethynyl uridine (5-EU); EVs were isolated from culture media and incubated with recipient cells (chondrocytes [n=5] were incubated with synoviocyte-derived EVs, and synoviocytes [n=4] with chondrocyte-derived EVs). Total RNA was extracted from recipient cells; the 5-EU labelled RNA was recovered and sequenced. Differential expression analysis (using edgeR v3.32), pathway analysis, and miRNA target prediction were performed. Overall, 198 and 213 miRNAs were identified in recipient synoviocytes and chondrocytes, respectively. The top five most abundant miRNAs were similar for synoviocytes and chondrocytes (eca-miR-21, eca-miR-221, eca-miR-222, eca-miR-100, eca-miR-26a), and appear to be linked to joint homeostasis. There were nine differentially expressed (p<0.05) miRNAs (eca miR-27b, eca-miR-23b, eca-miR-31, eca-miR-191a, eca-miR-199a-5p, eca-miR-143, eca-miR-21, eca-miR-181a and eca-miR-181b) between chondrocytes and synoviocytes, which appear to be linked to migration of cells, apoptosis, cell viability of connective tissue cell, and inflammation. In conclusion, the reported technique was effective in recovering and characterizing the EV-derived miRNA crosstalk between equine chondrocytes and synoviocytes and allowed the identification of EV communicated miRNA patterns potentially related to cell viability, inflammation, and joint homeostasis. Overall design: This study investigated the extracellular vesicle (EV)-derived microRNA crosstalk between equine chondrocytes and synoviocytes. Donor cells (chondrocytes, n=8; synoviocytes, n=9) were labelled with 5-ethynyl uridine (5-EU); EVs were isolated from culture media and incubated with recipient cells (chondrocytes [n=5] were incubated with synoviocyte-derived EVs, and synoviocytes [n=4] with chondrocyte-derived EVs). EV-donor (chondrocytes, n=2; synoviocytes, n=2) and recipient (chondrocytes, n=1; synoviocytes, n=1) control cells were collected from six additional horses and run in parallel. Total RNA was extracted from recipient cells; the 5-EU labelled RNA was recovered and sequenced.