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

作　　者：喻丽 凌志明 谷晓丽 杨秀鹏[1] 陈朋杰 许勇钢[1] YU Li;LING Zhiming;GU Xiaoli;YANG Xiupeng;CHEN Pengjie;XU Yonggang(Xiyuan Hospital,China Academy of Chinese Medical Sciences,Beijing 100091,China;School of Life Sciences,Beijing University of Chinese Medicine,Beijing 100029,China)

机构地区：[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

出　　处：《中华中医药杂志》2024年第10期5448-5452,共5页China Journal of Traditional Chinese Medicine and Pharmacy

基　　金：国家自然科学基金项目(No.82274346);北京市自然科学基金项目(No.7242256);中国中医科学院科技创新工程重大攻关项目(No.CL2021A01706)。

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48 h,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mi RNA-125a-5p的表达。结论:靛玉红可能通过mi RNA-29b-3p/TET2/5hm C轴促进SKM-1细胞DNA去甲基化。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 mi RNA 

分 类 号：R285[医药卫生—中药学]