Artificial MicroRNA-Mediated Tgfbr2 and Pdgfrb Co-Silencing Ameliorates Carbon Tetrachloride-Induced Hepatic Fibrosis in Mice
Artificial MicroRNA-Mediated Tgfbr2 and Pdgfrb Co-Silencing Ameliorates Carbon Tetrachloride-Induced Hepatic Fibrosis in Mice
复制标题
人工 MicroRNA 介导的 Tgfbr2 和 Pdgfrb 共沉默可改善四氯化碳诱导的小鼠肝纤维化
DOI：
10.1089/hum.2018.047
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发表时间：
2018-11-13
影响因子：
4.2
通讯作者：
Wang, Haijian
中科院分区：
文献类型：
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作者：
Jiang, Yan;Zhao, Yuanyuan;Wang, Haijian
肝星状细胞(HSCs)是导致肝纤维化的主要细胞类型。转化生长因子β1(TGF-1)和血小板衍生生长因子(PDGF)是调节HSC活化和增殖的关键促纤维化细胞因子，具有功能趋同作用。针对其受体的双重RNA干扰可能达到治疗效果。设计了一种新的基于HSC特异性GFAP启动子驱动和慢病毒表达的人工microRNAs(AmiRNAs)的RNAi策略，该策略由microRNA-30a骨架和针对小鼠PDGFR和TGFBR2的有效shRNAs组成。然后，在原代和培养的HSC以及受四氯化碳诱导的肝纤维化影响的小鼠中测试其抗肝纤维化的效果。研究表明，AmRNA介导的PDGFR和TGFBR2共沉默抑制HSC的激活和增殖。重组慢病毒颗粒经尾静脉注射进入肝脏后，治疗性amiRNAs在HSC中优先表达，并有效地协同下调Tgfbr2和PDGFR的表达，这与其信号转导的靶或效应基因如PAI-1、p70S6K和D-Cyclins的表达下调有关。TGFBR2和PDGFR联合沉默TGFBR2和PDGFR显著抑制肝纤维化标志物Sma和Col1a1、细胞外基质调节因子MMPs和TIMP1的表达，并明显改善肝纤维化，表现为血清丙氨酸转氨酶活性降低、胶原沉积和-Sma阳性染色减少。这一发现为在肝纤维化治疗中使用amiRNA介导的两条促纤维化通路的共沉默提供了概念证据，并突出了串联amiRNA用于基因治疗的治疗潜力。
Hepatic stellate cells (HSCs) are the primary cell type responsible for liver fibrogenesis. Transforming growth factor beta 1 (TGF-1) and platelet-derived growth factor (PDGF) are key profibrotic cytokines that regulate HSC activation and proliferation with functional convergence. Dual RNA interference against their receptors may achieve therapeutic effects. A novel RNAi strategy based on HSC-specific GFAP promoter-driven and lentiviral-expressed artificial microRNAs (amiRNAs) was devised that consists of an microRNA-30a backbone and effective shRNAs against mouse Pdgfr and Tgfbr2. Then, its antifibrotic efficacy was tested in primary and cultured HSCs and in mice affected with carbon tetrachloride-induced hepatic fibrosis. The study shows that amiRNA-mediated Pdgfr and Tgfbr2 co-silencing inhibits HSC activation and proliferation. After recombinant lentiviral particles were delivered into the liver via tail-vein injection, therapeutic amiRNAs were preferentially expressed in HSCs and efficiently co-knocked down in situTgfbr2 and Pdgfr expression, which correlates with downregulated expression of target or effector genes of their signaling, which include Pai-1, P70S6K, and D-cyclins. amiRNA-based HSC-specific co-silencing of Tgfbr2 and Pdgfr significantly suppressed hepatic expression of fibrotic markers -Sma and Col1a1, extracellular matrix regulators Mmps and Timp1, and phenotypically ameliorated liver fibrosis, as indicated by reductions in serum alanine aminotransferase activity, collagen deposition, and -Sma-positive staining. The findings provide proof of concept for the use of amiRNA-mediated co-silencing of two profibrogenic pathways in liver fibrosis treatment and highlight the therapeutic potential of concatenated amiRNAs for gene therapy.