A3.15 A novel role of MIR-29a in inhibition of TAB1-mediated timp-1 production in dermal fibroblasts: implication for SSC pathogenesis

Background and Objectives Systemic sclerosis (SSc) is an autoimmune connective tissue disease characterised by fibrosis of skin and internal organs via accumulation of extra cellular matrix (ECM) proteins. This ECM deposition is observed when the homeostasis between matrix metalloproteinases (MMPs) and their natural inhibitors, eg. tissue inhibitor of metalloproteinases (TIMPs), is lost. MicroRNAs (miRs) are small regulatory RNAs that control gene expression by targeting 3’untranslated region (3’UTR) of various genes resulted in gene suppression. There is increasing evidence that miRs are key regulators of genes involved in fibrosis development. In particular, miR-29a was demonstrated to regulate ECM-associated genes in SSc patients. However, a better understanding of how epigenetic modulation by miR-29a prevents pathogenesis during SSc development is not fully defined. Materials and Methods Healthy control (HC) or SSc fibroblasts were cultured from skin biopsies. In some experiments, fibroblasts were transfected with miR-29a or control non-targeting miRNA and collagen, TIMP-1, MMP-1 and TGF-b activated kinase 1 binding protein 1 (TAB1) expression were measured using ELISA, qRT-PCR, Western Blotting, or functional collagen gel assay. In addition, HeLa cells (easy-to-transfect cells) were transfected with 3’UTR of TAB1 plasmid cloned downstream of firefly luciferase gene to assess TAB1 activity. HC fibroblasts and HeLa cells were also transfected with Target protectors. Target protectors are designed to specifically interfere with TAB1 seed region, therefore are able to block miR-29a activity. Results In this study, we found that TAB1 is a novel target gene of miR-29a that subsequently leads to decreased TIMP-1 expression. In particular, using computational approach and 3’UTR of TAB1 construct, we demonstrated that TAB1 is a bona fide target of miR29a. Furthermore, we showed that miR-29a not only leads to TIMP-1 downregulation, but also increased functional MMP-1 production resulting in collagen degradation. Blocking TAB1 activity by pharmacological inhibition (Oxozeanol) or TAB1 knockdown (siRNA against TAB1) resulted in TIMP-1 reduction, confirming TAB1-dependent TIMP-1 regulation. In addition, using Target protectors, TAB1 and TIMP-1 activity were derepressed due to miR-29a inhibition. Finally, miR-29a was able to reverse the profibrotic phenotype of SSc fibroblasts via down regulation of TIMP-1 and collagen expression. Conclusions This study demonstrates that miR-29a directly represses its novel target gene - TAB1 in dermal fibroblasts, which results in decreased TIMP-1. Exogenous miR-29a was also able to reverse profibrotic phenotype of SSc fibroblasts, thus highlighting the potential therapeutic role of miR-29a in SSc and fibrogenesis in general.