Background/Purpose:   Systemic sclerosis (SSc) is an autoimmune disease with a heterogenous and complex phenotype. Previously, our lab has identified four gene expression subsets (fibroproliferative, inflammatory, limited and normal-like) among SSc patients by their gene expression in skin using DNA microarrays. We have extended these findings by using RNA-Seq in a subset of SSc skin biopsies to detect mRNA levels, splice variants, novel non-coding RNAs, and coding region SNPs in a lower background signal over microarray. 

Methods: We performed RNA-Seq on eight SSc patients and four healthy controls and skin biopsies. The eight SSc patients included five individuals that mapped to the inflammatory subset and three from the fibroproliferative subset. We sequenced the small and large RNA fraction extracted from each biopsy by Illumina Solexa sequencing. We obtained 90-100 million 50 bp paired-end reads for the mRNA fraction and 25 – 50 million 36 bp reads for the miRNA fraction. 

Results:   Our analyses reveal significant (p<0.05) gene expression differences between healthy controls and SSc patients, as well as between intrinsic subsets. Specifically, we found >1000 genes are significantly expressed in the inflammatory and the fiboproliferative subsets of patients. Many of the significant genes are involved in cellular proliferation or immune responses, consistent with results found by DNA microarray hybridization. We did not observe any significant differential splicing between the healthy controls and the SSc patients. We identified 228 novel long non-coding RNAs (lncRNAs) that are significantly differentially expressed in the inflammatory subset and fibroproliferative subsets. The lncRNAs differentially expressed in the inflammatory subset map to Gene Ontology terms including inflammatory response, immune response, response to wounding, and defense response and those in the fibroproliferative group map to cell cycle, M phase, and RNA metabolism.  We also identified 54 miRs differentially expressed in the inflammatory subset of SSc patients.  These include the well-characterized miR21 that has previously been reported to be differentially expressed in SSc as well as many novel miRs.  We have previously shown CCL2 to be highly expressed in the inflammatory subset of SSc and inhibiting its function prevents development of disease in the sclGVHD mouse model.  We now identify a novel miR with decreased expression that is predicted to target the 3’UTR of CCL2.  We show that transfection of the CCL2-targeting miR, but not a negative control miR, into RAW264.7 macrophage cells expressing a heterologous Luciferase-CCL2 (3’UTR) reduced luciferase activity by 74%.

Conclusion:   To summarize, we conducted the first comprehensive RNA-Seq study in SSc skin and identified differentially expressed non-coding RNAs genome-wide.  Our findings show that a complex network of regulatory factors controls the disease specific gene expression subsets in SSc skin.

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ACR Meeting Abstracts - https://acrabstracts.org/abstract/rna-seq-and-mir-seq-analysis-of-ssc-skin-across-intrinsic-gene-expression-subsets-shows-differential-expression-of-non-coding-rnas-regulating-ssc-gene-expression/