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背景与目的:Smad7基因是转化生长因子(transforminggrouthfactor-茁,TGF-β)信号通路的抑制分子,有研究表明TGF-β通过活化SMAD通路和ras/MEK/ERK通路来诱导某些基因的表达。本研究旨在探讨在人永生化支气管上皮细胞BEP2D经辐射诱发恶性转化过程中,作为SMAD蛋白家族的抑制分子Smad7是否参与TGF-β对MAPK信号通路的调控。方法:将人工合成的Smad7siRNA及Smad7真核表达载体与pTet-Elk,pTet-Jun反式激活载体、报告基因荧光素酶共转染BEP2D细胞,TGF-β刺激,通过报告基因荧光素酶的表达丰度来检测Smad7对MAPK信号通路的调控。结果:永生化BEP2D细胞中,TGF-β刺激之后,Elk和Jun磷酸化活性升高(PElk=0.033,PJun=0.016);Elk或Jun同Smad7真核表达载体共转染之后,Elk磷酸化活性升高,Jun磷酸化活性降低(PElk=0.017,PJun=0.028);Elk或Jun同Smad7-siRNA共转染之后,Elk磷酸化活性降低,Jun磷酸化活性升高(PElk=0.018,PJun=0.005)。恶性化BERP35T2细胞中,TGF-β刺激之后,Elk磷酸化活性增强(P=0.006),Elk同Smad7siRNA共转染之后,Elk磷酸化活性降低(P=0.000);恶性化细胞中几乎检测不到Jun的活性。结论:在BEP2D细胞发生恶性转化过程中,Smad7基因干预MAPK信号通路,使ERK和JNK磷酸化活性的平衡失调,导致促增殖作用强于生长抑制作用,从而有助于细胞向恶性方向发展。
BACKGROUND & OBJECTIVE: Smad7 gene is a repressor of transforming growth factor-β (TGF-β) signaling pathway. It has been reported that TGF-β induces the expression of certain genes by activating SMAD pathway and ras / MEK / ERK pathway. The purpose of this study was to investigate whether Smad7, a SMAD protein family, is involved in the regulation of MAPK signaling through transforming growth factor-β (TGF-β) in the process of radiation-induced malignant transformation of human immortalized bronchial epithelial cells BEP2D. METHODS: Synthesized Smad7 siRNA and Smad7 eukaryotic expression vector were co-transfected with pTet-Elk, pTet-Jun transactivator and reporter luciferase into BEP2D cells. The expression of luciferase Abundance to detect Smad7 MAPK signaling pathway regulation. RESULTS: The Elk and Jun phosphorylation activities were increased in immortalized BEP2D cells (PElk = 0.033, PJun = 0.016) after stimulation with TGF-β. After co-transfected with Elk or Jun and Smad7 eukaryotic expression vector, Elk phosphorylation activity (PElk = 0.017, PJun = 0.028). After co-transfected with Elk or Jun with Smad7-siRNA, Elk phosphorylation activity decreased and Jun phosphorylation activity increased (PElk = 0.018, PJun = 0.005 ). In malignant BERP35T2 cells, Elk phosphorylation activity was enhanced (P = 0.006) after TGF-β stimulation, Elk phosphorylation activity was decreased (P = 0.000) after co-transfected with Elk and Smad7 siRNA; almost undetectable in malignant cells Jun activity. CONCLUSIONS: In the process of malignant transformation of BEP2D cells, Smad7 intervenes the MAPK signaling pathway and imbalances the phosphorylation activity of ERK and JNK, leading to the effect of promoting proliferation more than the growth inhibition and thus contributing to the malignant development of cells.