Background/Purpose: MicroRNA-155 plays a critical role in various physiological and pathological processes including cell proliferation and migration, epithelial- mesenchymal transition, as well as inflammation. Previous research found that miR-155 could promote the development of murine pulmonary fibrosis. The aim of this study was to evaluate the role of miR-155 in a bleomycin induced mouse model of systemic sclerosis (SSc) and to investigate the potential mechanisms involved.

Methods: miR-155 knock-out (KO) C57BL/6 and background controlled mice were given bleomycin subcutaneously every other day for 21days to induce experimental fibrosis. Dermal thickness and collagen deposition were determined histologically by Sirius red dying. Tissue fibroblast number was measured through α-smooth muscle actin (α-SMA) staining by immunohistochemistry. For in vitro study, primary murine skin fibroblasts were cultured and stimulated with TGF-β.  siRNA was applied to  inhibit miR-155 expression. Real-time PCR was used to compare collagen synthesis and activation marker expressions in miR-155 knockdown fibroblasts with controls. Collagen release was analyzed by Sircol assay. Major TGF-β induced profibrotic pathways were analyzed by Western Blot. Targets of miR-155 were predicted by bio-informatics and were confirmed by using dual luciferase report assay and Western blot.

Results: The expression level of miR-155 was significantly elevated in mice injected with bleomycin, as well as in fibroblasts stimulated by TGF-β. Compared with control mice, miR-155 KO mice displayed significantly less fibrosis in both skins and lungs, as well as less number of activated fibroblasts in situ. Consistent with the in vivo findings, miR-155-knockdown primary fibroblasts showed significantly reduced collagen synthesis and lower activation markers (fibronectin and α-SMA) upon TGF-β stimulation. Analysis of major TGF-β downstream pathways including Smad2/3, JNK, ERK, Wnt and Akt revealed that the activation of Wnt and Akt pathway were notably suppressed in miR-155-knockdown fibroblasts. These two pathways were also attenuated in the skin of miR-155 KO mice injected with bleomycin. Further bio-informatics and literature research suggested that miR-155 could directly target casein kinase I (CK-1) and Src homology 2-containing inositol phosphatase-1 (SHIP-1), which are negative regulators of Wnt and Akt pathway, respectively.  We found miR-155 inhibition would result in an increased level of these two proteins in primary fibroblast.  Further studies using dual luciferase reporter assay confirmed the direct interaction between miR-155 and 3’-UTR of CK-1 and SHIP-1. 

Conclusion: Inhibition of miR-155 could ameliorate experimental fibrosis in a bleomycin induced SSc mouse model. miR-155 modulates profibrotic Wnt and Akt pathway by directly targeting CK-1 and SHIP-1, respectively. miR-155 is a potential target for therapeutic intervention in SSc.

---

« Back to 2013 ACR/ARHP Annual Meeting

ACR Meeting Abstracts - https://acrabstracts.org/abstract/inhibition-of-microrna-155-ameliorated-experimental-fibrosis-by-suppressing-wnt-and-akt-pathways/