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RNA-Seq approaches to understand the role of ARNO/Cyth2 on IL-1beta-stimulated synovial fibroblasts.

ABSTRACT: The aim of this study was to explore the role of ARNO, also known as Cyth2, in synovial fibroblasts using NGS-derived transcriptome analysis (RNA-seq). Synovial fibroblasts were isolated from mouse synovium, and stimulated with IL-1beta for 12 hours in healthy cells and cells where Cyth2 expression was knocked-down by silencing RNA. Methods: Synovial fibroblasts were extracted from the synovium of healthy mice and expanded in vitro, RNA was extracted from naïve, IL-1β stimulated and IL-1β stimulated ARNO knock-down synovial fibroblast. Methods: Whole Transcriptome Profiling of synovial fibroblasts were generated by deep sequencing, in triplicate, using Illumina NextSeq™ 500 platform. Libraries were prepared using polyA selection (TruSeq stranded mRNA kit). Methods: The sequence reads that passed quality filters were aligned to mouse reference genome (GRCM38) using Hisat2 version 2.1.0. we mapped about 30 million sequence reads per sample (75 bp, paired-end) Methods: Featurecounts version 1.4.6 was used to quantify reads counts. Data quality control, non-expressed gene filtering, median ratio normalization (MRN) implemented in DESeq2 package and identification of differentially expressed (DE) genes were done using the R bioconductor project DEbrowser. Results: Firstly, We detected differentially expressed (DE) genes among three conditions, which pass the threshold of >4 fold, adjp <0.01. Then, we detected DE genes in IL-1β stimulated ARNO knock-down synovial fibroblast compared to IL-1β stimulated synovial fibroblast, which pass the threshold of >2 fold, adjp <0.01. DE genes reflect decreased cellular inflammatory response after ARNO knockdown Conclusions: Our results reflect an ARNO-mediated inflammatory response in synovial fibroblast, provides new opportunitties for targeting fibroblast in chronic arthritis and joint disease.

ORGANISM(S): Mus musculus

PROVIDER: GSE192488 | GEO | 2021/12/25

REPOSITORIES: GEO

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Project description:Synovial fibroblasts in persistent inflammatory arthritis have been suggested to have parallels with cancer growth and wound healing, both of which involve a stereotypical serum response program. We tested the hypothesis that a serum response program can be used to classify diseased tissues, and investigated the serum response program in fibroblasts from multiple anatomical sites and two diseases. To test our hypothesis we utilized a bioinformatics approach to explore a publicly available microarray dataset including RA, OA and normal synovial tissue, then extended those findings in a new microarray dataset representing matched synovial, bone marrow and skin fibroblasts cultured from RA and OA patients undergoing arthroplasty. The classical fibroblast serum response program discretely classified RA, OA and normal synovial tissues. Analysis of low and high serum treated fibroblast microarray data revealed a hierarchy of control, with anatomical site the most powerful classifier followed by response to serum and then disease. In contrast to skin and bone marrow fibroblasts, exposure of synovial fibroblasts to serum led to convergence of RA and OA expression profiles. Pathway analysis revealed three inter-linked gene networks characterising OA synovial fibroblasts: Cell remodelling through insulin-like growth factors, differentiation and angiogenesis through ?3 integrin, and regulation of apoptosis through CD44. We have demonstrated that Fibroblast serum response signatures define disease at the tissue level, and that an OA specific, serum dependent repression of genes involved in cell adhesion, extracellular matrix remodelling and apoptosis is a critical discriminator between cultured OA and RA synovial fibroblasts. Fibroblasts were isolated from synovium, bone marrow and skin tissue samples taken at the time of knee or hip replacement surgery from 12 rheumatoid arthritis patients meeting the 1987 ACR criteria and 6 osteoarthritis patients diagnosed on the basis of characteristic x-ray findings and the absence of features suggestive of inflammatory arthritis. Only one hip sample was present in either disease group. Fibroblasts were maintained in fibroblast medium (consisting of 81.3% RPMI 1640, 10% FCS, 0.81x MEM non-essential amino acids, 0.81mM sodium orthopyruvate, 1.62mM glutamine, 810U/ml penicillin and 81?g/ml streptomycin) at 37°C in a humidified 5% CO2 atmosphere.

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RNA-Seq approaches to understand the role of ARNO/Cyth2 on IL-1beta-stimulated synovial fibroblasts.

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