小鼠感染肝毛细线虫肝脏microRNA的差异表达分析  

Differential expression of microRNA in the liver of mice infected by Capillaria hepatica

作　　者：张雅兰[1,2] 蒋甜甜 贺志权 邓艳 陈伟奇[1,2] 朱岩昆 张红卫[1,2] 赵东阳 ZHANG Ya-lan;JIANG Tian-tian;HE Zhi-quan;DENG Yan;CHEN Wei-qi;ZHU Yan-kun;ZHANG Hong-wei;ZHAO Dong-yang(Henan Center for Disease Control and Prevention,Zhengzhou 450016,China;Provincial Key Laboratory for Infectious Disease Prevention and Control,Zhengzhou 450016,China)

机构地区：[1]河南省疾病预防控制中心,郑州450016 [2]河南省传染病病原生物重点实验室,郑州450016

出　　处：《中国寄生虫学与寄生虫病杂志》2022年第1期56-60,67,共6页Chinese Journal of Parasitology and Parasitic Diseases

基　　金：河南省科技计划(202102310453);河南省医学科技攻关计划(LHGJ20200129)。

摘　　要：目的分析小鼠感染肝毛细线虫后肝脏microRNA (miRNA)的表达谱,筛选差异表达miRNA,为肝毛细线虫感染致肝纤维化机制研究提供资料。方法 6只雄性BABL/c小鼠随机分为肝毛细线虫感染组和对照组,每组3只。感染组小鼠经灌胃感染肝毛细线虫感染期虫卵(20个卵/鼠),对照组灌胃等量生理盐水。感染后35 d分别取感染组和对照组小鼠的肝组织,进行组织切片、苏木精-伊红(HE)染色后,镜下观察肝组织病变情况。提取和纯化小鼠肝组织总RNA进行文库构建和miRNA高通量测序。通过生物信息学分析筛选差异表达的mi RNA,预测其靶基因并进行基因本体论(GO)和京都基因与基因组百科全书(KEGG)富集分析。采用实时荧光定量PCR (qRT-PCR)对部分差异表达miRNA进行验证分析。结果肝组织切片的HE染色结果显示,感染组小鼠肝组织呈虫卵肉芽肿性病变,伴有明显的炎性细胞浸润;对照组肝细胞形态完整,未见炎性细胞浸润、变性坏死和纤维化。通过miRNA测序,筛选出差异表达的miRNA共16条,4条表达上调,且均上调2倍以上,其中mmu-miR-129-5p上调4倍以上;12条表达下调,均下调0.5倍以下,其中mmu-miR-21a-3p下调0.1倍以下。mi RNA靶基因GO功能分析显示,富集的基因主要参与了信号转导、蛋白连接、转化酶活性、 G蛋白偶联受体通路、细胞凋亡的负向调控、细胞增殖的正向调控、转录调控等;KEGG通路分析显示,富集的信号通路主要有Hippo信号通路、 Ras信号通路、 mTOR信号通路、磷脂类信号通路、 Wnt信号通路。qRT-PCR检测结果显示,感染组2个上调基因mmu-miR-21a-3p和mmu-miR-181a-1-3p的相对表达水平分别为0.70±0.30和0.68±0.27,2个下调基因mmu-miR-409-5p和mmu-miR-129-5p的相对表达水平分别为1.27±0.24和2.61±0.52,与参考基因相比表达水平均与测序结果趋势一致。结论肝毛细线虫感染导致小鼠肝组织miRNA表达谱发生了改变,mmu-miR-409-5p和Objective To analyze the microRNA (miRNA) expression profiles in the liver of mice infected with Capillaria hepatica,screening deferentially expressed miRNA,to provide basis for research on the mechanism of liver fibrosis caused by C.hepatica infection.Methods Six male BALB/c mice were randomly assigned to the C.hepatica infection group and control group,with 3 mice in each group.Each of the infection group mice was infected with 20 infective eggs of C.hepatica by gavage.For the control group,mice were given the same volume of saline.At 35 days post-infection,mice liver tissues from the two groups were collected for histo-section and hematoxylin-eosin (HE) staining to observe the pathological changes microscopically.Mice liver tissue total RNA was extracted and purified for construction of liberary and high throughput sequencing.Bioinformatics analysis was used to screen differentially expressed miRNA,estimate target genes and perform gene ontology (GO) analysis and Kyoto encyclopedia of genes and genomes (KEGG) enrichment.Real-time quantitative PCR (qRT-PCR) was used to validate part of differentially expressed miRNAs.Results The liver tissue sections stained with HE showed egg-granulomatous lesion in the livers of infection group,accompanying with significant inflammatory cell infiltration,while the hepatocytes in control group remained morphologically intact,with no inflammatory cell infiltration,degeneration,necrosis and fibrosis.Based on miRNA sequencing,the screening found 16 differentially expressed miRNAs,among them 4 were expression up-regulated with elevating over 2-fold,of which mmu-miR-129-5p expression was elevated4-fold;12 miRNAs expression were down-regulated with lowering more than 50%,of which mmu-miR-21 a-3p expression was lowered by more than 10%.The GO analysis of the miRNA target genes showed that the enriched genes were mainly involved in signal transduction,protein binding,transferase activity,G protein coupled receptor signaling pathway,negative regulation of apoptotic process,positive reg

关 键 词：肝毛细线虫 MIRNA 差异表达 肝纤维化 

分 类 号：R383.1[医药卫生—医学寄生虫学]