Session Title: Cytokines, Mediators, and Gene Regulation
Session Type: Abstract Submissions (ACR)
Background/Purpose: Long noncoding RNAs (lncRNA, >200nt) have recently emerged as regulators of gene expression functioning as signals, decoys, guides or scaffolds for transcription factors and/or the epigenetic machinery of the cell. The repressive epigenetic mark trimethylated histone 3 lysine 27 (H3K27me3) is transferred by the histone methyltransferase EZH2 which is up regulated in rheumatoid arthritis (RA) synovial fibroblasts (SF) and inducible by tumor necrosis factor (TNF)-α (Trenkmann M et al., ARD, 2011). The lncRNA HOX transcript antisense RNA (HOTAIR) has been associated with cancer and metastasis; it was shown to interact with EZH2 and proposed to guide H3K27 trimethylation of target genes (Wang KC and Chang HY, Mol Cell, 2011). Here, we studied expression, regulation and function of HOTAIR in SF.
Methods: Gene expression in SF was measured by SYBR Green or TaqMan real-time PCR with normalization to GAPDH. SF were stimulated with TNFα (10ng/ml; n=11) and interleukin (IL)-1β (1ng/ml; n=5). Osteoarthritis (OA)SF were transfected with a vector encoding EZH2 (n=4) or siRNA targeting HOTAIR (n=11). Chromatin from SF was precipitated with antibodies for histone 3 (H3), H3 methylation H3K4me3 and H3K27me3, and H3 acetylation (Ac), and the HOTAIR promoter was analyzed for epigenetic regulation.
Results: The expression of HOTAIR was strongly decreased (12.7-fold) in RASF (n=9) compared to OASF (n=13) (ΔCt 14.3±3.3 vs. 10.6±1.7, p=0.005). EZH2 over expression in OASF reduced the expression of HOTAIR by 30±17% (p<0.05) suggesting that EZH2-mediated methylation of H3K27 may regulate HOTAIR expression. Indeed, HOTAIR levels in RASF and OASF inversely correlated with the repressive H3K27me3 epigenetic mark in the promoter of HOTAIR (Spearman R=-0.8725, p<0.0001). In detail, the H3K27me3 to H3 ratio in RASF was 0.51±0.35 whereas the HOTAIR promoter in OASF showed much less H3K27 trimethylation (ratio to H3 0.25±0.23; p=0.0309). Interestingly, neither in RASF nor in OASF H3K4me3 or H3-Ac could be detected at the HOTAIR promoter. Stimulation of OASF with TNFα for 24h and 48h decreased HOTAIR levels by 63±16% and 56±26% (p<0.0001). IL-1β-stimulation reduced HOTAIR expression by 54±10% and 63±6%, respectively (p<0.0005). To identify a functional role for HOTAIR repression in RASF, we silenced HOTAIR in OASF using RNA interference. The silencing of HOTAIR significantly increased mRNA levels of the matrix metalloproteases (MMP) 3, 13 and 14 (MMP3 by 1.8±0.7-fold, MMP13 by 4.3±3.1-fold and MMP14 by 2.1±0.7-fold, p<0.05); in contrast, results for MMP1 were inconsistent. The TNFα-induced expression of MMP3 (51.6±21.2-fold) and MMP13 (6.9±2.6-fold) was further increased by silencing of HOTAIR (71.5±28-fold and 18.8±8.6-fold, p<0.05) whereas the IL-1β-stimulated expression was not significantly changed (n=5 each).
Conclusion: This is the first report of a differential expression of a lncRNA in RA. The expression of HOTAIR is strongly reduced in RASF via the repressive epigenetic mark H3K27me3. We demonstrate that HOTAIR silencing may account for the up regulation of matrix-degrading enzymes indicating a potential role for HOTAIR repression in the activated, matrix-destructive phenotype of RASF.
Masterswitch-FP7, IMI-BTCure, IAR,
R. E. Gay,
B. A. Michel,
L. C. Huber,
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ACR Meeting Abstracts - https://acrabstracts.org/abstract/the-epigenetically-repressed-long-noncoding-rna-hotair-influences-the-expression-of-matrix-metalloproteases-in-synovial-fibroblasts/