Detection of Oncofetal H19 RNA in Rheumatoid Arthritis Synovial Tissue

Abstract

The expression of oncofetal H19 RNA and its localization/cellular source was analyzed in synovial tissue (ST) and isolated synovial macrophages (Mphi) or synovial fibroblasts (SFBs) by reverse transcriptase-polymerase chain reaction (RT-PCR), in situ hybridization, and immunohistochemistry. RT-PCR showed significantly higher H19 expression in ST from patients with rheumatoid arthritis (RA) (P = 0.000) and osteoarthritis (OA) (P = 0.009) than in normal/joint trauma controls (N/JT), but comparable levels in reactive arthritis. In situ hybridization demonstrated strong signals in all RA-ST samples (n = 8), with > or =85% positive cells in the lining layer, diffuse infiltrates, and stroma regions. In lymphoid aggregates and endothelial cells only 20% were positive. RA-ST contained a significantly higher percentage of strongly positive lining cells than OA-ST and N/JT-ST. H19 RNA was expressed in both Mphi and SFBs, as confirmed by RT-PCR in isolated RA Mphi and SFBs (n = 3). In RA-SFBs, low constitutive H19 RNA expression in culture (10% fetal calf serum) was strongly increased on starvation (3.5-fold, 1% fetal calf serum), with or without the addition of interleukin-1beta (10 to 100 U/ml), tumor necrosis factor-alpha (1 to 25 ng/ml), or platelet-derived growth factor-BB (2.5 to 10 U/ml). In OA-SFBs, this starvation-induced increase was lower (twofold), reaching significant differences compared with RA-SFBs after stimulation with interleukin-1beta and platelet-derived growth factor-BB. In both RA- and OA-SFBs, the MAP-kinase ERK-1/2 pathway and the phosphatidylinositol-3 kinase pathway influenced H19 RNA expression, as shown by inhibitor studies. Significant overexpression of H19 RNA and its increased sensitivity to starvation/cytokine regulation in RA suggests a pathogenetic role of this oncofetal gene, possibly reflecting embryonal dedifferentiation of the adult ST and/or ongoing inflammatory/oxidative stress.

Predominant activation of MAP kinases and pro-destructive/pro-inflammatory features by TNF in early-passage synovial fibroblasts via TNF receptor-1: failure of p38 inhibition to suppress matrix metalloproteinase-1 in rheumatoid arthritis

“…Previous reports have shown a much weaker induction of IL6 protein by TNFa, possibly owing to the concurrent use of 10% FCS in cell culture (known to pre-stimulate cells). 35 The present results, therefore, indicate that TNF-R1 plays a pivotal part also for the induction of pro-inflammatory mediators.…”

Long non‐coding RNAs in rheumatoid arthritis

“…55 They also used in situ hybridization to confirm that H19 was strongly expressed in the diffuse infiltrates, lining layer and stroma regions of synovial tissues from RA patients. In particular, H19 was expressed in synovial macrophages and fibroblasts.…”

The Increasing Complexity of the Oncofetal H19 Gene Locus: Functional Dissection and Therapeutic Intervention

“…One model that was thoroughly examined for H19 expression in this context is rheumatoid arthritis (RA) [31], an inflammatory disease resembling cancer in many features, like, for example, the metastatic-like invasiveness of the synovial tissue into cartilage and bone, expression of several proto-oncogenes and mutations in p53. As mentioned above, H19 is upregulated in hypoxic cancerous cells, as well as upon starvation stress and reacts to several stress related factors, such as thioredoxin, MMK1, NF-Kb, JNK2, TNF-α and IL-6.…”