目的:探讨鼻咽癌相关基因6(NGX6)对结肠癌Wnt/β-catenin信号转导通路下游靶分子的影响,明确NGX6与Wnt通路之间是否存在反馈调节.方法:将pCMV-myc-NGX6真核表达载体及pCMV-myc空白载体瞬时转染结肠癌细胞系SW620,Western印迹检测转染NGX6前后Wnt/β-catenin信号转导通路下游靶分子c-Jun,c-myc,细胞周期蛋白D1(cyclin D1)的表达.构建LEF1缺失突变体(缺乏N末端,即β-catenin结合区域)pcDNA3.1(+)-LEF1DN瞬时转染SW620细胞,阻断Wnt通路,并通过Western印迹及T细胞转录因子4(TCF-4)荧光素酶报告质粒检测确定阻断效果.通过RT-PCR检测Wnt通路阻断后NGX6的表达.结果:NGX6基因瞬时转染结肠癌细胞系SW620后,可明显下调Wnt/β-catenin信号转导通路下游靶分子c-Jun,c-myc,cyclin D1的表达.成功构建真核表达载体pcDNA3.1(+)-LEF1DN,Western印迹及TCF-4荧光素酶报告质粒均证实pcDNA3.1(+)-LEF1DN能够下调Wnt通路下游靶分子c-Jun,c-myc,cyclin D1的表达及TCF/LEF的转录活性,对Wnt通路具有阻断作用.瞬时转染pcDNA3.1(+)-LEF1DN抑制Wnt通路后,NGX6在转录水平的表达有上升.结论:NGX6对Wnt/β-catenin信号转导通路下游靶分子具有抑制作用.阻断Wnt通路对NGX6的表达有影响,Wnt通路与NGX6之间可能存在反馈调节作用.%Objective To explore the role of nasopharyngeal carinoma associated gene 6 (NGX6) in Wnt/β-catenin signaling pathway and to identify whether there is feedback regulation between NGX6 and Wnt signal pathway once the Wnt signal is blocked.Methods pCMV-mycNGX6 and pCMV-myc were transfected to colon cancer cell line SW620 and the expression of downstream target molecules of Wnt/β-catenin signaling pathway such as c-myc, cyclinD1, and c-jun were detected by Western blot.The eukaryotic expression vector pcDNA3.1 ( ± )-LEF1 DN(without N-terminal β-catenin binding domain) was constructed to inhibit Wnt signaling pathway, and the effect was confirmed by Western blot and TCF-4 luciferase report system.The mRNA expression of NGX6 in SW620 was detected by RT-PCR after pcDNA3.1 ( ± ) -LEF1 DN transient transfection.Results The expression of above molecules were obviously down-regulated by NGX6 transient transfection in SW620.The eukaryotic expression vector pcDNA3.1 ( ± )-LEF1DN showed an inhibitory effect on Wnt/β-catenin signaling pathway.The mRNA expression of NGX6 in SW620 was up-regulated after pcDNA3.1 ( ± )-LEF1DN transient transfection.Conclusion NGX6 could inhibit the expression of Wnt signaling downstream target genes, which maybe related to the potential feedback regulation between NGX6 and Wnt signal pathway.
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