p53与Numb蛋白在结直肠癌中相互作用分子机制研究

Molecular Mechanism of the Interaction between p53 and numb protein in colorectal cancer

目的探讨结直肠癌中p53蛋白与Numb蛋白的相互作用及共定位,观察两者相互作用的结构域及及对结直肠癌细胞增殖周期的影响。方法以人结肠癌细胞系HCT116(+)及HCT116(-)(分别为p53野生型及p53缺失型)作为研究对象,分别采用基因过表达(Transfection)及RNA干扰(RNAi),用免疫共沉淀实验(CoIP)检测p53与Numb蛋白内源性的相互作用,用GST-Pull down检测两者直接相互作用的结构域,用流式细胞术检测p53/Numb对结肠癌细胞增殖周期的影响。结果分别用p53、Numb抗体做免疫共沉淀实验,p53和Numb蛋白可相互共沉淀。为了进一步明确Numb介导与p53相互作用的区段,构建4段带有GST标签的Numb分段质粒,采用GST-Pull down技术验证了它们与p53的结合情况。分别是1~592全长质粒以及1~264、92~353、352~592分段质粒。结果显示全长的Numb、92-353都可以和p53相互作用,1~264则和p53有较弱的结合能力,提示Numb的92~353段的结构域对于Numb和p53的结合是必须的。而在HCT116(p53^(+))中p53 RNA干扰后作用则与之相反;p53^(+)细胞中,转入Numb后其细胞周期出现G_(2)-M期阻滞,实验组G_(2)/M期DNA含量高于对照组[1.62倍(35.1%/19.66%),t=4.116,P<0.05]增殖抑制率实验组高于对照组(4.2%比15.6%,t=3.821,P<0.01),而在p53^(-)中转入Numb后也出现G_(2)-M期阻滞,增殖水平下降,但较在HCT116(+)中作用弱。结论结肠癌细胞中p53通过与Numb蛋白的相互作用及共定位,可能影响结肠癌细胞增殖周期,为探索两者在结肠癌中的相互作用的功能学机制奠定了新的实验基础。

Objective To explore the interaction and co localization of p53 protein and Numb protein in colorectal cancer,and to observe the structural domains of their interaction and their impact on the proliferation cycle of colorectal cancer cells.Methods Colon cancer cell lines HCT116(+)and HCT116(-)(p53 wild-type and p53 deletion type,respectively)were used as research subjects.Gene overexpression(Transfer)and RNA interference(RNAi)were used,respectively.Immunoprecipitation assay(CoIP)was used to detect the endogenous interaction between p53 and Numb protein,and GST Pull down was used to detect the domains of direct interaction between the two,Use flow cytometry to detect the effect of p53/Numb on the proliferation cycle of colon cancer cells.Results Immunoprecipitation experiments were conducted using p53 and Numb antibodies,and p53 and Numb proteins could co precipitate with each other.In order to further clarify the segments of Numb mediated interaction with p53,four segments of Numb segmented plasmids with GST tags were constructed,and their binding to p53 was verified using GST Pull down technology.They are full length plasmids 1-592 and segmented plasmids 1-264,92-353,and 352-592,respectively.The results showed that the full-length Numb and 92-353 can interact with p53,while 1-264 has weak binding ability with p53,indicating that the domain of the 92-353 segment of Numb is necessary for the binding of Numb to p53.In HCT116(p53^(+)),the effect of p53 RNA interference is opposite;In p53^(+)cells,after transferring to Numb,there was a G_(2)-M phase arrest in the cell cycle.The percentage content of DNA in the S and G_(2)/M phases of the control group was 1.78 and 1.62(t=4.116,P<0.05),respectively,compared to the experimental group.The proliferation was inhibited(4.2%vs.15.6%,t=3.821,P<0.01).However,after transferring to Numb in p53^(-),there was also a G_(2)-M phase arrest,and the proliferation level decreased,but the effect was weaker than in HCT116(+).Conclusion Through the interaction and co localization with Numb protein,p53 in colon cancer cells may affect the proliferation cycle of colon cancer cells,laying a new experimental foundation for exploring the functional mechanism of their interaction in colon cancer.