National Demonstration Center for Experimental Fisheries Science Education, Key Laboratory of Freshwater Aquatic Genetic Resources, Ministry of Agriculture and Rural Affairs, Shanghai Ocean University, Shanghai 201306, PR China.
National Demonstration Center for Experimental Fisheries Science Education, Key Laboratory of Freshwater Aquatic Genetic Resources, Ministry of Agriculture and Rural Affairs, Shanghai Ocean University, Shanghai 201306, PR China.
Gen Comp Endocrinol. 2022 Sep 1;325:114054. doi: 10.1016/j.ygcen.2022.114054. Epub 2022 May 14.
The insulin-like androgenic gland hormone gene (IAG) of crustaceans plays pivotal roles in the regulation of sex differentiation. MicroRNAs (miRNAs) are a class of short, non-coding RNAs that function as post-transcriptional gene regulators. However, little information about the regulatory relationship between miRNA and Macrobrachium rosenbergii IAG (MrIAG) were exposed. In this study, we used the 3' untranslated region (UTR) of MrIAG to predict potential target sites of miRNAs. The results showed that miR-184 has one target site in the 3'UTR of MrIAG. Dual-luciferase report assay in vitro confirmed that miR-184 can significantly down-regulate MrIAG expression. Besides, we constructed mutant plasmids of 3'UTR of MrIAG. The result displayed that after co-transfection of mutant plasmids and miR-184 agomir, the activity of luciferase was not affected compared to the control. These results indicated that miR-184 could directly regulate MrIAG. In addition, we found that overexpression of miR-184 in M. rosenbergii can lead to significant changes in the transcription level of genes. Compared with control group, we identified 1510 differentially expressed genes (DEGs) in the miR-184 injection group. Some DEGs were involved in sex differentiation, gonad development, growth and molting were found. qRT-PCR verification was performed on eight DEGs randomly, and the results showed that the expression level of sex-, growth-, and metabolism-related genes changed significantly after MrIAG gene knockdown. Collectively, findings from this study suggest that miR-184, by mediating IAG expression, may be involved in many physiological processes in M. rosenbergii. The current study lays a basic understanding for short-term silencing of MrIAG with miR-184, and facilitates miRNA function analysis in M. rosenbergii in future.
甲壳动物的胰岛素样雄激素腺激素基因（IAG）在性别分化的调节中起着关键作用。microRNAs（miRNAs）是一类短的非编码 RNA，作为转录后基因调控因子发挥作用。然而，关于 miRNA 与罗氏沼虾 IAG（MrIAG）之间的调控关系的信息很少。在这项研究中，我们使用 MrIAG 的 3'非翻译区（UTR）来预测 miRNAs 的潜在靶位点。结果表明，miR-184 在 MrIAG 的 3'UTR 中有一个靶位点。体外双荧光素酶报告实验证实，miR-184 可以显著下调 MrIAG 的表达。此外，我们构建了 MrIAG 3'UTR 的突变质粒。结果显示，突变质粒与 miR-184 agomir 共转染后，与对照相比，荧光素酶的活性没有受到影响。这些结果表明，miR-184 可以直接调控 MrIAG。此外，我们发现罗氏沼虾中 miR-184 的过表达会导致基因转录水平的显著变化。与对照组相比，我们在 miR-184 注射组中鉴定出 1510 个差异表达基因（DEGs）。一些 DEGs 参与性别分化、性腺发育、生长和蜕皮。随机选择 8 个 DEGs 进行 qRT-PCR 验证，结果显示 MrIAG 基因敲低后，与性别、生长和代谢相关的基因表达水平发生显著变化。综上所述，本研究表明，miR-184 通过调节 IAG 的表达，可能参与罗氏沼虾的许多生理过程。本研究为今后利用 miR-184 对 MrIAG 进行短期沉默奠定了基础，并促进了罗氏沼虾中 miRNA 功能的分析。
