Department of Aquatic Life Medicine, Pukyong National University, Busan, 48513, South Korea.
Department of Integrative Bio-industrial Engineering, Sejong University, Seoul, 05006, South Korea.
Fish Shellfish Immunol. 2019 Nov;94:381-388. doi: 10.1016/j.fsi.2019.09.026. Epub 2019 Sep 12.
MicroRNAs (miRNAs) are non-coding small RNAs involved in the regulation of gene expression. In the present study, we firstly reported the use of a fish RNA virus, viral hemorrhagic septicemia virus (VHSV), as a delivery vehicle of a miRNA-30e, and the effect of miR-30e produced by the recombinant VHSV on the immune responses of Epithelioma papulosum cyprini (EPC) cells was investigated. The expression of functional miR-30e using a CMV promoter-driven vector was verified by the significantly lower eGFP expression in cells transfected with a vector containing miR-30e sponge sequence than that in cells transfected with a control vector that had mutated miR-30e sponge sequence. Furthermore, the down-regulation of reporter gene containing 3'-UTR of NF-κb inhibitor α-like protein B (NFκbiαb) by miR-30e was demonstrated, suggesting that miR-30e overexpression can increase immune responses related to NF-κB activation through inhibition of IκB. A miR-30e-expressing recombinant VHSV (rVHSV-A-miR30e) that had primary microRNA-30e sequence between N and P genes was rescued using the reverse genetic method, and the successful expression of miR-30e in the cells infected with rVHSV-A-miR30e was demonstrated using Northern blot and qRT-PCR. Cells infected with rVHSV-A-miR30e showed the increase of NF-κB activation and type I interferon induced genes expression, suggesting that rVHSV-A-miR30e can produce functional miR-30e in fish cells, and VHSV can be used as a vehicle to deliver functional microRNAs in fish.
微小 RNA（miRNA）是参与基因表达调控的非编码小 RNA。本研究首次报道了将鱼类 RNA 病毒，病毒性出血性败血症病毒（VHSV）作为 miRNA-30e 的递送载体，并研究了重组 VHSV 产生的 miR-30e 对鲤鱼上皮瘤细胞（EPC）免疫反应的影响。通过使用 CMV 启动子驱动的载体表达功能性 miR-30e，与转染含有 miR-30e 海绵序列突变的对照载体的细胞相比，转染含有 miR-30e 海绵序列的载体的细胞中 eGFP 表达明显降低，证实了 miR-30e 的表达。此外，通过 miR-30e 下调包含 NF-κB 抑制剂 α 样蛋白 B（NFκbiαb）3'-UTR 的报告基因，表明 miR-30e 过表达可以通过抑制 IκB 增加与 NF-κB 激活相关的免疫反应。通过反向遗传学方法拯救了具有 N 和 P 基因之间初级 miRNA-30e 序列的 miR-30e 表达重组 VHSV（rVHSV-A-miR30e），并通过 Northern blot 和 qRT-PCR 证明了感染 rVHSV-A-miR30e 的细胞中 miR-30e 的成功表达。感染 rVHSV-A-miR30e 的细胞显示 NF-κB 激活和 I 型干扰素诱导基因表达增加，表明 rVHSV-A-miR30e 可以在鱼类细胞中产生功能性 miR-30e，并且 VHSV 可作为递送功能性 microRNAs 的载体在鱼类中使用。
