GSE192488
RNA-Seq approaches to understand the role of ARNO/Cyth2 on IL-1beta-stimulated synovial fibroblasts.
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-1ß stimulated and  IL-1ß 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-1ß stimulated ARNO knock-down synovial fibroblast compared to IL-1ß 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. Overall design: Our results show the transcriptome profile of naïve, IL-1ß stimulated and IL-1ß stimulated ARNO knock-down synovial fibroblast expanded in vitro