Fisheries College, Jimei University, Xiamen, China.
College of Animal Science, Fujian Agriculture and Forestry University, Fuzhou, China.
Mol Reprod Dev. 2019 Feb;86(2):122-131. doi: 10.1002/mrd.23063. Epub 2018 Nov 25.
Mud crab Scylla paramamosain is a commercially important species widely cultured in China. It is well known that the eyestalk regulates reproductive activities in crustaceans. In our previous research, we found that the miR-34 expression level in male eyestalk was significantly higher than that in females. Thus, we assumed that it may play an important role in regulating reproduction. In this study, we used bioinformatic tools to identify the target genes of miR-34 in eyestalk. Six reproduction-related genes with an intact 3'-untranslated region (UTR), including molt-inhibiting hormone (MIH), crustacean hyperglycemic hormone (CHH), vitellogenesis-inhibiting hormone, red pigment concentrating hormone, ecdysone receptor (EcR), and farnesoic acid methyltransferase (FAMeT) were identified. When the 3'-UTR plasmid vectors of the six genes were cotransfected with miR-34 mimics into 293FT cells, respectively, the luciferase activities of four genes (MIH, CHH, EcR, and FAMeT) were significantly decreased compared with that in the control group; on the contrary, when the six plasmid vectors were cotransfected with the miR-34 inhibitor respectively, the luciferase activities of four genes (MIH, CHH, EcR, and FAMeT) were significantly higher than that in the control group. When agomiR-34 and antagomiR-34 were injected into the eyestalk respectively in vivo, the expression levels of the MIH, CHH, EcR, and FAMeT genes were detected by a quantitative real-time polymerase chain reaction. The results showed that agomiR-34 suppressed the expression of the four genes, whereas antagomiR-34 enhanced their expression. These experimental results confirmed our hypothesis that miR-34 may indirectly regulate reproduction via binding to the 3'-UTRs of MIH, CHH, EcR, and FAMeT genes and suppressing their expression.
拟穴青蟹（Scylla paramamosain）是中国广泛养殖的一种具有商业价值的物种。众所周知，眼柄调节甲壳动物的生殖活动。在我们之前的研究中，发现雄性眼柄中的 miR-34 表达水平明显高于雌性。因此，我们假设它可能在调节生殖方面发挥重要作用。在这项研究中，我们使用生物信息学工具来鉴定眼柄中 miR-34 的靶基因。我们鉴定了六个具有完整 3'非翻译区（UTR）的与生殖相关的基因，包括蜕皮抑制激素（MIH）、甲壳动物高血糖激素（CHH）、卵黄生成抑制激素、红色素浓缩激素、蜕皮激素受体（EcR）和法呢酸甲基转移酶（FAMeT）。当将这六个基因的 3'UTR 质粒载体与 miR-34 模拟物分别共转染到 293FT 细胞中时，与对照组相比，四个基因（MIH、CHH、EcR 和 FAMeT）的荧光素酶活性显著降低；相反，当将六个质粒载体分别与 miR-34 抑制剂共转染时，四个基因（MIH、CHH、EcR 和 FAMeT）的荧光素酶活性显著高于对照组。当体内分别注射 agomiR-34 和 antagomiR-34 到眼柄时，通过定量实时聚合酶链反应检测 MIH、CHH、EcR 和 FAMeT 基因的表达水平。结果表明，agomiR-34 抑制了四个基因的表达，而 antagomiR-34 增强了它们的表达。这些实验结果证实了我们的假设，即 miR-34 可能通过与 MIH、CHH、EcR 和 FAMeT 基因的 3'UTRs 结合并抑制其表达，间接调节生殖。
