Background/Purpose:

Microvesicles (MVs) (100-1000 nm diameter) are subcellular particles that are enriched in nucleic acid, including microRNA (miR), which may be transferred from cell to cell as a novel form of intercellular communication.  In this study, we wished to characterize the microRNA content of microvesicles purified from the synovial fluid of patients with osteoarthritis or rheumatoid arthritis.  Furthermore, we wished to determine whether predominant miR species identified in RA SF MVs may be transferred to recipient fibroblast-like synoviocytes (FLS) in vitro.

Methods:

Discarded synovial fluid (SF) from patients with established diagnoses of osteoarthritis (OA) (n=5) or rheumatoid arthritis (RA) (n=5) obtained during routine clinical care was transferred into tubes containing acid-citrate-dextrose and stored at 4°C until further processing.  SF was treated with thromboplastin D, centrifuged at 300 g x 10 minutes and 3000 g x 15 minutes to remove cells and cellular debris, respectively, and filtered through a 0.8 mm filter.  Next, SF was placed into ultracentrifuge tubes diluted with sterile PBS in a 1:8 ratio and ultracentrifuged at 100,000 g x 90 min using a Beckman 70.1Ti rotor.   Purified MV pellets were resuspended in sterile PBS at one-fifth the original volume and quantified using a Nanosight LM10 instrument.  MicroRNA from purified MV pellets was purified using a miRNeasy Serum/Plasma Kit (Qiagen), reverse transcribed, and subject to miRNA analysis using miScript miRNA PCR Serum/Plasma Arrays.  PCR array data were analyzed using the Qiagen GeneGlobe Data Analysis Center online software tool, normalizing to global CT mean of expressed miRNAs, using a cutoff of CT=37.  Purified RA SF MVs were incubated with OA FLS for 16 hours, and pri-miR, pre-miR, and mature miR expression by FLS was determined by qPCR.

Results: Purified RA SF MVs purified in this manner were significantly greater than OA SF MVs (OA vs. RA 24.95 vs. 53.55 million/ml, p=0.0001).  A total of 39 miRs showed greater than 2-fold statistically significant differences in expression between groups (p<0.05).  Of these miRs, hsa-mir-223-3p expression was significantly upregulated in the RA SF MV group (102.4 fold regulation, p=0.005) when compared with the OA SF MV group.  Addition of purified RA SF MVs to cultured human FLS induced expression of mir-223, but not pri-miR-223 or pre-mir-223, indicating that mir-223 in FLS was of exogenous (i.e. microvesicle) origin.

Conclusion: Microvesicle numbers in RA synovial fluid are greater than in OA synovial fluid, consistent with other previously published reports.  We found significantly increased expression of mir-223-3p in RA SF MV, likely reflecting myeloid cell activation in the inflamed joint, and demonstrated that mir-223 may be transferred to recipient FLS in vitro.

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ACR Meeting Abstracts - https://acrabstracts.org/abstract/microvesicle-associated-hsa-mir-223-3p-is-elevated-in-rheumatoid-synovial-fluid-compared-with-osteoarthritis-synovial-fluid/