Sun Tongzhen, Huang Jinqiang, Li Yongjuan, Wu Shenji, Zhao Lu, Kang Yujun
College of Animal Science and Technology, Gansu Agricultural University, Lanzhou, 730070, China.
College of Animal Science and Technology, Gansu Agricultural University, Lanzhou, 730070, China.
Fish Shellfish Immunol. 2025 Mar;158:110157. doi: 10.1016/j.fsi.2025.110157. Epub 2025 Jan 27.
MicroRNAs (miRNAs) are highly conserved endogenous non-coding RNAs that play a crucial role in fish immune response by regulating gene expression at the post-transcriptional level. In recent years, the viral diseases caused by infectious hematopoietic necrosis virus (IHNV) have caused significant economic losses in rainbow trout (Oncorhynchus mykiss) aquaculture, whereas the immune regulatory mechanisms of miRNAs involved in rainbow trout resistance to IHNV infection remains largely undefined. In this study, we analyzed the structural characteristics of Oncorhynchus mykiss tumor necrosis factor receptor-associated factor 3 (OmTRAF3) by bioinformatics software and explored the molecular mechanism of miR-203-3p in rainbow trout resistance to IHNV by regulating OmTRAF3 in vivo and in vitro. The open reading frame (ORF) of OmTRAF3 gene was 1731 bp and encoded 576 amino acids including an N-terminal RING finger domain, two zinc finger domains, a coiled-coil domain, and a C-terminal MATH domain. The expression pattern analysis showed that the expression of miR-203-3p and OmTRAF3 in immune-related tissues (head kidney, spleen, and liver) and liver cells of rainbow trout infected with IHNV varied with certain regularity and had opposite trends at key time points, and a targeting relationship between miR-203-3p and OmTRAF3 was confirmed using a dual luciferase reporter gene assay. Further, we found that in vivo and in vitro overexpression of miR-203-3p significantly reduced the expression of OmTRAF3, downstream immune-related genes (OmTANK, OmIKKε, OmIFN1, and OmISG15) and promoted IHNV copy number replication, while silencing of miR-203-3p yielded opposite results. More importantly, OmTRAF3 and downstream genes as well as IHNV copy number changed accordingly with the silencing of OmTRAF3. The above results revealed that miR-203-3p participates in the immune response against IHNV by targeting OmTRAF3, and provides a theoretical basis for the screening of antiviral drugs in rainbow trout.
微小RNA（miRNA）是高度保守的内源性非编码RNA，通过在转录后水平调节基因表达在鱼类免疫反应中发挥关键作用。近年来，传染性造血坏死病毒（IHNV）引起的病毒性疾病给虹鳟（Oncorhynchus mykiss）养殖造成了重大经济损失，然而，参与虹鳟抵抗IHNV感染的miRNA的免疫调节机制仍 largely未明确。在本研究中，我们通过生物信息学软件分析了虹鳟肿瘤坏死因子受体相关因子3（OmTRAF3）的结构特征，并在体内和体外通过调节OmTRAF3探索了miR-203-3p在虹鳟抵抗IHNV中的分子机制。OmTRAF3基因的开放阅读框（ORF）为1731 bp，编码576个氨基酸，包括一个N端RING指结构域、两个锌指结构域、一个卷曲螺旋结构域和一个C端MATH结构域。表达模式分析表明，miR-203-3p和OmTRAF3在感染IHNV的虹鳟的免疫相关组织（头肾、脾脏和肝脏）和肝细胞中的表达随一定规律变化，在关键时间点呈相反趋势，并且使用双荧光素酶报告基因测定法证实了miR-203-3p与OmTRAF3之间的靶向关系。此外，我们发现体内和体外过表达miR-203-3p显著降低了OmTRAF3、下游免疫相关基因（OmTANK、OmIKKε、OmIFN1和OmISG15）的表达并促进了IHNV拷贝数的复制，而沉默miR-203-3p则产生相反的结果。更重要的是，随着OmTRAF3的沉默，OmTRAF3和下游基因以及IHNV拷贝数相应改变。上述结果表明，miR-203-3p通过靶向OmTRAF3参与针对IHNV的免疫反应，并为虹鳟抗病毒药物的筛选提供了理论依据。
