Background Long noncoding RNA (lncRNA) have emerged as key regulators of gene expression. Recently, new susceptibility loci were identified for severity of rheumatoid arthritis (RA), mapping to different lncRNA genes (Knevel et al., Ann Rheum Dis 2013). In addition, we have shown that the global profile of lncRNA is altered in rheumatoid arthritis synovial fibroblasts (RASF) compared to osteoarthritis synovial fibroblasts (OASF).
Objectives To determine the influence of pro-inflammatory cytokines on the expression and function of lncRNA in RASF.
Methods The global lncRNA profile of synovial fibroblasts (SF) from 3 RA and 3 OA age-matched female patients was determined using human LncRNA Array v3.0 (Arraystar). Selected differentially expressed lncRNA were validated by qPCR in RASF and OASF (n=10 each) with normalization to GAPDH. RASF were stimulated with TNFα (10ng/mL) or IL1β (1ng/mL) for 24h. The expression of lncRNA, including the lncRNA ANRIL (antisense non-coding RNA in the INK4 locus), was measured by real time PCR. For functional studies, ANRIL was silenced in RASF (72h) by transfecting ANRIL GapmeR (10nM) using Lipofectamine 2000 and, after stimulation with TNFα (24h), the levels of interleukin 6 (IL6), IL8, matrix metalloproteinase 1 (MMP1) and MMP3 were measured by real-time PCR and ELISA. Proliferation and apoptosis of RASF were determined by the BrdU assay and flow cytometry using Annexin V/PI staining, respectively.
Results The microarray analysis identified 36 significantly downregulated and 64 significantly upregulated lncRNA in RASF compared to OASF. So far, small nucleolar RNA host gene 1 was validated by PCR as a novel lncRNA being upregulated in RASF (dCt±SD OASF: 6.92±0.29; RASF: 6.53±0.45, p=0.038), its expression, however, was not affected by TNFα or IL1β. In contrast, the levels of lncRNA MALAT1, GAS5, MEG3 and ANRIL, although not different in RASF vs. OASF, were significantly influenced by pro-inflammatory cytokines. Whereas MALAT1 (x-fold mean±SD: 0.58±0.19, p=0.0005, n=8) and GAS5 (0.52±0.11, p<0.0001, n=7) were downregulated, ANRIL (x-fold median: 1.61, range: 1.34-1.72, p=0.008, n=8) was upregulated by TNFα. Furthermore, MEG3 levels (1.62±0.25, p=0.002, n=7) were increased and MALAT1 levels were decreased (0.66±0.33, p=0.02, n=8) by IL1β, the expression of GAS5 and ANRIL, however, was not altered by IL1β. Silencing of ANRIL (by 66±15%, p=0.02, n=3) in RASF (n=5) significantly decreased the levels of TNFα-induced MMP1 (by 56±19%, p=0.003) and MMP3 (by 52±10%, p=0.036) mRNAs and inhibited TNFα-induced secretion of MMP1 (by 42±17%, p=0.002) and MMP3 (by 34±29%, p=0.06), but did not alter the levels of IL6 and IL8. Proliferation and apoptosis of TNFα-stimulated RASF (48h) were not affected by ANRIL silencing.
Conclusions TNFα and IL1β, the major pro-inflammatory cytokines in RA, significantly affect the lncRNA expression in RASF exhibiting lncRNA specific regulatory effects. Furthermore, the TNFα-upregulated lncRNA ANRIL enhances TNFα-induced expression of MMPs, thereby contributing to the matrix-destructive properties of RASF. These data indicate an important and novel role of lncRNA in the pathogenesis of RA.
Acknowledgements IMI-BTCure, IAR Epalinges, EURO-TEAM, EMDO
Disclosure of Interest None declared