人SMAD4全长及剪接体在HEK-293T细胞中的亚细胞定位及转录活性分析

Subcellular Localization and Transcriptional Activity Analysis of Full-length Human SMAD4 and Its Splicing Variants in HEK-293T Cells

为了观察人SMAD4全长及剪接体在HEK-293T中的亚细胞定位,并分析它们在HEK-293T细胞中的转录活性差异,将SMAD4全长及剪接体的pEGFP-C1重组质粒转染至HEK-293T细胞中,在荧光显微镜下观察含GFP标签的SMAD4全长及其剪接体蛋白的亚细胞定位,并通过双荧光素报告酶实验在HEK-293T细胞中对SMAD4全长及其剪接体激活SBE报告基因的情况进行比较。亚细胞定位观察显示,与pEGFP-C1空载组中GFP在HEK-293T中呈现核质均匀分布不同,GFP-SMAD4和GFP-SMAD4△4、GFP-SMAD4△6、GFP-SMAD4△5-6、GFP-SMAD4△4-6、GFP-SMAD4△4-7五种剪接体均分布于HEK-293T的细胞质中;而GFP-SMAD4△3则主要分布于HEK-293T的细胞核中。双荧光素酶实验结果显示,与空载组相比,在6种剪接体过表达组中,只有SMAD4△3过表达组能对SBE报告基因的荧光素酶活性起到一定的上调作用;但与SMAD4△3过表达组比较,SMAD4全长过表达组能对SBE报告基因的荧光素酶活性起到更明显的上调作用。本文观察了人SMAD4全长及剪接体在HEK-293T中的亚细胞定位,并分析了其对下游信号通路的转录激活情况,可为后续SMAD4剪接体蛋白的功能研究提供一定的基础。

To observe the subcellular localization of full-length and splicing variants of human SMAD4 and analyze their transcriptional activity, pEGFP-C1 recombinant plasmids containing SMAD4 full-length gene and splicing variants were transfected separately into HEK-293T cells, and the subcellular localization of the GFP-tagged SMAD4 and its splicing variants was observed. Activation of SMAD binding element(SBE)by full-length SMAD4 and splicing variants in HEK-293 T cells was compared using dual-luciferase reporter experiment. The fluorescence results showed that the full length SMAD4 and five kinds of SMAD4 splicing variants, including SMAD4△4, SMAD4△6, SMAD4△5-6, SMAD4△4-6 and SMAD4△4-7, were mainly distributed in the cytoplasm, while the GFP-tagged SMAD4△3 was mainly localized in the nucleus, compared with the uniform distribution of GFP across both nucleus and cytoplasm in pEGFP-C1 empty vectortransfected group. The dual-luciferase reporter experiment showed that, among the six variants, only SMAD4 △3 could up-regulate the luciferase activity of the SBE reporter gene when compared with the vector group;however, the full-length SMAD4 can significantly up-regulate the luciferase activity of the SBE reporter gene when compared with SMAD4 △3. In summary, observation of subcellular localization and analysis of the transcriptional activity of full-length human SMAD4 and its splicing variants could provide the basis for further functional study of SMAD4 splicing variants.