ABSTRACT

Systemic Sclerosis (SSc) is an autoimmune connective tissue disease that leads to skin and lung fibrosis. The Wnt pathway is clearly elevated in SSc and is pro-fibrotic via activation of canonical Wnt signalling. sFRP-1 is a Wnt antagonist that acts as a negative regulator of Wnt signalling. We sought to measure the levels of serum sFRP-1 in early diffuse SSc patients compared to healthy controls and if this is regulated by microRNA27a-3p. Ten early diffuse SSc patients and healthy controls sera were taken and sFRP-1 quantified by ELISA. Skin biopsies were also taken in five SSc patients and controls. Fibroblasts were quantified for microRNA27-3p expression by Taqman qRT-PCR with an internal microRNA to normalize. 3ʹUTR luciferase assays were performed to confirm direct targets of microRNA27a-3p with microRNA overexpression. Fibroblasts were transfected with microRNA27a mimics or scramble controls and using ELISA sFRP-1 was quantified. Furthermore, Collagen, Axin-2, TIMP-1 and MMP-1 were measured. Serum sFRP-1 was significantly reduced in early diffuse SSc patients. We identified microRNA27a-3p-3p as regulating sFRP-1 in dermal fibroblasts. We found significantly elevated microRNA27a-3p in isolated dermal fibroblasts from SSc patients. We confirmed that sFRP-1 is a direct target of microRNA27a-3p through cloning of the 3ʹUTR into a luciferase vector. ECM genes were also upregulated by microRNA27a-3p-3p and the matrix-degrading enzyme MMP-1 was suppressed. Serum sFRP-1 is reduced in diffuse SSc patients and is regulated by microRNA27a-3p and this is a direct regulation. Modulation of microRNA27a-3p levels could mediate fibrosis regression.

摘要

系统性硬化症 (SSc) 是一种自身免疫性结缔组织病，可导致皮肤和肺纤维化。Wnt 通路在 SSc 中明显升高，并通过激活经典 Wnt 信号传导促进纤维化。sFRP-1 是一种 Wnt 拮抗剂，可作为 Wnt 信号传导的负调节剂。我们试图测量早期弥漫性 SSc 患者与健康对照相比的血清 sFRP-1 水平，以及这是否受 microRNA27a-3p 的调节。采集 10 名早期弥漫性 SSc 患者和健康对照血清，并通过 ELISA 对 sFRP-1 进行定量。还对五名 SSc 患者和对照组进行了皮肤活检。通过 Taqman qRT-PCR 对成纤维细胞的 microRNA27-3p 表达进行量化，并使用内部 microRNA 进行标准化。进行 3'UTR 荧光素酶测定以确认 microRNA27a-3p 的直接靶标与 microRNA 过表达。用 microRNA27a 模拟物或乱序对照转染成纤维细胞，并使用 ELISA 对 sFRP-1 进行量化。此外，还测量了胶原蛋白、Axin-2、TIMP-1 和 MMP-1。早期弥漫性 SSc 患者的血清 sFRP-1 显着降低。我们确定 microRNA27a-3p-3p 在真皮成纤维细胞中调节 sFRP-1。我们发现 SSc 患者分离的真皮成纤维细胞中的 microRNA27a-3p 显着升高。我们通过将 3ʹUTR 克隆到荧光素酶载体中证实了 sFRP-1 是 microRNA27a-3p 的直接靶标。ECM 基因也被 microRNA27a-3p-3p 上调，基质降解酶 MMP-1 被抑制。弥漫性 SSc 患者的血清 sFRP-1 降低，并受 microRNA27a-3p 的调节，这是一种直接调节。调节 microRNA27a-3p 水平可以介导纤维化消退。