Session Title: Rheumatoid Arthritis - Human Etiology and Pathogenesis
Session Type: Abstract Submissions (ACR)
Comprehensive analysis of 260 miRNAs suggested a downregulation of miR-196a in rheumatoid arthritis (RA) synovial fibroblasts (SF) compared with osteoarthritis (OA) SF. Mature miR-196a originates from 2 regions in human genome: MIR-196-A1 (chromosome 17) and MIR-196-A2 (chromosome 12) both located in HOX- gene clusters. The altered expression of miR-196a has been reported in multiple conditions, such as cancer. Our aim is to analyze the expression and regulation of miR-196a in cells from patients with RA.
Methods Expression of pri-miRNA precursor and mature miR-196a was analyzed in RA/OA synovial tissue, SF under proinflammatory and hypoxic conditions, peripheral blood mononuclear cells (PBMC), synovial fluids and sera using TaqMan RealTime-PCR. Chromatin immunoprecipitation (ChIP) was used to analyze histone methylation and acetylation within both promoters MIR-196A1 and MIR-196A2 in SF. Ilumina sequencing with subsequent single assay verification was performed following Lipofectamine transfection with pre-miR-196a or anti-miR-196a to identify miR-196a targets genes.
Expression of miR-196a is significantly lower in RA synovial tissues (n=6) compared with OA (n=4, p=0.01) as well as RASF (n=19) compared with OASF (n=15, p<0.0001). No difference was observed in PBMC or synovial fluids while a lack of cell-free miR-196a was detected in RA and OA sera. The precursor pri-miR-196A1 was expressed neither in RA nor OA SF while pri-miR-196A2 was significantly downregulated in RASF vs. OASF (n=4 each, p<0.05). Expression of miR-196a in SF was not affected by proinflammatory cytokines (TNFα, IL-1β), TLR ligands (LPS), 1% hypoxia or 5’AZA mediated DNA demethylation. ChIP of MIR-196A2 promoter revealed significantly higher methylation of repressive H3K27me3 (p=0.005), lower methylation of activating H3K4me3 (p=0.001) and hypoacetylation of H3 (p=0.008) in RASF (n=13) compared to OASF (n=10) explaining the downregulation of the mature miR-196a in RASF. Using Ilumina sequencing after pre-miR transfection of RASF (n=2) HOXC8 and HOXA7 were identified as most likely direct miR-196a targets. Downregulation HOXC8 (p=0.01) as well as HOXA7 (p<0.0001) pre-miR-196a transfection (n=5), and upregulation of HOXC8 (p=0.11) and HOXA7 (p=0.56) and upon anti-miR-196a transfection (n=5) suggest these HOX genes as direct targets. However, unexpectedly, significant downregulation of HOXC8 was observed in RASF (n=17) vs. OASF (n=18, p=0.004) while the expression HOXA7 was not significantly different between RASF and OASF (p=0.91).
MiR-196a is one of the few miRNA showing a significant dowregulation in resident cells of synovial tissue in RA patients regulated by histone modifications. Although HOXC8 and HOXA7 are suggestive of being direct targets of miR-196a, their distinctive role in unique RASF behavior remains to be investigated. Given low expression of miR-196a in RASF and location within HOXC cluster, we hypothesize that expression of HOXC8, except regulation by miR-196a, may be influenced by additional epigenetic features similar to those regulating miR-196a.
MHCR no. 23728 ,
B. A. Michel,
MHCR no 23728,
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ACR Meeting Abstracts - https://acrabstracts.org/abstract/downregulation-of-mirna-196a-and-its-downstream-hoxc8-target-gene-in-rheumatoid-arthritis-synovial-fibroblasts/