PPARγ在系统性硬化症中的表达调控及抗纤维化的新机制探讨

Progressive fibrosis replacement of normal tissue architecture in multiple organs is the main cause of death in systemic sclerosis. The expression of peroxisome proliferator-activated receptor γ (PPARγ) is decreased in lesional skin and fibroblasts from patients with systemic sclerosis, but it’s reason and anti-fibrogenic mechanism is still unclear. Our previous study demonstrated that miR-130b was up-regulated in systemic sclerosis, and the 3'untranslated region of PPARγ contained its conserved potential binding sequences. In systemic sclerosis and bleomycin-induced skin fibrosis animal models, the expression of PPARγ was decreased, however, the expression of TLR4 was increased. In bortezomib treated group, the expression of PPARγ was increased and the expression of TLR4 was decreased, the skin fibrosis was ameliorated. In this study, we will first comfirm that the expression of PPARγ was regulated by miR-130b via using miRNA-specific inhibitor or mimics and luciferase reporter. Then, we will investigate the mechanism of PPARγ anti-fibrogenesis effects via using PPARγ agonist or inhibitor,PPARγ expression vector, chromatin immunoprecipitation assay in parimary skin fibroblasts and bleomycin-induced skin fibrosis animal models. This study may suggest new idea for the therapy of systemic sclerosis via anti-fibrogenic treatment .

全身多器官进行性纤维化是系统性硬化症患者高死亡率的主要原因。过氧化物酶体增殖物激活受体-γ（PPARγ）在系统性硬化症中表达降低，具有重要的抗纤维化作用。但其表达降低的原因及抗纤维化机制不明确。申请者在前期工作中发现，miR-130b在系统性硬化症中表达增高，PPARγ3’UTR区含miR-130b的结合序列；在系统性硬化症和博莱霉素诱导的纤维化小鼠中，PPARγ表达降低，TLR4表达增高；使用硼替佐米治疗纤维化小鼠后，PPARγ表达升高，TLR4表达降低，纤维化程度减轻。本项目首先通过miR-130b过表达或低表达技术、荧光素酶报告基因等明确PPARγ受miR-130b的直接调控。进一步采用PPARγ激动剂或抑制物、PPARγ表达载体、染色质免疫共沉淀等阐明PPARγ抗维化的新机制。开展本项目，有望为系统性硬化症的抗纤维化治疗提供新的思路。

表观遗传学在系统性硬化症(SSc)纤维化中发挥着重要作用，但其机制未被阐明。本项目从三方面研究表观遗传学在SSc纤维化中的重要机制。①通过miR-130b过表达或低表达技术、荧光素酶报告基因等明确PPARγ受miR-130b的直接调控，在SSc中发挥抗纤维化作用。②利用miRNA芯片、GEO数据库、DAVID、IPA等生物信息学工具阐明整体水平miRNAs 和mRNA 的表达调控网络。研究发现：21个miRNAs 和2698个mRNA在SSc皮肤组织中表达异常。17个 miRNAs及其33个靶基因 (55 对miRNA-mRNA ) 参与了Toll样信号通路、TGF-β和Wnt信号通路。Real-time PCR验证发现：在SSc皮肤组织和原代皮肤成纤维细胞及被20%SSc患者血清刺激的正常皮肤成纤维细胞和内皮细胞中，miR-146b, miR-130b, miR-21, miR-31 和miR-34a表达增高，miR-145表达降低。在SSc原代皮肤成纤维细胞及被SSc血清刺激的正常成纤维细胞和内皮细胞中，ACVR2B, FZD2, FZD5和SOX2表达增高。6个miRNAs和4个mRNA可能在SSc病理过程中发挥重要作用。miR-21为SSc特异的血清标志物。③ 利用高通量芯片技术对SSc外周血单个核细胞进行DNA甲基化、mRNA和细胞因子表达谱整合研究。发现了SSc中4848个低甲基化位点和216个高甲基化位点，1023个表达上调的基因和1284个表达下调的基因，384个表达上调的基因甲基化水平降低，14个表达下调的基因甲基化水平增高。一些与自身免疫密切相关的基因如AIF1, ARID3A, IFI44L, PARP9和RSAD2 等均存在低甲基化和基因高表达。Wnt信号通路和趋化因子信号通路中多个基因存在低甲基化。细胞因子IFNγ, IL-1RA, IP-10, MCP-1, RANTES和 VEGF在SSc中表达增高，IL-2 和IL-5表达降低。IL2的甲基化位点 cg09526693和cg25065535呈高甲基化，IL1RA的甲基化位点 cg03989987, cg01467417和cg02543462，VEGF的甲基化位点cg21099624均呈低甲基化。本项目为SSc新的生物标志物筛选、纤维化机制探索及疾病防治提供新的理论基础和实验依据。