Smad4小分子干扰RNA对转化生长因子-β诱导的纤溶酶原激活物抑制剂-1表达的影响

目的 探讨Smad4小分子干扰RNA(siRNA)对转化生长因子(TGF)-β诱导纤溶酶原激活物抑制剂(PAI)-1的表达的影响及作用机制.方法 将Smad4 siRNA转染入肝星状细胞24h后行TGF-β(1μg/L)刺激,24、48 h后收集细胞.利用萤光素酶报告基因法测定Smad结合转写因子的活性;Northern印迹分析技术检测PAI-1 mRNA表达的变化;Western印迹分析观察Smad4蛋白、PAI-1蛋白的变化.结果 Smad4 siRNA能抑制Smad4蛋白(3.0±0.1比16.0±0.5)表达;Smad4 siRNA抑制TGF-β1和ALK5激活的4×SBE荧光素酶报告基因活性;TGF-β时间依赖性促进PAI-1 mRNA表达,24 h达高峰;Smad4 siRNA从mRNA(1.3±0.1比6.5±0.2)和蛋白(1.50±0.15比5.52±0.36)水平抑制TGF-β激活的PAL-1的表达.结论 Smad4 siRNA能够有效地阻断TGF-β诱导的PAI-1表达.

The effect of Smad4 siRNA on transforming growth factor-β-induced-PAI-1 expression

Objective The activation of transforming growth factor-β (TGF-β) has been implicated in liver fibrotic diseases by regulating plasminogen activator inhibitor (PAI-1) expression.This study aims at elucidating whether Smad4 small interference RNA (siRNA) can be used to suppress the TGF-β-induced PAI-1 in hepatic steUate cells (HSCs).Methods Smad4 siRNA was intreduced to HSCs by transient transfection for 24 h and stimulated by TGF-β (1 μg/L) for 24 or 48 h.The activities of Smad binding element (SBE) reporter was measured by a luciferase reporter assay.The expression of Smad4 protein was detected by immunoblot analysis.The expression of PAI-1 mRNA and protein was determined by Northern blot and immunoblot analysis,repsectively.Results Smad4 siRNA abrogated the Smad4 protein expression (3.0 ± 0.1 vs 16.0 ± 0.5).Smad4 siRNA inhibited both TGF-β1-and TGF-β type Ⅰ constitutively active receptor (ALK5) -induced 4 × SBE luciferase reporter activity, respectively.Smad4 siRNA inhibited TGF-β1-induced PAI-1 mRNA (1.3 ± 0.1 vs 6.5±0.2) and protein (1.50±0.15 vs 5.52 ± 0.36) expression.Conclusion These results indicate that Smad4 siRNA could provide a novel approach for therapeutic intervention to inhibit fibrotic liver diseases.