Department of Animal Science and Technology, Chung-Ang University, Anseong, 17546, Republic of Korea.
Department of Biochemistry and Immunology, National Institute of Veterinary Research, 86 Truong Chinh, Dong Da, Hanoi, 100000, Vietnam.
Vet Res. 2021 Mar 3;52(1):36. doi: 10.1186/s13567-021-00892-3.
Exosomes are membrane vesicles containing proteins, lipids, DNA, mRNA, and micro RNA (miRNA). Exosomal miRNA from donor cells can regulate the gene expression of recipient cells. Here, Ri chickens were divided into resistant (Mx/A; BF2/B21) and susceptible (Mx/G; BF2/B13) trait by genotyping of Mx and BF2 genes. Then, Ri chickens were infected with H5N1, a highly pathogenic avian influenza virus (HPAIV). Exosomes were purified from blood serum of resistant chickens for small RNA sequencing. Sequencing data were analysed using FastQCv0.11.7, Cutadapt 1.16, miRBase v21, non-coding RNA database, RNAcentral 10.0, and miRDeep2. Differentially expressed miRNAs were determined using statistical methods, including fold-change, exactTest using edgeR, and hierarchical clustering. Target genes were predicted using miRDB. Gene ontology analysis was performed using gProfiler. Twenty miRNAs showed significantly different expression patterns between resistant control and infected chickens. Nine miRNAs were up-regulated and 11 miRNAs were down-regulated in the infected chickens compared with that in the control chickens. In target gene analysis, various immune-related genes, such as cytokines, chemokines, and signalling molecules, were detected. In particular, mitogen-activated protein kinase (MAPK) pathway molecules were highly controlled by differentially expressed miRNAs. The result of qRT-PCR for miRNAs was identical with sequencing data and miRNA expression level was higher in resistant than susceptible chickens. This study will help to better understand the host immune response, particularly exosomal miRNA expression against HPAIV H5N1 and could help to determine biomarkers for disease resistance.
外泌体是一种含有蛋白质、脂质、DNA、mRNA 和 microRNA（miRNA）的膜囊泡。供体细胞的外泌体 miRNA 可以调节受体细胞的基因表达。在这里，通过 Mx 和 BF2 基因的基因型分析，将 Ri 鸡分为抗性（Mx/A；BF2/B21）和易感（Mx/G；BF2/B13）性状。然后，用 H5N1（一种高致病性禽流感病毒（HPAIV））感染 Ri 鸡。从抗性鸡的血清中纯化外泌体进行小 RNA 测序。使用 FastQCv0.11.7、Cutadapt 1.16、miRBase v21、非编码 RNA 数据库、RNAcentral 10.0 和 miRDeep2 对测序数据进行分析。使用包括倍数变化、edgeR 中的 exactTest 以及层次聚类在内的统计方法确定差异表达的 miRNA。使用 miRDB 预测靶基因。使用 gProfiler 进行基因本体论分析。20 个 miRNA 在抗性对照和感染鸡之间表现出明显不同的表达模式。与对照鸡相比，感染鸡中有 9 个 miRNA 上调，11 个 miRNA 下调。在靶基因分析中，检测到各种免疫相关基因，如细胞因子、趋化因子和信号分子。特别是，丝裂原活化蛋白激酶（MAPK）途径分子受到差异表达 miRNA 的高度调控。miRNA 的 qRT-PCR 结果与测序数据一致，并且抗性鸡中的 miRNA 表达水平高于易感鸡。本研究将有助于更好地了解宿主免疫反应，特别是针对 HPAIV H5N1 的外泌体 miRNA 表达，并有助于确定疾病抗性的生物标志物。
