p53/Numb蛋白功能复合体对结肠癌细胞凋亡的影响  

Effect of p53/Numb protein functional complex on apoptosis of colon cancer cells

作　　者：张辉[1] 祝丽宇 叶颖江[1] 周静[1] Zhang Hui;Zhu Liyu;Ye Yingjiang;Zhou Jing(Department of Gastroenterology Surgery,Peking University People's Hospital,Beijing 100044,China)

机构地区：[1]北京大学人民医院胃肠外科,北京100044

出　　处：《中华实验外科杂志》2024年第3期436-440,共5页Chinese Journal of Experimental Surgery

基　　金：国家重点研发项目(2017YFC0908300);国家自然科学基金(81172272)。

摘　　要：目的探讨结直肠癌中p53蛋白对Numb蛋白表达的影响及其作用机制,观察两者相互作用对Numb蛋白泛素化降解影响以及对结肠癌细胞凋亡的影响。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,美国典型菌种保藏中心(ATCC)来源]作为研究对象,分别采用基因过表达(Transfection)及RNA干扰(RNAi),再用蛋白酶体抑制剂MG132分别处理或不处理细胞12 h,用蛋白质免疫印迹法(Western blot)检测了p53蛋白对Numb蛋白表达量及其泛素-蛋白酶体途径稳定性的影响,用流式细胞术检测p53/Numb对结肠癌细胞凋亡的影响。组间比较采用方差分析(ANOVA),独立样本t检验比较蛋白表达量差异,统计学方法分别采用独立样本t检验、独立样本t检验及单因素方差分析。结果Numb的蛋白水平随着p53的蛋白表达水平的逐渐升高而升高,呈剂量依赖性,空白组与p53转染组(2μg和4μg)Numb蛋白表达水平比值分别为0.632(0.633±0.008)和0.137(0.136±0.003),(t=5.332,P<0.01)而Numb的mRNA水平随着p53的mRNA表达水平的升高无明显变化[0.892(0.890±0.003)比0.826(0.825±0.006),t=1.232,P>0.05];p53 RNA干扰或不干扰48 h后,Numb的蛋白水平低于对照组(0.167(0.163±0.001)和0.536(0.532±0.005),t=3.374,P<0.05),Numb的mRNA水平随着p53的mRNA表达水平的降低无明显变化;同时p53 RNA干扰后,Numb蛋白表达水平降低,而蛋白酶体抑制剂MG132可使Numb的蛋白水平趋于一致[0.753(0.750±0.009)比0.742(0.744±0.006),t=1.532,P>0.05];当没有MG132时,p53存在情况下的Numb蛋白水平要比p53不存在时的Numb蛋白水平要高,但是此时Numb蛋白水平仍然比MG132存在时的Numb蛋白水平要低;共聚焦显微镜结果示当p53不存在时,Numb主要定位在胞膜及胞质,呈弥散分布;而当有p53存在时,Numb定位于胞质,呈聚集分布并且表达量增多,但p53也有部分是定位在胞质,说明Numb和部分p53共定位于胞质。而在HCT116(p53+)中p53 RNA干扰后�Objective To explore the effect of p53 protein on the expression of Numb protein in colorectal cancer and its action mechanism,observe the interaction between the two on the ubiquitination degradation of Numb protein and the impact on apoptosis of colon cancer cells.Methods Human colon cancer cell lines HCT116(+)and HCT116(-)[p53 wild-type and p53 deletion type,American Center for the Collection of Typical Strains(ATCC)source]were used as research subjects.Gene overexpression(Transfer)and RNA interference(RNAi)were used respectively,and the cells were treated or not treated with proteasome inhibitor MG132 for 12 h.The effect of p53 protein on the expression level of Numb protein and the stability of its ubiquitin proteasome pathway was detected using Western blotting,and the effect of p53/Numb on apoptosis of colon cancer cells was detected using flow cytometry.Analysis of variance(ANOVA)was used for inter group comparison,and independent sample t-test was used to compare protein expression differences.Statistical methods included independent sample t-test,independent sample t-test,and one-way analysis of variance.ResultsThe protein level of Numb increased in a dose-dependent manner as the protein expression level of p53 gradually increased.In the blank group and p53 transfection group(2μg and 4μg),the ratio of Numb protein expression levels was 0.632(0.633±0.008)and 0.137(0.136±0.003),respectively(t=5.332,P<0.01),while the mRNA level of Numb showed no significant change with the increase of p53 mRNA expression level[0.892(0.890±0.003)vs.0.826(0.825±0.006),t=1.232,P>0.05].After 48 h of p53 RNA interference or no interference,the protein level of Numb was lower than that in the control group[0.167(0.163±0.001)and 0.536(0.532±0.005),t=3.374,P<0.05],and the mRNA level of Numb showed no significant change with the decrease of p53 mRNA expression level.After p53 RNA interference,the expression level of Numb protein decreased,while the proteasome inhibitor MG132 could make the protein level of Numb tend to be c

关 键 词：结直肠肿瘤 P53 NUMB 泛素-蛋白酶体途径 凋亡 

分 类 号：R735.35[医药卫生—肿瘤]