miR-330 targeting BCO2 is involved in carotenoid metabolism to regulate skin pigmentation in rainbow trout (Oncorhynchus mykiss)

Abstract

Background: MicroRNAs (miRNAs) play a critical role in regulating skin pigmentation. As a key economic trait, skin color directly affects the market value of rainbow trout (Oncorhynchus mykiss), however, the regulatory mechanism of most miRNAs in fish skin color is still unclear. Results: In this study, the full-length cDNA sequence of β-carotene oxygenase 2 (BCO2, a key regulator of carotenoid metabolism) from the rainbow trout was obtained using rapid-amplification of cDNA ends (RACE) technology, and qRT-PCR was used to investigate the differential expression of miR-330 and BCO2 in 14 developmental stages and 13 tissues between wild-type rainbow trout (WTrt) and yellow mutant rainbow trout (YMrt). Additionally, the function of miR-330 was verified by overexpression and silencing in vitro and in vivo. The results showed that the complete cDNA sequence of BCO2 was 2057 bp with a 1707 bp ORF, encoding a 568 amino acid protein having a molecular weight of 64.07 kD. Sequence alignment revealed that higher conservation of BCO2 protein amongst fishes than amongst other vertebrates, which was further confirmed by phylogenetic analysis. The analysis of spatial and temporal expression patterns suggested that BCO2 and miR-330 were abundantly expressed from fertilized-stage to multi-cell as well as in the dorsal and ventral skin of WTrt and YMrt, and their expression patterns were opposite in most of the same periods and tissues. In vitro, luciferase reporter assay confirmed that BCO2 was a direct target of miR-330, and transfection of miR-330 mimics into rainbow trout liver cells resulted in a decrease in the expression of BCO2; conversely, miR-330 inhibitor had the opposite effect to the miR-330 mimics. In vivo, miR-330 agomir significantly decreased BCO2 expression in dorsal skin, tail fin, and liver. Furthermore, overexpression of miR-330 could suppress cell proliferation and induce apoptosis. Conclusion: Our results showed that miR-330 is involved in the regulation of skin pigmentation in rainbow trout by targeting BCO2 and shows its promise as a potential molecular target to assist the selection of rainbow trout with better skin color patterns.