Regulation Of Key Genes In ERK Pathway Of Mud Crab Scylla Paramamosain Via MiR-9 And MiR-263

Mud crab Scylla paramamosain is one of the most important economic crabs in China.It is widely cultivated on the southeast coast of China.High quality eggs are in urgent need for the breeding of the mud crab in the aquaculture industry.Furthermore,the economical and nutritional value of crabs with mature ovary is much higher than that of immature crabs Therefore,it is of great significance in theory and practice to investigate the mechanism of ovarian development in the mud crab.ERK(extracellular signal-regulated protein kinase,ERK)signaling pathway is one of the most important cell signal transduction pathways.Many studies have shown that ERK signaling pathway has the functions of regulating proliferation,differentiation,and apoptosis of cell.Some studies found that ERK signaling pathway plays an important role in the ovarian development of the mud crab.Therefore,it has a great significance to study the molecular regulatory mechanism of ERK signaling pathway.MicroRNAs(miRNA)are a class of noncoding RNA molecules that negatively regulate gene expression through base pairing interactions between 3’-UTR of the target mRNAs and seed sequence of miRNA,and are one of the factors affecting the signal transduction of ERK signaling pathway.In this study,we used the bioinformatics tools to predict the target genes of selected miRNAs in the ovary of mud crab.The results showed that miR-9,miR-9c,miR-263a,and miR-263b can regulate the ERK signaling pathway-related genes:ERK2,MEK2,and Rap-b.The regulation of miRNA on the 3’-UTR of target genes were verified by dual luciferase reporter plasmid vector construction,cell culture,cell transfection,and dual luciferase reporter gene assay.Subsequently,in vivo experiments were also carried out to confirm the role of predicted miRNAs.The miRNA reagents were injected into the living mud crabs,and the expression levels of miRNAs and target genes after the injection were analyzed by quantitative real-time PCR to further validate the regulation of miRNAs on target genes.The experimental results are summarized as follows:1)The results of target gene prediction showed that:ERK2 is a target gene of miR-9c,miR-263a,and miR-263b,MEK2 is a target gene of miR-263a,and Rap-lb is a target gene of miR-9,miR-9c and miR-263a.2)The 3’-UTR sequences of ERK2,MEK2 and Rap-lb genes were obtained from NCBI,and the double luciferase reporter gene plasmids containing the 3’-UTR of these three genes were successfully constructed.They are pmir-RB-REPORTTM-ERK2-3’UTR,pmir-RB-REPORTTM-MEK2-3’UTR,and pmir-RB-REPORTTMRap-1b-3’UTR.3)To detect miRNA-mRNA interactions in vitro,miRNA and a double luciferase reporter gene plasmid were co-transfected into HEK293T cells under a dual-luciferase assay system.(1)The experimental groups were co-transferred with the miRNA mimics and the dual luciferase reporter gene plasmid into the cells,and the control groups were co-transferred with the miRNA mimics NC and the dual luciferase reporter gene plasmid into the cells.The results showed that the fluorescence value of the experimental groups were significantly lower than the control groups.(2)the experimental groups were co-transferred with the miRNA inhibitor and the dual luciferase reporter gene vector into the cells,and the control groups were co-transferred with the miRNA inhibitor NC and the dual luciferase reporter gene vector into the cell.The results showed that the fluorescence value of the experimental groups was significantly higher than the control groups.All these results preliminarily verified that miR-9,miR-9c,miR-263a and miR-263b have regulatory effects on ERK2,MEK2,and Rap-1b genes of ERK signaling pathway.4)To detect miRNA-mRNA interactions in vivo,the miR-9c enhancers/inhibitors or the miR-263a enhancers/inhibitors were injected into the mud crab,and the control group was injected with saline.The results showed that:(1)the expression of miR-263a and miR-9c was up-regulated in ovary and hepatopancreas respectively after injected with miR-263a enhancer(agomiR-263a)and miR-9c enhancer(agomiR-9c)respectively;the expression of miR-263a and miR-9c was down-regulated respectively after injected with miR-263a inhibitor(antagomiR-263a)and miR-9c inhibitor(antagomiR-9c)respectively.(2)In ovarian and hepatopancreatic tissues,the expression of ERK2,MEK2 and Rap-lb genes after injection of agomiR-263a was significantly lower than that of the control group;the expression of ERK2,MEK2 and Rap-1b genes after injection of antagomiR-263a was significantly higher than that of the control group.The expression level of ERK2 and Rap-lb genes was significantly lower than that of the control group after injection of agomiR-9c.The expression level of ERK2 and Rap-lb genes after injection of antagomiR-9c was significantly higher than that of the control group.These results further confirmed that miR-9c can regulate the expression of ERK2 and Rap-1b genes,and miR-263a can regulate the expression of ERK2,MEK2,and Rap-1b genes.