Rui Hong-Bing, Zhang Han-Hua, Shi Li-Fang, Ma Zhi-Yong
Department of Hematology,The First Affiliated Hospital, Fujian Medical University, Fuzhou 350005, Fujian Province, China. E-mail:
Department of Hematology,The First Affiliated Hospital, Fujian Medical University, Fuzhou 350005, Fujian Province, China.
Zhongguo Shi Yan Xue Ye Xue Za Zhi. 2014 Oct;22(5):1256-60. doi: 10.7534/j.issn.1009-2137.2014.05.013.
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 Notch1-ICD and target gene Hes1. 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 Hes1 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 C-MYC genes, which induces apoptosis and reduces proliferation.
本研究旨在探讨DLL4/Notch1配体对白血病细胞系K562细胞生长的影响及其相关机制。采用脂质体2000将pBudCE4.1-DLL4质粒转染至K562细胞，运用RT-PCR和蛋白质印迹法监测外源性DLL4基因的mRNA和蛋白表达，以及Notch1-ICD和靶基因Hes1的表达。通过蛋白质印迹法检测K562细胞中Rb、YY1和C-MYC蛋白的表达水平。采用细胞计数试剂盒-8检测K562细胞的增殖情况，运用Annexin V染色流式细胞术检测细胞凋亡情况。结果显示，实验组细胞中DLL4、Notch1-ICD和Hes1的mRNA和蛋白表达水平显著高于对照组（P<0.05），表明Notch1信号通路被成功激活。与对照组细胞相比，实验组细胞中Rb、YY1和C-MYC的蛋白表达水平显著升高（P<0.05）。转染后，K562细胞的增殖明显受到抑制，DLL4转染细胞的凋亡率显著提高。DLL4转染显著增加了G1期细胞数量，减少了S期细胞数量。结论：K562细胞中DLL4配体基因的过表达导致Notch1信号通路被成功激活，增加了Rb、YY1和C-MYC基因的表达，从而诱导细胞凋亡并减少细胞增殖。
