全反式维甲酸下调NB4细胞膜联蛋白Ⅱ表达及其机制探讨

Down-regulation of Annexin Ⅱ Expression in NB4 Cells Induced by all-trans Retinoic Acid and Its Mechanisms

目的:研究全反式维甲酸(ATRA)对急性早幼粒细胞白血病NB4细胞膜联蛋白Ⅱ(AnnexinⅡ)的表达影响,分析在ATRA的作用下AnnexinⅡ启动子的荧光素酶活性。方法:培养NB4细胞,用RT-PCR和Western blot技术分别检测1μmol/L ATRA作用的NB4细胞在不同时间点的AnnexinⅡ表达。构建AnneixnⅡ启动子,用电转染将重组质粒p GL4.15-AnnexinⅡ-promoter瞬时转染NB4细胞,在24 h后用1μmol/L ATRA处理转染的NB4细胞,分析AnnexinⅡ启动子的荧光素酶活性。结果:在转录水平,AnnexinⅡ的表达在48 h时逐渐下降;在蛋白水平,AnnexinⅡ的表达在24 h开始下降,而在48 h时下降明显。同时通过ATRA作用AnnexinⅡ启动子,发现ATRA可使AnnexinⅡ启动子活性下降,且随时间的延长荧光素酶活性越来越弱。结论:ATRA可诱导NB4细胞中AnnexinⅡ下调;ATRA可使转染细胞NB4中Annexin启动子荧光素酶活性随时间的延长而逐渐减弱,本研究为深入探讨其分子机制奠定了基础。

Objective:To explore effect of all-trans retinoic acid(ATRA) on annexin Ⅱ expression in NB4 cells and to analyze the luciferase activity of annexin Ⅱ promoter in condition of ATRA-induced treatment.Methods:NB4 cells were cultured in vitro,the transcriptional or translational expression levels of Annexin Ⅱ in NB4 cells treated with 1μmol/L ATRA at different time points were detected by RT-PCR or Western blot respectively.Annexin Ⅱ-promoter was constructed,the recombinant plasmids pGL4.15 Annexin Ⅱ-promoter were transfected into NB4 cells with electroporation,and after being treated with 1 μmol/L ATRA for 24 hours the luciferase acttivity of Annexin Ⅱpromoter was determined by luciferase activity assay.Results:The transcriptional expression of Annexin Ⅱ was downregulated after 48 h.The translation expression of Annexin Ⅱ was slowly weakened after 24 h,and it was seriously reduced after 48 h.Further,Luciferase activity of Annexin Ⅱ promoter in NB4 cells treated with 1 μmol/L ATRA was down-regulated,and showed a decreased tendency at indicated time points.Conclusion:All-trans retinoic acid can induce the down-regulation of Annexin Ⅱ expression on the membrane of NB4 cells,and the activity of Annexin Ⅱpromoter is down-regulated too.This study provide a basis for further study of molecular mechanism.