Regulation of VIH by miR-277 in the eyestalk of mud crab Scylla paramamosain

Abstract

Abstract The regulatory role of microRNAs in gonad development and reproduction is attracting more and more attention in recent years.Vitellogenesis−/Gonadal-inhibiting hormone (VIH/GIH) is a member of the crustacean hyperglycemic hormone (CHH) family. It plays a key role in regulating ovarian development and gonadal maturation in crustaceans. In this study, based on our preliminary studies of Spvih (Scylla paramamosain vih, Spvih), the regulation of expression of Spvih gene was further investigated. Predicted by bioinformatics software, we found that miRNA-34/miRNA-277 may play a regulatory role in Spvih expression. Analysis of luciferase activity of HEK293T cells transfected with the synthetic miRNA-34/miRNA-277 mimics and miRNA-34/miRNA-277 inhibitor revealed that miRNA-277 mimics significantly reduced the activity and miRNA-277 inhibitor significantly increased the activity compared to the control group. However, there was no significant difference in activity between miRNA-34 mimics and miRNA-34 inhibitor. The relative expression of Spvih and miRNA-277 was detected in the eyestalk, which had been injected with stroke-physiological saline solution(SPSS)and chemically synthesized miRNA-277 agomir and miRNA-277 antagomir, respectively. The results showed that compared with the control group SPSS, the expression of miR-277 in the eyestalk was increased significantly after miRNA-277 agomir injection, while the expression of Spvih was decreased significantly. After the injection of miRNA-277 antagomir, the expression of miR-277 in the eyestalk decreased significantly, while the expression of Spvih increased significantly. The inhibitory effect of miRNA-277 on Spvih expression was further verified. Based on miR-277 agomir and miRNA-277 antagomir injected into the base of the crab, the relative expression of the Spvtg gene in the ovarian and hepatopancreas was detected. The results showed that compared with the control group SPSS, the expression of Spvtg in the ovary and hepatopancreas were significantly increased after injection of miRNA-277 agomir, while the expression of Spvtg was significantly decreased after injection of miRNA-277 antagomir. Our study may provide an evidence of how miRNA works on the regulation of Spvih, which indirectly affects the ovarian development of mud crab, and suggest a new strategy for controlling crab reproduction.