沉默Notch1基因促进人乳腺癌MCF-7细胞JNK1和p53磷酸化

 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。

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

AIM: To investigate the effect of Notch1 gene silencing on phosphorylations of JNK1 and p53 in human breast cancer MCF-7 cells.METHODS: shRNA-Notch1 eukaryotic expression plasmid was constructed and transfected into MCF-7 cells. The expression of Notch1 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 Notch1 significantly reduced the expression of Notch1 and Hes-1 in MCF-7 cells (P<0.01). In shNotch1 group, the number of apoptotic cells was much higher (P<0.01) and mitochondrial membrane potential 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).CONCLUSION: 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.