The Regulation Of Cyclins And CDKs In Ovary Via MiR-9c And MiR-263a Of Scylla Paramamosain

Cyclin and cyclin-dependent kinase(CDK)are key regulatory factors in the cell cycle signaling pathway,and are also important genes for regulating ovarian development.Micro RNAs(miRNAs)are a class of non-coding single-stranded RNA molecules that can negatively regulate gene expression at the post-transcriptional level by base complementary pairing with the3’untranslated region(3’-UTR)of target genes.Mud crab Scylla paramamosain is one of the most important economically cultured crab species in China.However,few studies about the effects of miRNAs on ovarian development regulate cyclins and CDKs in crabs.Based on previous research by our laboratory,we selected miR-9c and miR-263 a suitable for this study from the miRNAs of Scylla paramamosain ovary by bioinformatics analysis.The target genes of this study were obtained by software prediction: cyclin A,cyclin B,cyclin E,cyclin H,CDK1,and CDK2.Simultaneously,combined with plasmid construction,cell transfection,and quantitative real-time PCR methods,the regulatory effect of miRNA on target genes was verified in vitro and in vivo.The main experimental results are as follows:(1)Cyclin A,cyclin B,cyclin E,cyclin H,CDK1,and CDK2 were predicted to be the target genes of miR-9c and miR-263 a in this study through online software.(2)We constructed the double luciferase reporter gene plasmids containing the 3’-UTR of the target gene,and the regulatory effect of miRNA on the target gene was tested by cell transfection experiment.The double luciferase reporter plasmid of the target gene and miRNA reagent were co-transfected into HEK293 T cells.The transfection experiments were divided into experimental group and the treatment group.The experimental group was transfected with miRNA mimics/inhibitor and target plasmid,respectively.While the control group was transfected with miRNA scrambled sequence(miRNA mimics/ inhibitor NC)and target plasmid.The results of double luciferase reporter gene detection showed that the fluorescence value of the experimental group transfected with miRNA mimics and the target gene plasmid was significantly lower than that of the control group transfected with miRNA mimics NC and the target gene plasmid.The fluorescence value of the experimental group transfected with miRNA inhibitor and target gene plasmid was significantly higher than that of the control group transfected with miRNA inhibitor NC and target gene plasmid.The results preliminarily verified that miR-9c could regulate five target genes,including cyclin A,cyclin B,cyclin H,CDK1,and CDK2,while miR-263 a had regulatory effects on all of the six target genes.(3)The synthetic miRNA enhancers(agomiR-9c/263a),miRNA inhibitors(antagomiR-9c/263a),random miRNA sequences(miR-9c/263a-NC,which were predicted to have no regulatory effect on target genes),and saline were injected into the crab,respectively.There were nine crabs in each group,the regulation effect of miRNA on target genes was verified by quantitative real-time PCR.The result showed that in the ovary tissue of the crab,the expression of target genes in the experimental group injected with miRNA enhancer was significantly lower than that in the control group,while the expression of target genes in the experimental group injected with miRNA inhibitor was significantly higher than that in the control group.It was further verified that miR-9c could regulate the expression of cyclin A and CDK1,and miR-263 a could regulate the expression of cyclin A,cyclin B,cyclin E,cyclin H,CDK1 and CDK2 genes.The results of our study provide a new way to explore the reproductive regulation mechanism in mud crab and further enrich the knowledge of cell cycle and ovarian development regulation in invertebrates.