Session Type: Abstract Submissions (ACR)
Transforming growth factor beta (TGFβ) modulates microRNA (miRNA) biogenesis in a variety of cell types. The expression of miRNAs is deregulated in the synovial fibroblasts from patients with rheumatoid arthritis (RASF). However, the role of TGFβ in the regulation of miRNAs in RASF was not investigated so far. The aim of the current study was to investigate the role of TGFβ in the regulation of miRNAs and in the inflammatory and matrix-destructive properties of RASF. Moreover, the effects of TGFβ on TNFα-induced signaling were analyzed.
Synovial tissues were obtained from RA patients undergoing joint replacement surgery. RASF (n=6) were stimulated with 10 ng/ml TGFβ, 10 ng/ml TNFα or with TGFβ and TNFα together in cultivation medium supplemented with 5% FCS for 24, 48 or 72 hours. Total RNA was isolated using Qiagen miRNeasy Kit. Global expression of miRNAs was analyzed by human miRNA Array analysis (Card A, Life Technologies) and verified by measurement of single miRNAs using real-time PCR with miRNA-specific TaqMan primers. The levels of interleukins (ILs) and matrix metalloproteinases (MMPs) were detected by Real-time TaqMan and SYBR green PCR.
The global miRNA expression profile was altered by stimulation of RASF with TGFβ after 48h in all patients. The expression of 29 miRNAs was downregulated by 25-70% whereas the levels of 32 miRNAs were upregulated by 50-800%. TGFβ induced changes in several miRNAs that were previously reported to be deregulated in RASF. In particular, the levels of miR-155, miR-221, miR-222 and miR-335 were downregulated, while the expression of miR-18a, miR-22, miR-145 and miR-203 was significantly increased. We then investigated whether TGFβ can modify the effects of TNFα. Stimulation with TNFα alone increased the levels of miR-155 by 8.2-fold±1.2 (p=0.01), while the co-stimulation with TNFα and TGFβ resulted only in 4.3-fold±0.5 (p=0.03) increase in miR-155 levels. Treatment with TNFα decreased the expression of miR-145 by 0.7-fold±0.1 (p=0.03). However, co-stimulation with TNFα and TGFβ reversed this effect completely and increased the levels of miR-145 by 2.0-fold±0.3 (p=0.03). In contrast, TGFβ amplified TNFα-induced reduction in the expression of miR-222 and miR-335. Stimulation with TGFβ or TNFα alone already significantly reduced the expression of these miRNAs and co-stimulation with TGFβ and TNFα together led to a further decrease in the levels of miR-222 (by 0.35-fold±0.05, p=0.03) and miR-335 (by 0.4-fold±0.08, p=0.02). Moreover, TGFβ mitigated the matrix-destructive activities of RASF that were induced by TNFα by strongly decreasing TNFα-induced MMP1 expression (by 16-fold, p=0.04). In contrast TNFα-initiated IL6 production was increased by TGFβ(by 1.7-fold, p=0.03).
In the current study we found that TGFβ modulates the expression of miRNAs involved in the pathogenesis of RA. Moreover, TGFβ influences the inflammatory and matrix-destructive activities of RASF by reduction of TNFα-induced MMP1 expression and by enhancement of TNFα-initiated IL6 production. Thus, TGFβ has dual effects in the regulation of RASF and exhibits pro-inflammatory as well as anti-matrix-destructive properties.
R. E. Gay,
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ACR Meeting Abstracts - https://acrabstracts.org/abstract/transforming-growth-factor-beta-is-a-major-regulator-of-micro-rna-synthesis-in-rheumatoid-arthritis-synovial-fibroblasts/