基于DNA甲基化组学技术分析TET 1基因对小鼠uNK细胞DNA甲基化的影响  

Analysis of the Effect of TET 1 Gene on Methylation of Mouse uNK Cells based on DNA Methylation Histology Technique

作　　者：赵静贤 杨晓伟[1,2,3] 刘言言 赵自亮 赵光伟 赵永聚[2,3] ZHAO Jingxian;YANG Xiaowei;LIU Yanyan;ZHAO Ziliang;ZHAO Guangwei;ZHAO Yongju(College of Veterinary Medicine,Southwest University,Chongqing 402460,China;College of Animal Science and Technology,Southwest University,Chongqing 400715,China;Chongqing Key Laboratory of Herbivore Science,Chongqing 400715,China;National Pig Technology Innovation Center,Chongqing 402460,China)

机构地区：[1]西南大学动物医学院,重庆402460 [2]西南大学动物科学技术学院,重庆400715 [3]草食动物科学重庆市重点实验室,重庆400715 [4]国家生猪技术创新中心,重庆402460

出　　处：《畜牧兽医学报》2025年第2期912-924,共13页ACTA VETERINARIA ET ZOOTECHNICA SINICA

基　　金：国家自然基金(32002356);重庆市自然基金(cstc2019jcyj-msxmX0140);重庆市科企联合体种质资源收集利用与品种试验项目(草食牲畜)。

摘　　要：本研究旨在探究去甲基化酶1(ten eleven translocation,TET 1)对小鼠子宫内自然杀伤细胞(uterine natural killer,uNK)DNA甲基化的影响,深入了解其分子调控机制。无菌采集妊娠10 d小鼠子宫蜕膜,分离纯化uNK细胞进行培养,利用RNA干扰技术敲低TET 1基因的表达,提取TET 1干扰组和正常对照组细胞的总DNA,利用简化基因组DNA甲基化测序(reduced representation bisulfite sequencing,RRBS)技术进行测序,测序结果经生物信息学软件分析进行两组样本差异甲基化区域(differentially methylated region,DMR)的统计与注释,并进一步对DMR相关基因进行GO数据库分析及注释,了解相关基因的功能,利用KEGG数据库对其调控的信号通路进行富集分析。结果显示,TET 1干扰组相较对照组共有14120个DMRs,其中高甲基化的DMR有4897个,低甲基化的DMR有9223个,分布在基因体(genebody)上的DMR最多,共9762个,占总数的69.14%。DMR广泛分布于基因组的不同元件,且有些基因不同元件同时存在高甲基化和低甲基化的DMR。GO注释结果显示,存在DMR的基因主要集中在ATP结合、核酸结合、细胞组建、细胞分化、胚胎发育、RNA聚合酶Ⅱ转录调控、细胞增殖负调控等方面。KEGG数据库分析显示,DMR主要在代谢通路呈现显著富集,其中丙酮酸代谢通路共有12个参与代谢的关键分子出现了54个DMR,是出现DMR显著富集的代谢通路,其中乙酰辅酶A合成酶(Acss)、乳酸脱氢酶B(Ldhb)和丙酮酸激酶(Pklr)的DMR呈现单一高甲基化状态。此外,PI3K/AKT信号通路和HIF-1信号通路在介导丙酮酸代谢过程中发挥重要作用,且在基因体和启动子上的DMR也出现显著富集。综上,TET 1对小鼠uNK细胞具有甲基化调控作用,丙酮酸代谢是其发挥调控作用的主要途径,Acss、Ldhb和Pklr是其潜在的调控靶分子,PI3K/AKT和HIF-1是参与调控的重要信号通路。The purpose of this study was to investigate the effect of ten eleven translocation 1(TET 1)on the methylation of the DNA of mouse uterine natural killer(uNK)cells,and to gain insight into its molecular regulatory mechanism.Mouse uterine metaphase was aseptically collected on the 10th day of gestation,and the uNK cells were isolated and purified for culture.The expression of TET 1 gene was knocked down by RNA interference technology.Then,total DNA from cells of the interference group and the normal control group were extracted,respectively.Sequencing was performed using reduced representation bisulfite sequencing(RRBS)method,and the results were analyzed by bioinformatics software for the statistics and annotation of differentially methylated region(DMR)in the two group samples,and further analyzed by the GO database for DMR-related genes to understand their functions.Also,the sequencing results were analyzed for the differentially methylated region(DMR)annotation,and the enriched signaling pathways in which regulated by the KEGG database.The results showed that there were 14120 DMRs in the TET 1 interference group when compared with the control group,of which 4897 were hypermethylated DMRs and 9223 were hypomethylated DMRs.The largest number of DMRs were distributed in the genebody,with a total of 9762 DMRs,accounting for 69.14%of the total number of DMRs.DMRs were widely distributed in the different elements of the genome,and some genes had DMRs with both hypermethylated and hypomethylated in different elements.GO annotation results showed that the DMRs were mainly concentrated in ATP binding,nucleic acid binding,cell formation,cell differentiation,embryonic development,RNA polymerase II transcriptional regulation and negative regulation of cell proliferation,etc.KEGG database analysis results revealed that the DMRs were significantly enriched in the metabolic pathways,with a total of 12 key molecules involved in metabolism in the pyruvate metabolic pathway,and 54 DMRs appeared,which was the most significant en

关 键 词：TET 1基因 UNK细胞 甲基化组学 丙酮酸 小鼠 

分 类 号：S857.2[农业科学—临床兽医学]