^(137)Csγ射线全身照射对小鼠骨髓细胞中circRNA m^(6)A修饰谱的影响

Effect of total body^(137)Csγ-irradiation on the m^(6)A modification profile of circRNA in mouse bone marrow cells

目的研究电离辐射对小鼠骨髓细胞中环状RNA(circular RNA,circRNA)的N^(6)-甲基腺嘌呤(N^(6)-methyladenine,m^(6)A)修饰谱的影响,为揭示RNA表观遗传修饰与放射性造血系统损伤之间的关系提供科学依据。方法将24只C57BL/6 J小鼠按随机数表法分为健康对照组和照射组,每组12只。照射组用4 Gy^(137)Csγ射线对小鼠进行全身照射。照后将两组小鼠脱颈处死,收集股骨中的骨髓细胞。提取总RNA,通过m^(6)A RNA免疫沉淀-高通量测序(MeRIP-Seq)技术和生物信息学分析方法,探究小鼠受照后骨髓细胞中circRNA m^(6)A修饰谱的变化。运用MeRIP-qPCR实验对变化显著的m^(6)A修饰位点进行验证。结果健康对照组和照射组中各鉴定出325和455个(其中两组共有178个,健康对照组特有147个,照射组特有277个)circRNA的m^(6)A修饰位点。健康对照组和照射组中各鉴定出1275和1017个(其中两组共有767个,健康对照组特有508个,照射组特有250个)发生m^(6)A修饰circRNA的来源基因。与健康对照组相比,照射组中有414个m^(6)A位点的富集倍数显著上调,178个m^(6)A位点的富集倍数显著下调,差异具有统计学意义(P<10^(-10);倍数筛选阈值>5)。基因本体论(GO)分析显示,电离辐射后小鼠骨髓细胞中m^(6)A修饰水平显著改变的circRNA来源基因涉及染色质相关调控、纤毛转换纤维和多聚(A)特异性核糖核酸酶活性等多种功能。京都基因与基因组百科数据库(KEGG)分析显示,电离辐射后小鼠骨髓细胞中m^(6)A修饰水平显著改变的circRNA来源基因涉及血小板激活、FcγR-介导的吞噬作用和B细胞受体信号通路等多种通路。结论电离辐射可引起小鼠骨髓细胞中circRNA的m^(6)A修饰谱的迅速改变,差异甲基化的circRNA的来源基因涉及多种造血系统放射生物学相关的功能通路。该研究为从表观遗传水平揭示造血系统辐射损伤的分子机制奠定基础。

Objective To investigate the effect of ionizing radiation on the N^(6)-methyladenine(m^(6)A)modification profile of circular RNA(circRNA)in mouse bone marrow cells and provide scientific basis for revealing the relationship between RNA epigenetic modification and hematopoietic radiation injury.Methods A total of twenty four C57BL/6 J mice were randomly divided into two groups:the healthy control group(n=12),and ionizing radiation group(n=12)irradiated in total body with 4 Gy of^(137)Csγ-rays.At 5 min after irradiation,mice were killed and bone marrow cells were collected from the femur.Total RNAs were extracted and the changes in circRNA m^(6)A modification profiles were investigated by RNA immunoprecipitation-high-throughput sequencing(MeRIP-Seq)technology and bioinformatics analysis.The representative alterations of m^(6)A peaks were validated by MeRIP-PCR assay.Results 325 and 455 m^(6)A sites were identified on circRNAs in the healthy control group and ionizing radiation group(178 common sites,147 specific sites in the healthy control group and 277 specific sites in ionizing radiation group),respectively.1275 and 1017 deriving genes of m^(6)A-circRNAs were identified in the healthy control group and ionizing radiation group(767 common genes,508 specific genes in the healthy control group and 250 specific genes in ionizing radiation group),respectively.Compared with the control healthy group,414(178)m^(6)A peaks was significantly up-(down-)regulated in the ionizing radiation group(P<10-10;fold-change cut-off>5).Moreover,Gene Ontology(GO)assay revealed that the deriving genes of circRNAs with differentially methylated m^(6)A sites between two groups involves various functions including chromatin regulation,ciliary transition fiber and poly(A)-specific ribonuclease activity.Kyoto Encyclopedia of Genes and Genomes(KEGG)assay revealed that the deriving genes of circRNAs with differentially methylated m^(6)A sites between two groups included numerous pathways such as platelet activation,FcγR-mediated phagocytosis and B cell receptor signaling pathway.Conclusions Ionizing radiation triggers rapid alterations in the m^(6)A modification profile of circRNA in mouse bone marrow cells.The deriving genes of differentially methylated circRNAs are associated with a variety of functions and signaling pathways of hematopoietic radiobiology.