靛玉红通过miRNA-29b-3p/TET2/5hmC轴促进骨髓增生异常综合征细胞株SKM-1细胞DNA去甲基化
Indirubin promoting DNA demethylation of myelodysplastic syndrome cell line SKM-1 cells by the miRNA-29b-3p/TET2/5hmC axis
喻丽 1凌志明 2谷晓丽 1杨秀鹏 1陈朋杰 1许勇钢1
作者信息
- 1. 中国中医科学院西苑医院,北京 100091
- 2. 北京中医药大学生命科学学院,北京 100029
- 折叠
摘要
目的:探索青黄散有效成分靛玉红对骨髓增生异常综合征(MDS)的去甲基化作用机制.方法:以MDS细胞株SKM-1细胞为研究对象,以不同浓度的靛玉红干预细胞48h,采用DNA斑点印记方法,检测5-羟甲基胞嘧啶(5hmC)、5-甲基胞嘧啶(5mC)等DNA甲基化指标的变化情况;通过qPCR、蛋白免疫印迹检测SKM-1细胞TET2基因表达;通过转录组高通量测序分析靛玉红调控SKM-1细胞miRNA表达的情况,并通过miRNA靶基因预测工具miranda工具筛选参与靛玉红促进MDS的TET2表达的候选miRNAs,随后使用qPCR进行实验验证.结果:DNA斑点印记结果显示靛玉红显著提高了SKM-1细胞DNA中5hmC的水平而降低了5mC的表达水平.qPCR与蛋白免疫印迹结果显示靛玉红没有显著改变TET2 mRNA表达,但是显著提高了TET2蛋白表达.转录组高通量测序结果显示靛玉红干预后有35个miRNA存在表达差异,其中17个miRNA上调,18个miRNA下调.Miranda结果显示差异miRNA中miRNA-29b-3p、miRNA-125a-5p可能作用于TET2基因.qPCR结果显示,靛玉红下调了miRNA-29b-3p的表达而上调了miRNA-125a-5p的表达.结论:靛玉红可能通过miRNA-29b-3p/TET2/5hmC轴促进SKM-1细胞DNA去甲基化.
Abstract
Objective:To explore the mechanism of demethylation of indirubin,the active ingredient of Qinghuang Powder in myelodysplastic syndrome(MDS).Methods:The MDS cell line SKM-1 was used as the research object,and different concentrations of indirubin were used to intervene the cells for 48 h.The changes of DNA methylation indexes,such as 5-hydroxymethylcytosine(5hmC)and 5-methylcytosine(5mC),were detected using DNA spot blotting method;the effects of TET2 gene expression in SKM-1 cells were detected by qPCR and protein immunoblotting;the effects of TET2 gene expression in SKM-1 cells were analyzed by transcriptional group high-throughput sequencing to analyze the miRNA expression in SKM-1 cells regulated by indirubin and the miRNA target gene.Results:DNA spot blotting showed that indigo red significantly increased the level of 5hmC and decreased the expression level of 5mC in the DNA of SKM-1 cells.qPCR and protein immunoblotting showed that indirubin did not significantly alter the expression of TET2 mRNA but significantly increased the expression of TET2 protein.High-throughput sequencing of the transcriptome showed that 35 miRNAs were differentially expressed after indirubin intervention,of which 17 miRNAs were up-regulated and 18 miRNAs were down-regulated.Miranda results showed that among the differentiated miRNAs,miRNA-29b-3p and miRNA-125a-5p.Conclusion:Indirubin may promote DNA demethylation in SKM-1 cells through the miRNA29b-3p/TET2/5hmC axis.
关键词
骨髓增生异常综合征/SKM-1细胞/DNA甲基化/靛玉红/TET2/miRNAKey words
Myelodysplastic syndrome(MDS)/SKM-1 cell/DNA methylation/Indirubin/TET2/miRNA引用本文复制引用
基金项目
国家自然科学基金项目(82274346)
北京市自然科学基金项目(7242256)
中国中医科学院科技创新工程重大攻关项目(CL2021A01706)
出版年
2024
NETL
NSTL
万方数据