Evolution, target genes certification and expression analysis of microRNA-137 in Japanese ornamental carp (Cyprinus carpio var. koi)

MicroRNAs (miRNAs) play an crucial role in post-transcriptional regulation of most transcripts in almost all animal species. Researchers have discovered that many miRNAs and a subset of their targets are conserved across deeply diverged phyla. miR-137 has been identified to function in melanogenesis in mammals. Considering the relation of miR-137 with melanin synthesis and conservation in species evolution, it might exert efforts in fish pigment synthesis. However, the function of miR-137 in fish color formation is still unclear. The Japanese ornamental carp (C. carpio var. koi) is famous for various colors and widely cultured all over the world. Due to its fast establishment of various colors under cultivation, C. carpio var. koi is a distinctive specie for genetics research in pigmentation. In order to explore the role of miR-137 in the color formation of C. carpio var. koi, the precursor sequences of miR-137 in 14 fish species were aligned and phylogenetic tree was constru-cted by Maximum Likelihood Estimate. Besides, the expression levels of miR-137 in different developmental stages and tissues also detected. Then, the target genes of miR-137 were predicted and analyzed by GO and KEGG analysis. In addtion, the luciferase reporter was used to verify the availability of miR-137 with its targets, and the expression levels of miR-137 and target genes in different development stages and tissues were checked and the correlation analysis between them was also conducted. The results showed that the precursor sequence similarity of miR-137 between C. carpio var. koi and C. carpio was the highest, and miR-137 sequences exhibited high conservation among all fish species. A great number of predicted target genes were enriched in melanogenesis, pigment cell differentiation, pteridine-containing compound metabolic process and so on. Real time PCR results showed the expression of miR-137-3p Feached the peak on 11 dph and rapidly dicreased in the following stages. miR-137 was expressed in all kinds of tissues, especially significantly in eye and muscle. Besides, the skin and fins, pigment cell enriched tissues, also had a higher expression and the expression of miR-137 in white tissues was higher than the red. The luciferase reporter analysis certified that miR-137 bound with the mitfa 3′-UTR to inhibit its expression but did not present significant inhibitory effect on sprb, and miR-137 was negatively correlated with the expression of mitfa and sprb in different developmental stages, but not in tissues. All the above results confirmed miR-137 was highly conserved during fish species evolution and displayed correlation with the koi color formation, in which miR-137 might regulate mitfa involved in color formation. This study provides basic research data for further exploring the role of miR-137 in C. carpio var. koi color formation.