MicroRNA-146a is linked to pain-related pathophysiology of osteoarthritis

Abstract

Because miR-146a is linked to osteoarthritis (OA) and cartilage degeneration is associated with pain, we have characterized the functional role of miR-146a in the regulation of human articular cartilage homeostasis and pain-related factors. Expression of miRNA 146a was analyzed in human articular cartilage and synovium, as well as in dorsal root ganglia (DRG) and spinal cord from a rat model for OA-related pain assessment. The functional effects of miR-146a on human chondrocytic, synovial, and microglia cells were studied in cells transfected with miR-146a. Using real-time PCR, we assessed the expression of chondrocyte metabolism-related genes in chondrocytes, genes for inflammatory factors in synovial cells, as well as pain-related proteins and ion channels in microglial cells. Previous studies showed that miR-146a is significantly upregulated in human peripheral knee OA joint tissues. Transfection of synthetic miR-146a significantly suppresses extracellular matrix-associated proteins (e.g., Aggrecan, MMP-13, ADAMTS-5, collagen II) in human knee joint chondrocytes and regulates inflammatory cytokines in synovial cells from human knee joints. In contrast, miR-146a is expressed at reduced levels in DRGs and dorsal horn of the spinal cords isolated from rats experiencing OA-induced pain. Exogenous supplementation of synthetic miR-146a significantly modulates inflammatory cytokines and pain-related molecules (e.g., TNFα, COX-2, iNOS, IL-6, IL8, RANTS and ion channel, TRPV1) in human glial cells. Our findings suggest that miR-146a controls knee joint homeostasis and OA-associated algesia by balancing inflammatory responses in cartilage and synovium with pain-related factors in glial cells. Hence, miR-146a may be useful for the treatment of both cartilage regeneration and pain symptoms caused by OA.

Fig 1

Human articular chondrocyte isolated from knee cartilage (grade 0–1) in monolayer were transfected with synthetic miR-146a(10 and 20 pmol). IL-1 was administered in parallel to provoke a catabolic response. After 48 hours of tansfection, the total RNA was isolated and analyzed of MMP13, ADANTS5, Aggrecan and collage II gene were analyzed by real-time RT-PCR(A,B,C,D). The total protein was isolated and analyzed for MMP13 protein expression by western blotting (E).

Fig 2

Human articular synovial cells from knee cartilage (grade 0–1) were transfected with synthetic miR-146a(10 and 20 pmol). IL-1 was administered in parallel to provoke a catabolic response. After 48 hours of transfection, the total RNA was isolated and COX2 (A), TRAF6(B) IRAK1(C), IL-8(D), ADAMTS5(E) and MMP13(F) gene were analyzed by real-time RT-PCR

Fig 3

Rats received a single intra-articular injection of MIA (0.5 mg) or saline (control) were euthanized at week 2 and week 4. Left lumbar DRGs at levels of 3/4, 4/5 and 5/6 ( A ) and dorsal horn of the spinal cords (B) were harvested and relative expression of micro-RNA 146a were analyzed using real-time PCR

Fig 4

Human astroglial cells were transfected with micro RNA 146a (10 and 20 pmol). After 48 hours of transfection, the total RNA was isolated and COX2 (A), iNOS (B), TNF-α (C), IL-6(D), IL-8(E) RATNS(F) and TRPV1(G) gene were analyzed by real-time RT-PCR.