【摘要】 目的探讨TGF-β1诱导的肿瘤细胞中CSRNP1/AXUD1基因的表达及其调控机制。方法分别用不同浓度的TGF-β1或相同浓度下不同处理时间处理指数生长期的人肺腺癌细胞A549和人前列腺癌细胞PC-3,荧光定量RT-PCR检测TGF-β1对两种肿瘤细胞中半胱氨酸-丝氨酸蛋白(cystein-serine-rich nuclear protein,CSRNP1)基因表达的剂量和时间依赖性效应;培养的A549细胞血清饥饿后,分别在有无TGF-β1(20 ng/ml)和环己亚胺(30μg/ml)作用下处理24 h,检测TGF-β1诱导的CSRNP1基因的表达是否需要新蛋白的合成;最后,分别将SMAD3表达质粒flag-SMAD3和显性失活突变体表达质粒(flag-SMAD3-mu转染A549细胞中,并以空质粒pcDNA3.1作为对照。血清饥饿后,进行有无TGF-β1(20 ng/ml)处理24 h,Western blot确定外源性SMAD3的过表达,荧光定量RT-PCR检测SMAD3的过表达对TGF-β1诱导的CSRNP1基因表达的影响。结果 TGF-β1以浓度和时间依赖的方式诱导肿瘤细胞中CSRNP1基因的表达,并需要新的蛋白合成。SMAD3的过表达增加TGF-β1诱导的CSRNP1的表达,而显性失活的SMAD3突变体(SMAD3-mu)的过表达则显著降低TGF-β1诱导的CSRNP1的表达。结论肿瘤细胞中TGF-β1可能通过激活SMAD3及其下游信号通路而诱导CSRNP1基因的表达。更多还原

【Abstract】 Objective To investigate AXIN1-related CSRNP1 gene expression and the mechanism of its transcriptional regulation in TGF-β1-induced tumor cells.Methods Human lung carcinoma A549 cells or human prostate cancer PC3 cells were treated with TGF-β1 at different doses(0,20,40,and 80 ng/ml) or at 20 ng/ml for 0,8,12,or 24 h,and the dose and time effect of TGF-β1 on CSRNP1 mRNA expression in the tumor cells were evaluated with real-time RT-PCR.A549 cells were also treated with TGF-β1 and cycloheximide to clarify whether CSRNP1 expression induced by TGF-β1 required de novo protein synthesis.A549 cells transfected with pcDNA3.1,flag-SMAD3,or flag-SMAD3-mu,after serum starvation,were treated with or without TGF-β1(20 ng/mL) for 24 h,and the overexpression of wild-type SMAD3 and dominant negative SMAD3-mu mutant were confirmed by Western blotting.The effect of SMAD3 or SMAD3-mu overexpression on CSRNP1 mRNA expression was also measured by real-time RT-PCR.Results In both A549 and PC3 cells,TGF-β1 dose-and time-dependently stimulated CSRNP1 expression,which required de novo protein synthesis in A549 cells.Overexpression of wild-type SMAD3 significantly increased the expression of CSRNP1 mRNA induced by TGF-β1,while overexpression of dominant negative SMAD3 mutant remarkably reduced CSRNP1 mRNA expression in response to TGF-β1 in A549 cells.Conclusion TGF-β1 may contribute to CSRNP1 expression through SMAD3 activation and downstream signaling in tumor cells.更多还原