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. 2011 Feb;63(2):373-81.
doi: 10.1002/art.30115.

Altered expression of microRNA-203 in rheumatoid arthritis synovial fibroblasts and its role in fibroblast activation

Joanna Stanczyk  1 , Caroline Ospelt, Emmanuel Karouzakis, Andrew Filer, Karim Raza, Christoph Kolling, Renate Gay, Christopher D Buckley, Paul P Tak, Steffen Gay, Diego Kyburz
Affiliations

Altered expression of microRNA-203 in rheumatoid arthritis synovial fibroblasts and its role in fibroblast activation

Joanna Stanczyk et al. Arthritis Rheum. 2011 Feb.

Abstract

Objective: MicroRNA (miRNA) are recognized as important regulators of a variety of fundamental biologic processes. Previously, we described increased expression of miR-155 and miR-146a in rheumatoid arthritis (RA) and showed a repressive effect of miR-155 on matrix metalloproteinase (MMP) expression in RA synovial fibroblasts (RASFs). The present study was undertaken to examine alterations in expression of miR-203 in RASFs and analyze its role in fibroblast activation.

Methods: Differentially expressed miRNA in RASFs versus osteoarthritis synovial fibroblasts (OASFs) were identified by real-time polymerase chain reaction (PCR)-based screening of 260 individual miRNA. Transfection of miR-203 precursor was used to analyze the function of miR-203 in RASFs. Levels of interleukin-6 (IL-6) and MMPs were measured by real-time PCR and enzyme-linked immunosorbent assay. RASFs were stimulated with IL-1β, tumor necrosis factor α (TNFα), lipopolysaccharide (LPS), and 5-azacytidine (5-azaC). Activity of IκB kinase 2 was inhibited with SC-514.

Results: Expression of miR-203 was higher in RASFs than in OASFs or fibroblasts from healthy donors. Levels of miR-203 did not change upon stimulation with IL-1β, TNFα, or LPS; however, DNA demethylation with 5-azaC increased the expression of miR-203. Enforced expression of miR-203 led to significantly increased levels of MMP-1 and IL-6. Induction of IL-6 by miR-203 overexpression was inhibited by blocking of the NF-κB pathway. Basal expression levels of IL-6 correlated with basal expression levels of miR-203.

Conclusion: The current results demonstrate methylation-dependent regulation of miR-203 expression in RASFs. Importantly, they also show that elevated levels of miR-203 lead to increased secretion of MMP-1 and IL-6 via the NF-κB pathway and thereby contribute to the activated phenotype of synovial fibroblasts in RA.

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Figures

Figure 1
Figure 1
Increased basal expression of microRNA-203 (miR-203) in synovial fibroblasts from patients with established rheumatoid arthritis (RA). A, Basal expression of miR-203 in synovial fibroblasts from normal synovium (NSF), from patients with osteoarthritis (OASF), from patients with early RA (eRASF), and from patients with RA at a later stage of the disease (RASF). Bars show the mean. P values were determined by Mann-Whitney test with Bonferroni correction. B, Basal expression of miR-203 in OA synovial tissue (n = 6) and RA synovial tissue (n = 5). Data are presented as box plots, where the boxes represent the 25th to 75th percentiles, the lines within the boxes represent the median, and the lines outside the boxes represent the minimum and maximum values.
Figure 2
Figure 2
Epigenetic regulation of the expression of miR-203. A, Changes in the expression of miR-203 by RASFs (n = 6) after stimulation for 12 hours with interleukin-1 (IL-1), tumor necrosis factor (TNF), and lipopolysaccharide (LPS). Expression was measured by real-time polymerase chain reaction (PCR). Values are the mean ± SEM. B, CpG content (CG%) in 2,000 bp of miR-203 promoter and let-7a promoter. Gray shading shows particularly high density of CpG sites in the 680 bp adjacent to the miR-203 gene transcription initiation site (arrows). C, Changes in the expression of let-7a and miR-203 in healthy synovial fibroblasts (n = 2) after incubation for 1 week with 1 μM 5-azacytidine (5-aza). Expression was measured by real-time PCR. See Figure 1 for other definitions.
Figure 3
Figure 3
Increased levels of matrix metalloproteinase 1 (MMP-1) associated with increased expression of microRNA-203 (miR-203) in rheumatoid arthritis synovial fibroblasts (RASFs). A, RASFs were transfected with miR-203 precursor (pre–miR-203) or control pre-miR. MMP-1 transcripts were measured by real-time polymerase chain reaction after 24, 48, and 72 hours. Bars show the mean. P values (versus levels in control pre-miR–transfected cells [reference; set at 1]) were determined by Wilcoxon’s matched pairs test. B, MMP-1 levels in the supernatants of control pre-miR–transfected and pre–miR-203–transfected RASFs (both n = 5) were measured by enzyme-linked immunosorbent assay after 72 hours. Data are presented as box plots, where the boxes represent the 25th to 75th percentiles, the lines within the boxes represent the median, and the lines outside the boxes represent the minimum and maximum values. P value was determined by Wilcoxon’s matched pairs test. C, MMP-3, MMP-9, and MMP-13 transcripts were measured 48 and 72 hours after transfection of RASFs with control pre-miR or pre–miR-203. Levels in control pre-miR–transfected cells (set at 1) were used as the reference.
Figure 4
Figure 4
Increased levels of interleukin-6 (IL-6) associated with increased expression of miR-203 in RASFs. A, RASFs were transfected with pre–miR-203 or control pre-miR. IL-6 transcripts were measured by real-time polymerase chain reaction after 12, 24, 48, and 72 hours. Bars show the mean. P values (versus levels in control pre-miR–transfected cells [reference; set at 1]) were determined by Wilcoxon’s matched pairs test. B, IL-6 levels in the supernatants of control pre-miR–transfected and pre–miR-203–transfected RASFs (both n = 9) were measured by enzyme-linked immunosorbent assay (ELISA) after 48 hours. Data are presented as box plots, where the boxes represent the 25th to 75th percentiles, the lines within the boxes represent the median, and the lines outside the boxes represent the minimum and maximum values. P value was determined by Wilcoxon’s matched pairs test. C, The correlation between basal levels of IL-6 and basal levels of miR-203 in synovial fibroblasts was determined by Spearman’s rank correlation test. D, RASFs were transfected with control pre-miR or pre–miR-203. Twelve hours after transfection, RASFs were left without further treatment or were treated for a further 12 hours with the IκB kinase 2 inhibitor SC-514. IL-6 levels in the supernatants (n = 6) were measured by ELISA. Values are the mean ± SEM. P values were determined by Wilcoxon’s matched pairs test. See Figure 3 for other definitions.
Figure 5
Figure 5
Expression of predicted targets of miR-203 after miR-203 overexpression. RASFs were transfected with pre–miR-203 or control pre-miR. Transcripts of the genes for nuclear factor of κ light polypeptide gene enhancer in B cells inhibitor (NFkBia), NF-κB p100, NF-κB–repressing factor (NkRF), tumor necrosis factor α–induced protein 3 (TNFAIP3), Tax1-binding protein 1 (TaxBP1), ring finger protein 11 (RNF11), suppressor of cytokine signaling 3 (SOCS-3), SOCS-6, and methyl CpG binding protein 2 (MECP2) were measured by real-time polymerase chain reaction after 12, 24, 48, and 72 hours. Levels in control pre-miR–transfected cells (set at 1) were used as the reference. Bars show the mean. See Figure 3 for other definitions.
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