Fujian Engineering Research Center of Aquatic Breeding and Healthy Aquaculture, Fisheries College, Jimei University, Xiamen, 361021, China.
College of Animal Science, Fujian Agriculture and Forestry University, Fuzhou, 350002, China.
Mar Biotechnol (NY). 2020 Aug;22(4):594-606. doi: 10.1007/s10126-020-09981-4. Epub 2020 Jul 11.
Mud crab Scylla paramamosain is one of the most important economic crabs in China. The molecular regulatory mechanism of ovarian development has received considerable attention in recent years. Some studies found that ERK (extracellular signal-regulated protein kinase) signaling pathway plays an important role in ovarian development and is negatively regulated by microRNAs (miRNAs). However, the study about the regulation of miRNA on the ERK pathway in crustacean's ovary remains unknown. In this study, the target genes of the ERK signaling pathway regulated by selected miRNAs identified from the ovary of mud crab in our previous research were predicted by using bioinformatics tools. The results showed that the ERK2 might be a target gene of miR-9c, miR-263a, and miR-263b; MEK2 may be a target gene of miR-263a; and Rap-1b may be a target gene of miR-9, miR-9c, and miR-263a. Results of in vitro dual-luciferase reporter assay showed that the relative luciferase activities were significantly lower in HEK293T cells co-transfected with the combination of miRNA mimics and pmir-RB-REPORTTM-target gene-3'UTR than those with the combination of mimics NC and pmir-RB-REPORTTM-target gene-3'UTR. In contrast, the relative luciferase activities were significantly higher in HEK293T cells co-transfected with miRNA inhibitor than those with inhibitor NC. To further validate in vitro results, the miRNA reagents were injected into the living female mud crabs, and the expression levels of miRNAs and target genes after the injection were analyzed by quantitative real-time PCR. The in vivo experimental results showed that miRNAs (miR-9c/miR-263a) agomir (enhancers)/antagomir (inhibitors) can enhance/decrease the expression of two miRNAs, respectively, and the expression of target genes in the ovary was declined/increased after injection of agomir/antagomir reagent. In conclusion, miR-9/miR-263 can negatively regulate the expression of the ERK pathway genes (ERK2, MEK2, and Rap-1b) in the ovary of mud crab.
拟穴青蟹（Scylla paramamosain）是中国最重要的经济蟹类之一。近年来，卵巢发育的分子调控机制受到了广泛关注。一些研究发现，ERK（细胞外信号调节蛋白激酶）信号通路在卵巢发育中起重要作用，并受到 microRNAs（miRNAs）的负调控。然而，甲壳动物卵巢中 miRNA 对 ERK 通路的调控研究仍不清楚。本研究利用生物信息学工具预测了从前期研究中筛选出的 Mud crab 卵巢中 ERK 信号通路的靶基因。结果表明，miR-9c、miR-263a 和 miR-263b 可能靶向 ERK2；miR-263a 可能靶向 MEK2；miR-9、miR-9c 和 miR-263a 可能靶向 Rap-1b。体外双荧光素酶报告基因检测结果显示，miRNA 模拟物与 pmir-RB-REPORTTM-靶基因-3'UTR 共转染 HEK293T 细胞后，荧光素酶相对活性明显低于 miRNA 模拟物 NC 与 pmir-RB-REPORTTM-靶基因-3'UTR 共转染组。相反，miRNA 抑制剂共转染 HEK293T 细胞后，荧光素酶相对活性明显高于抑制剂 NC 共转染组。为进一步验证体外结果，将 miRNA 试剂注入活体雌性青蟹体内，注射后通过 qRT-PCR 分析 miRNA 和靶基因的表达水平。体内实验结果表明，miR-9c/miR-263a 激动剂（增强剂）/拮抗剂（抑制剂）分别能增强/降低两种 miRNA 的表达，注射激动剂/抑制剂试剂后，卵巢中靶基因的表达下降/增加。综上所述，miR-9/miR-263 可以负调控拟穴青蟹卵巢中 ERK 通路基因（ERK2、MEK2 和 Rap-1b）的表达。
