Key Laboratory of Qinghai-Tibetan Plateau Animal Genetic Resource Reservation and Utilization, Ministry of Education, Southwest Minzu University, Chengdu, 610041, China; College of Life Science and Technology, Southwest Minzu University, Chengdu, 610041, China.
Department of Animal Sciences, Purdue University, West Lafayette, IN, 47907, USA.
Fish Shellfish Immunol. 2019 Nov;94:142-148. doi: 10.1016/j.fsi.2019.09.002. Epub 2019 Sep 2.
MiR-155 regulates the development of germinal-center and the generation of immunoglobulin class-switched plasma cells. However, whether miR-155 is involved in immune response in fish is still unclear. Here, CIK cells transfected with miR-155 overexpressed plasmid inhibited mRNA expression of mIg and Rag2 (P < 0.05). Interestingly, mIg was predicted as a potential target gene of miR-155 by RNAhybrid, with a putative binding site in its CDS. Further, mIg luciferase reporter vectors with successive deletions of mIg cDNA sequence were constructed and dual luciferase reporter assay showed that vectors containing the sequence from 318 to 347 in CDS exhibited lower relative luciferase activity than others without predicted binding region (P < 0.05), which indicated mIg is the target gene of miR-155 and reveal bona fide targeted binding site of mIg for miR-155 in fish. In vivo, the zebrafish were respectively injected with miR-155 overexpressed and empty vector, and showed that miR-155 efficiently expressed in zebrafish (P < 0.01), which consistently decreased mRNA level of immune-related genes, including mIg (P < 0.01), sIg (P < 0.05), AID (P < 0.01), PU.1 (P < 0.05) and Rag2 (P < 0.05) at d 3 and d 6 post injection, comparing to control. Collectively, this work indicates that overexpression of miR-155 suppresses the mRNA level of immune-related genes in CIK cells and zebrafish, and mIg is a novel target gene of miR-155 in fish. These findings provide an insight into the miR-155 modulating adaptive immunity in grass carp and zebrafish.
miR-155 调节生发中心的发育和免疫球蛋白类别转换浆细胞的产生。然而，miR-155 是否参与鱼类的免疫反应尚不清楚。本研究中，过表达 miR-155 的 CIK 细胞转染抑制了 mIg 和 Rag2 的 mRNA 表达（P<0.05）。有趣的是，RNAhybrid 预测 mIg 是 miR-155 的一个潜在靶基因，其 CDS 中存在一个潜在的结合位点。进一步构建了 mIg 荧光素酶报告载体，该载体连续缺失 mIg cDNA 序列，双荧光素酶报告基因检测表明，含有 CDS 中 318 到 347 位序列的载体表现出比没有预测结合区的载体更低的相对荧光酶活性（P<0.05），这表明 mIg 是 miR-155 的靶基因，并揭示了鱼类 mIg 与 miR-155 之间的真实靶结合位点。在体内，分别向斑马鱼注射 miR-155 过表达和空载体，结果表明 miR-155 在斑马鱼中高效表达（P<0.01），这一致地下调了免疫相关基因的 mRNA 水平，包括 mIg（P<0.01）、sIg（P<0.05）、AID（P<0.01）、PU.1（P<0.05）和 Rag2（P<0.05），在注射后第 3 天和第 6 天与对照组相比。总之，本研究表明，miR-155 的过表达抑制了 CIK 细胞和斑马鱼中免疫相关基因的 mRNA 水平，而 mIg 是鱼类 miR-155 的一个新靶基因。这些发现为 miR-155 调节草鱼和斑马鱼适应性免疫提供了新的见解。
