沉默Notch1基因促进人乳腺癌MCF-7细胞JNK1和p53磷酸化  

Notch-1gene silencing promotes phosphorylations of JNK1 and p53 in human breast cancer MCF-7 cells

作　　者：袁磊[1] 陈旭东[1] 范文娟[1] 杨旭光[1] 王建国[1] 

机构地区：[1]漯河医学高等专科学校,河南漯河462002

出　　处：《中国病理生理杂志》2013年第6期1014-1019,共6页Chinese Journal of Pathophysiology

基　　金：河南省基础与前沿技术研究计划项目(No.122300410277);漯河医学高等专科学校科研基金资助项目(No.2010-S10)

摘　　要：目的:探究沉默Notch1基因对人乳腺癌MCF-7细胞JNK1和p53磷酸化的影响。方法:选取人乳腺癌MCF-7细胞作为研究对象,构建shRNA-Notch1真核表达质粒用于转染MCF-7细胞使Notch1基因沉默。采用Western blotting方法检测MCF-7细胞Notch1、Hes-1、PUMA和NOXA蛋白的表达,JNK1和p53蛋白磷酸化水平以及caspase-3活化水平的改变。应用流式细胞术检测细胞凋亡和线粒体膜电位的变化。结果:人乳腺癌MCF-7细胞Notch1基因被沉默后,Notch1和Hes-1蛋白表达量明显减少(P<0.01),细胞凋亡率显著升高(P<0.01),JNK1和p53的磷酸化水平明显高于对照组(P<0.01),PUMA和NOXA表达量显著升高(P<0.05),cleaved caspase-3蛋白明显多于对照组(P<0.01),线粒体膜电位明显下降(P<0.05)。结论:沉默Notch1基因可能通过激活JNK1信号通路活化p53,促进PUMA和NOXA蛋白表达,进而通过线粒体途径导致人乳腺癌MCF-7细胞凋亡。AIM： To investigate the effect of Notchl gene silencing on phosphorylations of JNK1 and p53 in human breast cancer MCF-7 cells. METHODS： shRNA-Notchl eukaryotic expression plasmid was constructed and trans- fected into MCF-7 cells. The expression of Notchl and Hes-1 was observed by Western blotting after transfction. Apoptosis and mitochondrial membrane potential were detected by flow cytometry. Western blotting was also used to determine the protein levels of p-JNK1, p-p53, PUMA, NOXA and cleaved caspase-3 after Notch1 silencing was performed in MCF-7 cells. RESULTS： Silencing of Notchl significantly reduced the expression of Notchl and Hes-1 in MCF-7 cells （P 〈 0. 01 ）. In shNotchl group, the number of apoptotic cells was much higher （P 〈0. 01 ） and mitochondrial membrane poten- tial was much lower （P 〈 0. 05） than those in shControl group. The protein levels of p-JNK1, p-p53, PUMA, NOXA and cleaved caspase-3 increased obviously after silencing of Notch1 was performed in MCF-7 cells （P 〈 0. 05 ）. CONCLU- SION ： Notch1 silencing induces apoptosis of human breast cancer MCF-7 cells through promoting phosphorylations of JNK1 and p53, and increasing the production of PUMA, NOXA and cleaved caspase-3.

关 键 词：NOTCH1蛋白 短发夹RNA JNKI蛋白 p53蛋白 MCF-7细胞 

分 类 号：R737.9[医药卫生—肿瘤]