摘要
目的研究Notch1、Notch2受体对胰腺癌HPAC细胞Notch信号通路下游Hes家族靶基因的影响。方法运用siRNA干扰技术分别干扰HPAC细胞中Notch1和Notch2基因,用Western blotting法检测Notch1和Notch2的干扰效率,用real-time PCR检测siRNA干扰后Notch下游靶基因Hes1、Hes2、Hes4和Hes6的mRNA表达水平;同时用Western blotting法检测Hes1的蛋白表达变化。结果与空白对照组(1.000±0.019)和siRNA对照组(0.908±0.039)相比,Notch1-siRNA转染组(0.124±0.005)的Notch1蛋白表达量显著降低。Notch1敲减降低了Hes1和Hes6的mRNA表达,Hes1与空白对照组和siRNA对照组相比,差异有统计学意义(P<0.05);Hes6与空白对照组和siRNA对照组相比,差异有统计学意义(P<0.05),对Hes2和Hes4的mRNA则没有影响。Hes2与空白对照组siRNA对照组相比,差异无统计学意义(P>0.05);Hes4与空白对照组和siRNA对照组相比,差异无统计学意义(P>0.05)。与空白对照组(1.000±0.015)和siRNA对照组(0.990±0.017)相比Notch2-siRNA转染组(0.350±0.009)的Notch2蛋白表达量显著降低。Notch2敲减降低了Hes1的mRNA表达,Hes1与空白对照组和siRNA对照组相比,差异有统计学意义(P<0.05),而对Hes2、Hes4和Hes6的表达没有影响,Hes2与空白对照组和siRNA对照组相比,差异无统计学意义(P>0.05);Hes4与空白对照组和siRNA对照组相比,差异无统计学意义(P>0.05);Hes6与空白对照组和siRNA对照组相比,差异无统计学意义(P>0.05)。Notch1和Notch2下调均不能引起Hes1蛋白水平变化。结论 HPAC细胞中Notch1的靶基因是Hes1和Hes6,而Notch2的靶基因是Hes1,提示不同Notch家族成员调控的靶基因有交叉但是不同。
Objective To explore the effect of silencing Notch1 and Notch2 on the gene expression of Hes family target genes in HPAC pancreatic cancer cells. Methods Notch1-siRNA and Notch2-siRNA were transfected as a liposomal formulation into HPAC cells, respectively, the protein expression of Notch1 and Notch2 were detected by Western blotting, and the mRNA levels of Hes1, Hes2, Hes4 and Hes6 were detected by real-time PCR, and the protein expression level of Hes1 was detected by Western blotting. Results The protein level of Notch1 in Notch1-siRNA group （0. 124±0. 005） was significantly decreased compared to that of the blank group （1. 000± 0. 019） and RNAi control group （0. 908±0. 039）. Down-regulation of Notch1 expression decreased the mRNA expression of Hes1 and Hes6（Hes1 vs that of the blank group, P=0. 025, Hes1 vs RNAi control group, P=0. 024;Hes6 vs the blank group, P=0. 018, Hes6 vs RNAi control group, P=0. 012）, but has no effect on the expression of Hes2 and Hes4 mRNA（Hes2 vs the blank group, P=0. 064, Hes2 vs RNAi control group, P=0. 059;Hes4 vs the blank group, P=0. 069, Hes4 vs RNAi control group, P=0. 071. The protein expression of Notch2 in Notch2-siRNA group（0. 350±0. 009） was significantly decreased, compared to the blank group （1. 000±0. 015） and RNAi control group（0. 990±0. 017）. Down-regulation of Notch2 decreased the mRNA expression of Hes1（Hes1 vs the blank group, P=0. 015,Hes1 vs RNAi control group, P=0. 014）, but has no effect on the expression of Hes2, Hes4 and Hes6（Hes2 vs the blank group, P=0. 178;Hes2 vs RNAi control group, P=0. 121;Hes4 vs the blank group, P=0. 097, Hes6 vs RNAi control group, P=0. 100; Hes6 vs the blank group, P=0. 053, Hes6 vs RNAi control group, P=0. 088. Neither of down-regulation of Notch1 and Notch2 down-regulate the protein expression of Hes1. Conclusion The downstream target gene of Notch1 is Hes1 and Hes6, and the downstream target gene of Notch2 is Hes1. Our results indicate that Notch1 and Notch2 have different regulatory effects on the expressions of Hes family genes.
出处
《首都医科大学学报》
CAS
北大核心
2015年第2期244-250,共7页
Journal of Capital Medical University
基金
国家自然科学基金(81372156)
北京市教育委员会科技计划重点项目(KZ201410025020)~~
作者简介
Corresponding author，E-mail：yxzhang@ccmu．edu．cn
