Background/Purpose: Systemic sclerosis (SSc) is a complex autoimmune disease involving the immune system, vasculature and extracellular matrix [1]. Dysregulation of the Wnt pathway has been implicated in the development of fibrosis in SSc and is proposed to contribute to a failure to maintain tissue homeostasis and appropriate immune response [2]. The objective of this research study was to explore the role of altered microRNA expression in the skin of affected individuals and their role in the dysregulation of Wnt pathway in the development of fibrosis in black South African SSc patients with early, diffuse disease (dcSSc).

Methods:  Skin biopsies from eight black South African patients with dcSSc who fulfilled the 1987 ACR classification criteria, samples from both the forearm (affected skin) and the back (unaffected skin), and eight ethnically matched healthy control skin samples were examined. sRNA sequencing libraries were prepared for 50bp single-end sequencing on the Illumina HiSeq machine. The alignment software used was TopHat and Cufflinks [3], with downstream analyses done using mirDeep and SMARTAR [4]. Count data was analysed using DESeq2 [5]. Differential expression was considered significant if the adjusted p-value was <0.05 (Benjamini–Hochberg rule). For this study both TargetScan and miRanda were used to identify the Wnt pathway gene targets of the significantly differentially expressed miRNAs.

Results: The sRNA-seq data showed differential expression of 31 miRNAs that target the Wnt pathway genes, including miR-335 and miR-204 that are important regulators of normal tissue development. Ten miRNAs were differentially expressed only in the affected SSc skin samples when compared to controls. One of these miRNAs, miR-194- 5p was predicted to target 6 Wnt pathway genes, CCND2, EP300, FRZB, MMP7, PRICKLE1 and WIF1. Another, miR-326 was predicted to target five of the Wnt pathway genes, WNT5A, LEF1, NKD1, TCF7L1 and WIF1.Other significantly differentially expressed miRNAs included miR-15a-5p, miR-15b-5p, miR-375, miR-18b-5p and miR20b-5p, all of which are predicted to target Wnt pathway genes and have previously associated with fibrosis, autoimmunity or SSc. Table 1 summarises the main miRNA differential expression data. Experimental evidence suggests that perfect seed-region paring of the miRNA with the target mRNA is not necessarily a reliable predictor for miRNA interactions and could explain why some of the sites predicted in-silico are non-functional [6] and why validation of the outcome pf differential miRNA expression is essential.

Conclusion:  This study revealed a number of miRNAs were differentially expressed between SSc patients and controls indicating that epigenetic changes play an important role in the pathogenesis, progression and clinical features of the disease. These changes may be triggered by environmental changes and in the context of a disease susceptible genotype. This is consistent with the multifactorial nature of scleroderma. Thedifferentially expressed miRNAs are predicted to target genes within the Wnt pathway and could result in dysregulated gene expression disrupting the Wnt pathway. This highlights the potential for these miRNAs to be investigated as biomarkers of SSc and as potential novel targets for therapeutic intervention. References:

1. Abraham, D.J., et al., Overview of pathogenesis of systemic sclerosis. Rheumatology (Oxford), 2009. 48 Suppl 3: p. iii3-7.

2. Bhattacharyya, S., J. Wei, and J. Varga, Understanding fibrosis in systemic sclerosis: shifting paradigms, emerging opportunities. Nat Rev Rheumatol, 2012. 8(1): p. 42-54.

3. Trapnell, C., et al., Differential gene and transcript expression analysis of RNA-seq experiments with TopHat and Cufflinks. Nat Protoc, 2012. 7(3): p. 562-78.

4. Friedlander, M.R., et al., Discovering microRNAs from deep sequencing data using miRDeep. Nat Biotechnol, 2008. 26(4): p. 407-15.

5. Love, M.I., W. Huber, and S. Anders, Moderated estimation of fold change and dispersion for RNA-seq data with DESeq2 bioRxiv, 2014.

6. Kuhn, D.E., et al., Experimental validation of miRNA targets. Methods, 2008. 44(1): p. 47-54.

Table 1.Significantly differentially expressed miRNAs in affected and unaffected SSc skin compared to controls that are predicted to target Wnt pathway genes.

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