胶质纤维酸性蛋白启动子驱动的 RNA 干扰 TGF-β1 治疗肝纤维化。
GFAP promoter-driven RNA interference on TGF-β1 to treat liver fibrosis.
机构信息
Department of Pharmaceutical Sciences, University of Tennessee Health Science Center, 19 South Manassas, Memphis, Tennessee 38103-3308, USA.
出版信息
Pharm Res. 2011 Apr;28(4):752-61. doi: 10.1007/s11095-011-0384-y. Epub 2011 Feb 23.
PURPOSE
The objective was to determine the role of promoters and miRNA backbone in shRNA-based hepatic stellate cell (HSC)-specific transforming growth factor (TGF)-β1 gene silencing. This is expected to avoid the side effect of non-specific TGF-β1 gene silencing.
METHODS
Two most potent shRNAs targeting 769 and 1033 start sites of rat TGF-β1 mRNA were cloned into pSilencer 1.0 vector for enhanced TGF-β1 gene silencing. We then constructed HSC-specific pri-miRNA mimic and pri-miRNA cluster mimic expression plasmids in which shRNA expression was driven by a glial fibrillary acidic protein (GFAP) promoter to achieve HSC-specific TGF-β1 gene silencing to avoid nonspecific inhibition of TGF-β1 expression in other cells and organs.
RESULTS
These TGF-β1 pri-miRNA-producing plasmids showed the inhibition of proliferation and induced apoptosis of activated HSC-T6 cells. TGF-β1 pri-miRNA cluster mimic plasmids decreased TGF-β1 and collagen gene expression at both mRNA and protein levels.
CONCLUSIONS
GFAP promoter driven TGF-β1 pri-miRNA producing plasmids have the potential to be used for site-specific gene therapeutics to treat liver fibrosis.
目的
确定启动子和 miRNA 骨干在基于 shRNA 的肝星状细胞 (HSC) 特异性转化生长因子 (TGF)-β1 基因沉默中的作用。这有望避免非特异性 TGF-β1 基因沉默的副作用。
方法
将针对大鼠 TGF-β1 mRNA 的 769 和 1033 个起始位点的两个最有效的 shRNA 克隆到 pSilencer 1.0 载体中，以增强 TGF-β1 基因沉默。然后，我们构建了 HSC 特异性 pri-miRNA 模拟物和 pri-miRNA 簇模拟物表达质粒，其中 shRNA 表达由神经胶质纤维酸性蛋白 (GFAP) 启动子驱动，以实现 HSC 特异性 TGF-β1 基因沉默，从而避免 TGF-β1 在其他细胞和器官中的非特异性抑制。
结果
这些 TGF-β1 pri-miRNA 产生的质粒显示出对激活的 HSC-T6 细胞增殖的抑制和诱导凋亡。TGF-β1 pri-miRNA 簇模拟物质粒降低了 TGF-β1 和胶原基因在 mRNA 和蛋白质水平的表达。
结论
GFAP 启动子驱动的 TGF-β1 pri-miRNA 产生质粒有可能用于治疗肝纤维化的特定部位基因治疗。
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