Project description:Pathway expression analysis in systemic sclerosis revealed key mediators driving pathology within each of the intrinsic gene expression subsets. Genome-wide expression profiling in systemic sclerosis (SSc) has identified four 'intrinsic' subsets of disease (fibroproliferative, inflammatory, limited, and normal-like), each of which appear to be driven by distinct signaling pathways. Here we examine experimentally derived gene expression signatures for thirteen agonists to better understand the molecular mechanisms underlying each intrinsic subsets. Pathway-specific gene signatures were compared against skin biopsy microarray data consisting of 329 microarray hybridizations from 287 unique biopsies representing 111 patients. Hierarchical clustering recapitulated the four SSc 'intrinsic' gene expression subsets, along with an intermediate subset exhibiting both inflammatory and fibroproliferative gene expression signatures. The fibroproliferative subset is most strongly associated with the PDGF gene signature, while the inflammatory subset demonstrated strong activation of NF-kappaB, characterized by early induction of TH2 signals, which transitions to a more TH17-like immune response over time. The limited subset was associated with IFNalpha signaling, combined with strong downregulation of PDGF signaling. The inflammatory-proliferative subset shows downregulation of NF-kappaB-associated pathways in conjunction with increased PDGF signaling, suggestive of a transitional state linking the inflammatory and fibroproliferative subsets. IL-13 signaling was strongly associated with early disease, while TGFbeta signaling was associated with more severe disease. Together these data suggest a multi-step, progressive model of disease pathogenesis driven by distinct pathways. Targeting these pathways based upon a patient's underlying disease subset should improve therapeutic outcomes. In vitro treatment of human dermal fibroblasts was performed to identify pathway-specific gene signatures. These gene signatures were then compared against systemic sclerosis skin biopsy microarrays to determine the overall contribution of each pathway to disease. This study includes re-analysis of 247 skin biopsy samples from GSE9285, GSE32413, and GSE45485. Links to these Samples are provided below. The re-processed normalized data for these Samples are included in the supplementary file 'GSE56308_247_Skin_Biopsy_Reanalysis_Samples.txt'.

Project description:Purpose: The present study was carried out to investigate the global m6A modified RNA pattern and possible mechanisms underlying the pathogenesis of keloid. Method: 14 normal skin and 14 keloid tissue were firstly collected on clinic. Then 3 samples from each group were randomly selected to be verified with the Western Blot to determine the level of methyltransferase and demethylase. The total RNA of all samples in each group was isolated and subjected to the analysis of MeRIP-sequencing and RNA-sequencing. With the software of MeTDiff and htseq-count, the m6A peaks and differentially expressed genes (DEGs) were determined within the fold change > 2 and p -value < 0.05. The top 10 pathways of m6A modified genes in each group and the differentially-expressed genes were enriched by the Kyoto Encyclopedia of Genes and Genomes signaling pathways. Finally, the closely associated pathway was determined with the Western Blot and immunofluorescence staining. Results: There was a higher protein level of WTAP and Mettl3 in the keloid than in the normal tissue. 21,020 unique m6A peaks with 6573 unique m6A-associated genetic transcripts appeared in the keloid samples. In the normal tissue, 4028 unique m6A peaks with 779-associated modified genes appeared in the keloid. In the RNA-sequencing, there were 847 genes significantly changed between these groups, transcriptionally. The genes with m6A methylated modification and the upregulated differentially expressed genes between two tissues were both mainly related to the Wnt signaling pathway. Moreover, the hyper-m6A modified Wnt / β-catenin pathway in keloid was verified with western blotting. From immunofluorescence staining results, we found that the accumulated fibroblasts were under a hyper-m6A condition in the keloid and the Wnt/β-catenin signaling pathway was mainly activated in the fibroblasts. Conclusion: The fibroblasts in the keloid were under a cellular hyper-m6A methylated condition, and the hyper-m6A modified highly-expressed Wnt / β-catenin pathway in the dermal fibroblasts might promote the pathogenesis of keloid.

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STAT3 Signaling Pathway in Keloid Pathogenesis Implicated by Preliminary Transcriptome and ATAC Analyses

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