miR-9 and miR-263 Regulate the Key Genes of the ERK Pathway in the Ovary of Mud Crab Scylla paramamosain.

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.