DLL4/Notch1配体过表达抑制K562细胞生长机制研究

Over-Expression of DLL4 /Notch1 Ligand Inhibits Proliferation of K562 Cells

本研究旨在探讨Notch1信号通路的配体delta-like ligand 4(DLL4)基因过表达对K562细胞增殖的影响及其可能的作用机制。采用脂质体介导将含配体DLL4基因的质粒pBudCE4.1-DLL4转染K562细胞,通过RTPCR和Western blot检测转染48 h后DLL4基因的mRNA及蛋白表达,同样检测转染后Notch1受体胞内区(NICD)、下游靶基因Hes1的mRNA及蛋白表达;通过Western blot检测与Notch信号通路相关的细胞转录因子YY1、原癌基因C-MYC及抑癌基因Rb蛋白的表达水平;用CCK-8法检测转染后细胞的增殖情况;用流式细胞术检测转染质粒48 h后各组细胞的凋亡情况。结果表明,DLL4基因转染后的K562细胞中DLL4、NICD及下游靶基因Hes1的mRNA及蛋白表达量比对照组明显增多(P<0.05),说明DLL4的过表达激活了Notch1信号通路;Western blot方法检测显示,DLL4的转染增加了细胞转录因子YY1、原癌基因C-MYC及抑癌基因Rb蛋白的表达,从而抑制了K562细胞的生长,诱导细胞周期停滞于G1期及细胞凋亡的增加。结论:外源性DLL4基因过表达可有效活化K562细胞内Notch1信号通路,可能通过YY1、C-MYC及Rb等Notch信号通路相关基因的高表达诱导K562细胞的生长减慢及细胞凋亡。

This study was aimed to explore the effect of DLL4/Notch1 ligand on cell growth in leukemia cell line K562 and its relevant mechanism.The pBudCE4.1-DLL4 plasmid was transfected into K562 cells by lipofectamine 2 000,RT-PCR and Western blot were applied to monitor the mRNA and the protein expression of exogenous DLL4 gene,as well as the expression of Notchl-ICD and target gene Hesl.Expression levels of Rb,YY1 and C-MYC protein in K562 cells were also detected by Western blot.Cell counting Kit-8 was used to detect the proliferation of K562 cells,and flow cytometry with Annexin V staining was used to detect the cell apoptosis.The results showed that the mRNA and protein expression levels of DLL4,Notch1-ICD and Hesl in cells of experimental group were significantly higher than those of control groups （P 〈 0.05）,indicating the successful activation of the Notch1 signaling pathway.The protein expression levels of Rb,YY1 and C-MYC in cells of experimental group significantly increased when compared with that of control group cells （P 〈 0.05）.After transfection,the proliferation of K562 cells was obviously inhibited,and apoptosis rate in DLL4-transfected cells was significantly enhanced.DLL4 transfection significantly increased the number of cells in G1 phase and decreased that in S phase.It is concluded that the over-expression of DLL4 ligand gene in K562 cells results in successful activation of the Notch1 signaling pathway,increases expression of Rb,YY1 and CMYC genes,which induces apoptosis and reduces proliferation.