低叶酸联合甲氨蝶呤诱导神经管畸形胎鼠模型的lncRNA相关ceRNA网络构建及分析  

Construction and analysis of lncRNA-related ceRNA network in mouse model of methotrexate-induced neural tube defect under folate deficiency

作　　者：曾雨冰 刘帆 裴培[1] 王珊[1] ZENG Yubing;LIU Fan;PEI Pei;WANG Shan(Beijing Key Laboratory of Child Development and Nutriomics,Capital Institute of Pediatrics,Beijing,100020;Graduate School of Peking Union Medical College,Beijing,100020,China)

机构地区：[1]首都儿科研究所,北京市儿童发育组学重点实验室,北京100020 [2]北京协和医学院研究生院,北京100020

出　　处：《陆军军医大学学报》2023年第1期1-9,共9页Journal of Army Medical University

基　　金：国家自然科学基金面上项目(82071690);首都儿科研究所所级基金(FX-2020-05,CXYJ-2-21-09)。

摘　　要：目的 构建低叶酸联合甲氨蝶呤(methotrexate, MTX)诱导神经管畸形(neural tube defect, NTDs)的胎鼠模型中lncRNA相关的竞争性内源RNA(competing endogenous RNAs, ceRNA)调控网络,探讨低叶酸条件下NTDs中lncRNA可能的调控作用。方法 利用SPF级、7~8周龄的健康雌性C57BL/6J小鼠(体质量18~22 g)40只及健康雄性C57BL/6J小鼠(体质量20~22 g)20只,建立叶酸缺乏情况下的NTDs胎鼠模型为实验组,并设立对照组,每组选取3只胎鼠分离脑组织并提取总RNA,进行高通量RNA测序(RNA sequencing, RNA-seq),并对测序结果进行lncRNA-mRNA共表达网络构建、对共表达网络中mRNA进行GO功能富集及KEGG通路富集分析、构建lncRNA相关ceRNA调控网络,并用qPCR验证胎鼠、细胞样本中lncRNA Xist、Sema4f、Padi2的相对表达量。结果 测序共检测到差异lncRNA 47个(Log2FC>0.585或Log2FC<-0.585,FDR<0.05),其中上调38个,下调9个。构建lncRNA和mRNA基因间的共表达网络,并对共表达网络关系中的mRNA进行KEGG/GO功能富集分析,发现GO条目中管道发育(P<0.001)、胶原蛋白代谢过程(P=0.002)、系统发育(P=0.002)等显著富集,在KEGG通路分析中富集到与NTDs密切相关的PI3K-Akt通路;构建lncRNA相关ceRNA调控网络,发现lncRNA Xist、LOC102633899、1700086L19Rik等靶向差异基因Sema4f、Padi2、Lin28a等;选取与神经发育密切相关的lncRNA Xist及预测的靶基因Sema4f、Padi2,用qPCR验证胎鼠脑组织、细胞样本,结果与测序一致,相较对照组,均显著下调(P<0.05)。结论 在叶酸缺乏下的NTDs胎鼠模型中lncRNA Xist及预测的靶基因Sema4f、Padi2发生显著下调。Objective To construct a regulatory network of long noncoding RNA(lncRNA)-related competing endogenous RNAs(ceRNA)in low folate combined with methotrexate(MTX)induced neural tube defects(NTDs)mouse model,and to explore the potential lncRNAs regulation in low folate-induced NTDs.Methods Forty SPF-grade 7~8-week-old healthy female C57BL/6J mice(body weight 18~22 g)and 20 healthy male C57BL/6J mice(20~22 g)were selected to this experiment.After a folate deficiency induced fetal mouse NTD model was successfully established,the embryonic brain tissues were extracted for total RNA and RNA high-throughput sequencing(RNA-seq)was performed.The results of RNA-seq were used to carry out co-expression network construction,GO function enrichment of mRNA in co-expression network and KEGG pathway enrichment analysis,construction of lncRNA-related ceRNA regulation network and other bioinformatics analysis.And the relative expression of lncRNA Xist,Sema4f and Padi2 in the fetal mouse and cell samples was verified by qPCR.Results A total of 47 differential lncRNAs(Log2FC>0.585 or Log2FC<-0.585,FDR<0.05)were detected by RNA-seq,of which 38 were up-regulated and 9 were down-regulated.The co-expression network between lncRNA and mRNA was constructed.The results of KEGG/GO analysis on mRNAs in the co-expression network indicated that tube development(P<0.001),collagen metabolic process(P=0.002),system development(P=0.002)and other significant enrichments were highly relevant to NTDs.Besides,KEGG pathway analysis displayed that the PI3K-Akt pathway was closely related to NTDs;lncRNA Xist,LOC102633899,1700086L19Rik and other targeting differential genes Sema4f,Padi2,Lin28a were found in the lncRNA-related ceRNA network.qPCR confirmed that the above results in the fetal mice and cell samples under folic acid deficiency,with consistent corresponding sequences,and obviously down-regulated(P<0.05).Conclusion In folic acid deficiency-induced mouse NTD model,lncRNA Xist,and its targeting genes Sema4f and Padi2 are obviously down-regulated.

关 键 词：神经管畸形 表观遗传学 长非编码RNA 竞争性内源RNA 

分 类 号：R394.1[医药卫生—医学遗传学] R591.424[医药卫生—基础医学] R742.89