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Abstract Number: 2736

High Density Microarray Analysis Identifies Novel Differentially Expressed Long Noncoding RNAs In Rheumatoid Arthritis Synovial Fibroblasts

Mojca Frank Bertoncelj1, Michelle Trenkmann1, Christoph Kolling2, Beat A. Michel3, Renate E. Gay4 and Steffen Gay1, 1Center of Experimental Rheumatology, University Hospital Zurich and Zurich Center of Integrative Human Physiology (ZIHP), Zurich, Switzerland, 2Schultess Clinic, Zurich, Switzerland, 3Department of Rheumatology, University Hospital Zurich, Zurich, Switzerland, 4Center of Experimental Rheumatology, Zurich University Hospital, Zurich, Switzerland

Meeting: 2013 ACR/ARHP Annual Meeting

Keywords: Epigenetics, fibroblasts and rheumatoid arthritis (RA)

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Session Information

Title: Genetics and Genomics of Rheumatic Disease II

Session Type: Abstract Submissions (ACR)

Background/Purpose:

Long noncoding RNAs (lncRNAs) have emerged as key regulators of gene expression. Recently, several new susceptibility loci were identified for rheumatoid arthritis (RA), mapping to noncoding genomic regions (Eyre et al., Nat Gen 2012). Furthermore, we showed that the lncRNA HOTAIR is epigenetically repressed in RA synovial fibroblasts (SF), enhancing their activated phenotype. The aim of the present study was to determine the global lncRNA expression in RASF and to identify differentially expressed (DE) lncRNAs in RASF with a potential role in the pathogenesis of RA.

Methods: Total RNA was isolated from cultured synovial fibroblasts (SF), passages 5-6, from 3 RA and 3 osteoarthritis (OA) age-matched female patients. Sample labeling and array hybridization were performed according to the Agilent One-Color Microarray-Based Gene Expression Analysis protocol. Fluorescently-labeled cRNA was hybridized to human LncRNA Array v3.0 (Arraystar) detecting 30,586 lncRNAs and 26,109 mRNAs. The arrays were scanned by the Agilent Scanner G2505C and analyzed by the Agilent Feature Extraction software. The Agilent GeneSpring GX v12.0 software was used for data processing. To identify DE transcripts the absolute fold change of normalized signal intensities between RASF and OASF was calculated (cut-off value 1.5, p<0.05). Gene ontology (GO) and pathway analysis were performed. Fourteen selected DE lncRNAs were confirmed by qPCR in a larger cohort of age- and gender-matched RASF and OASF (n=10 each) with normalization to GAPDH.

Results:

The microarray analysis identified 225 DE RNA transcripts in RASF compared to OASF. Among these, 36 lncRNAs (x-fold range: 1.5-4.1) and 87 mRNAs (1.5-10.7) were significantly down-regulated, while 64 lncRNAs (1.51-3.5) and 38 mRNAs (1.5-3.3) were significantly upregulated in RASF. Among identified lncRNAs, small nucleolar RNA host gene 1 (SNHG1) (dCt±SD OASF: 6.92±0.29; RASF: 6.53±0.45, p=0.038) and RP11-39708.4 (dCt±SD OASF: 12.61±0.26; RASF: 12.07±0.69, p=0.042) were confirmed by PCR to be upregulated in RASF. Although their function is not known, it is of interest that RP11-39708.4 is a natural antisense transcript in the locus of fibroblast growth factor 14 and SNHG1 is a host gene for several small nucleolar RNAs, including SNORD22 and SNORD25-31. Among DE mRNAs, several transcripts linked to the pathogenesis of RA were found, such as interleukin 8, peroxisome proliferator-activated receptor (PPAR) gamma and sirtuin 1. Additionally, several novel DE mRNAs were identified, such as tumor protein p73, cell division cycle-associated protein 3 and chemokine-like receptor 1. DE mRNAs clustered to p53, Wnt and PPAR signaling and cell cycle-regulating pathways with significant enrichment of GO terms including cell adhesion molecules, cadherin and beta-catenin binding as well as mitosis and nuclear division.

Conclusion:

In this study we identified novel DE lncRNAs in RASF, including SNHG1 and RP11-39708.4. This is the first study determining the global lncRNA profile in combination with differential expression of mRNAs in RASF, which will be a powerful tool to identify novel gene regulating pathways in RA.


Disclosure:

M. Frank Bertoncelj,

IMI-BT Cure, IAR Epalinges, EURO-TEAM,

2;

M. Trenkmann,

EURO-TEAM, IMI BTCure, IAR Epalinges, KFSP USZ,

2;

C. Kolling,
None;

B. A. Michel,
None;

R. E. Gay,

EURO-TEAM, IMI BTCure, IAR Epalinges,

2;

S. Gay,

EURO-TEAM, IMI BTCure, IAR Epalinges,

2.

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