收稿日期: 2021-11-08
修回日期: 2021-12-23
网络出版日期: 2022-07-06
基金资助
西北民族大学预防兽医学创新团队项目(110130143);国家自然科学基金青年基金(31702224)
Identification and bioinformatics analysis of differentially expressed miRNAs in splenic lymphocytes in Echinococcus multilocularis-infected mice
Received date: 2021-11-08
Revised date: 2021-12-23
Online published: 2022-07-06
Supported by
Prevention Veterinary Innovation Team in Northwest Minzu University(110130143);National Natural Science Foundation of China-Young Science Foundation Project(31702224)
目的 筛选并鉴定多房棘球蚴感染的小鼠脾淋巴细胞中差异表达的微小RNA （miRNA）,分析其可能涉及的生物学过程及信号通路,为进一步研究miRNA在寄生虫感染中的作用提供实验依据。 方法 将12只小鼠随机分为两组,每组6只,感染组小鼠每只腹腔注射600个多房棘球蚴原头节,对照组注射等量PBS。感染后90 d,将小鼠安乐死后取脾组织,采用密度梯度离心法分离各组小鼠脾淋巴细胞,使用TRIzol法提取两组小鼠脾淋巴细胞的总RNA。利用高通量测序技术鉴定并筛选差异表达的miRNA;随机选取5个差异表达的miRNA进行实时荧光定量PCR（qRT-PCR）验证;利用miRanda和RNAhybrid两个数据库对差异表达的miRNA进行靶基因预测,并进行基因本体（GO）和京都基因与基因组百科全书（KEGG）富集分析;利用Cytoscape软件构建miRNA-mRNA相互作用网络图。 结果 通过高通量测序对照组和感染组分别获得12 104 631和10 856 249个纯净序列,其序列主要集中在20~24 nt。共筛选出69个差异表达2倍以上的miRNA,其中40个为上调表达的miRNA,29个为下调表达miRNA。随机选取的5个差异表达miRNA经qRT-PCR验证结果显示,miR-150-5p、miR-181a-5p、miR-467a-5p、miR-467b-5p表达下调,miR-223-3p表达上调,与高通量测序结果相一致。GO功能和KEGG通路分析结果显示,69个差异表达miRNA的靶基因与黏膜免疫、应激反应、细菌防御反应等相关,并且主要富集在丝裂原活化蛋白激酶（MAPK）、磷脂酰肌醇3激酶/蛋白激酶B（PI3K-Akt）、单磷酸腺苷激活的蛋白激酶（AMPK）等信号通路。miR-150-5p、miR-181a-5p、miR-467a-5p和miR-467b-5p与mRNA相互作用网络图显示,miRNA与多个mRNA相互作用,mRNA也可被多个miRNA所调控。 结论 小鼠在多房棘球蚴感染过程中,其脾淋巴细胞中的miRNA呈显著差异表达,差异表达的miRNA主要富集在一些免疫相关的信号通路,可能在对抗多房棘球蚴的免疫反应中发挥作用。
仲顺虎 , 孙玥 , 郭小腊 , 郑亚东 , 陈轶霞 . 多房棘球蚴感染小鼠脾淋巴细胞中差异表达miRNA的鉴定及其生物信息学分析[J]. 中国寄生虫学与寄生虫病杂志, 2022 , 40(3) : 288 -294 . DOI: 10.12140/j.issn.1000-7423.2022.03.003
Objective To screen and identify the differentially expressed microRNAs (miRNAs) in spleen lymphocytes of mice infected with Echinococcus multilocularis, analyze the possible biological processes involved and signalling pathways, and provide experimental basis for further study of the role of miRNAs in parasite infection. Methods Twelve mice were randomly divided into two groups with 6 mice in each group. Each mouse in the experimental group was intraperitoneally injected with 600 protoscoleces, while the control group was injected with the same amount of PBS. On the 90th day after infection, the spleen lymphocytes of the mice in each group were separated by density gradient centrifugation, and the total RNA of the spleen lymphocytes was extracted by the TRIzol method. The differentially expressed miRNAs were identified and screened by high-throughput sequencing. Five differentially expressed miRNAs were randomly selected for real-time quantitative PCR (qRT-PCR) validation. MiRanda and RNAhybrid databases were used to predict the target genes of differentially expressed miRNAs, and gene ontology (GO) and kyoto encyclopedia of genes and genomes (KEGG) enrichment analysis was performed. Cytoscape software was used to construct miRNA-mRNA interaction network diagrams. Results In the high-throughput sequencing, 12 104 631 and 10 856 249 clean reads were obtained in the control group and the experimental group, respectively. The sequences of clean reads were abundant in 20~24 nt in length. A total of 69 differentially expressed miRNAs (fold change> 2) were screened by high-throughput sequencing technology, of 40 were up-regulated, and 29 were down-regulated. The qRT-PCR results showed that the expression of miR-150-5p, miR-181a-5p, miR-467a-5p, and miR-467b-5p was down-regulated while the expression of miR-223-3p was up-regulated. GO enrichment results showed that the targets of 69 differentially expressed miRNAs were involved in mucosal immunity, stress response, and bacterial defence response. KEGG pathway analysis revealed that these genes were involved in the mitogen-activated protein kinase (MAPK) signalling pathway, phosphatidylinositol 3-kinase/protein kinase B (PI3K-Akt) signaling pathway, adenosine monophosphate-activated protein kinase (AMPK) signalling pathway, and other related pathways. The miR-150-5p, miR-181a-5p, miR-467a-5p, and miR-467b-5p and mRNA interaction network diagram shows that a miRNA interacted with multiple mRNAs, and a mRNA can also be regulated by multiple miRNAs. Conclusion The miRNA expression in mice spleen lymphocytes was significantly elevated during E. multilocularis infection, while the differentially expressed miRNAs were mainly enriched in some immune-related signalling pathways, which may play a role in the immune responses against E. multilocularis.
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