白花丹醌对白血病细胞去甲基化作用的实验研究及生物信息学分析  

Experimental Study and Bioinformatics Analysis of Demethylation in Leukemic Cells Induced by Plumbagin

作　　者：徐凯红[1] 杨澍均 张怡[1] 严笑[1] 欧阳桂芳[1] XU Kaihong;YANG Shujun;ZHANG Yi;YAN Xiao;OUYANG Guifang(Ningbo First Hospital,Ningbo 315000,Zhejiang,China)

机构地区：[1]宁波市第一医院,浙江宁波315000

出　　处：《中华中医药学刊》2022年第12期226-229,I0063,共5页Chinese Archives of Traditional Chinese Medicine

基　　金：浙江省中医药科技计划(2015ZZ018)。

摘　　要：目的探讨中草药提取物白花丹醌对人白血病细胞系HL-60细胞去甲基化的影响,并定位主要的作用途径和关键基因。方法白花丹醌0.8μmol/L作用于HL-60细胞不同时间,酶联免疫吸附(ELISA法)测定其甲基化水平;蛋白免疫印迹法测定DNA甲基转移酶:DNA甲基转移酶1(DNMT1),DNA甲基转移酶3a(DNMT3a)和DNA甲基转移酶3b(DNMT3b)相对于内参甘油醛-3-磷酸脱氢酶蛋白(GAPDH)的表达水平;应用Infinium 850K甲基化芯片筛选去甲基化作用的信号转导途径和关键基因。结果和对照组比较,白花丹醌作用18 h后HL-60细胞甲基化水平显著下降[(1.56±0.24)ng比(2.39±0.4)ng,(P<0.05)];经白花丹醌作用后DNMT1蛋白/GAPDH,DNMT3b蛋白/GAPDH表达下调[(0.41±0.06)比(0.79±0.17),(0.43±0.09)比(0.74±0.16),均P<0.05],而DNMT3a相对蛋白表达水平在研究时间内差异无统计学意义。甲基化芯片分析显示NUP93、CACNB2和ATP6V1B1等去甲基化明显的基因参与哺乳动物雷帕霉素靶蛋白(mTOR)信号通路,丝裂原活化蛋白激酶(MAPK)信号通路和癌症中的微小核糖核酸(MicroRNAs)信号通路,是基因调控网络的中心环节。结论低浓度白花丹醌能够诱导白血病细胞去甲基化,这种作用与抑制DNMT1和DNMT3b蛋白的表达有关,甲基化芯片技术对作用途径和关键环节进行定位,为下一步的研究确定了方向。Objective To investigate the effect of plumbagin on demethylation of human leukemia cell line HL-60 and identify the main pathways and key genes.Methods Being treated by 0.8μmol/L plumbagin,the methylation level of HL-60 cells was assayed by ELISA.The protein expression of DNA methyltransferase(DNMT1,DNMT3 a,DNMT3 b)was evaluated by Westen blot.The 850 K Illumina BeadChip was used to screen the signal transduction pathways and key genes of demethylation.Result A decrease in methylation level was observed after plumbagin treatment compared with the control group[(1.56±0.24)ng vs.(2.39±0.4)ng,(P<0.05)].The relative expression of DNMT1 protein/GAPDH and DNMT3 b protein/GAPDH decreased after plumbagin treatment(P<0.05),but there was no significant change in the expression of DNMT3 a.Illumina BeadChip showed that significantly demethylated genes such as NUP93,CACNB2 and ATP6 V1 B1 were involved in mTOR signaling pathway,MAPK signaling pathway and MicroRNAs in cancer pathway,which were important parts of the gene regulatory network.Conclusion Low concentration of plumbagin could induce demethylation of leukemia cells,which was associated with the decreased protein expression of DNMT1 and DNMT3 b.Methylation CHIP could identify the main pathways and key genes of demethylation,offering a clear direction for the next research.

关 键 词：白花丹醌 DNA甲基化 白血病细胞 甲基化芯片 

分 类 号：R282.71[医药卫生—中药学]