Abstract
MicroRNAs (miRNAs) are ∼22 nucleotide non-coding RNA molecules that act as crucial roles in plenty of biological processes. However, the molecular and cellular mechanisms of miRNAs to regulate skin color differentiation and pigmentation in fish have not been fully understood. Herein, we revealed that miR-206, a skin-enriched miRNA, regulates melanocortin 1 receptor (Mc1r, a key regulator of melanogenesis) expression by binding to its 3'-untranslated (UTR) region through bioinformatics and luciferase reporter assay in koi carp (Cyprinus carpio L.). The analysis of spatial and temporal expression patterns suggested that miR-206 is a potential regulator in the skin pigmentation process. Then, we silenced it in vivo with an antagomir method. The result showed a substantial increase of Mc1r mRNA expression and protein level, and also its downstream genes: tyrosinase (Tyr) and dopachrome tautomerase (Dct) that encoding key enzymes involved in melanin synthesis. Moreover, we constructed the miRNA-206 sponge lentivirus vector to transfect koi carp melanocytes in vitro, further checked the functions of melanocytes using Cck-8 and Transwell assays. As a result, inhibition of miR-206 significantly up-regulated Mc1r mRNA expression and protein level and accelerated the melanocyte proliferation and migration ability compared with the scrambled-sequence negative control group (miR-NC). Overall, these findings provide the evidence that miR-206 plays a regulatory role in the skin color pigmentation through targeting the Mc1r gene and would facilitate understanding the molecular regulatory mechanisms underlying miRNA-mediated skin color pigmentation in koi carp.
Highlights
MicroRNAs are single-stranded, evolutionary conserved ~22 nucleotides molecules that affect the gene expression by triggering either mRNA degradation or translational repression through combining the 3'-untranslated regions (3'-UTR) (Ambros, 2004; Krol et al, 2010)
We used a combination of TargetScan, RNAhybrid, and miRanda software to predict the skin color related-miRNA target genes by alignment with common carp (C. carpio) genome sequence, the final results must be consistent with the analysis of all three software in order to improve the accuracy (Additional file 3, Table S2)
The results indicated that melanocortin 1 receptor (Mc1r) may be regulated by more than one miRNA, including miR-206, miR-217, miR-125c, miR-181a-5p, miR196a, and miR-125b-5p
Summary
MicroRNAs (miRNAs) are single-stranded, evolutionary conserved ~22 nucleotides (nt) molecules that affect the gene expression by triggering either mRNA degradation or translational repression through combining the 3'-untranslated regions (3'-UTR) (Ambros, 2004; Krol et al, 2010). The researches about the interaction between miRNAs and mRNAs have gradually verified that it is crucial for transcriptional and signal transduction events like development, MiR-206 Regulation of Skin Pigmentation differentiation, cell proliferation, and apoptosis (Hwang and Mendell, 2006; Yan et al, 2013; Wang et al, 2017). Yan et al identified the miR-429 act as a crucial role in skin pigmentation by targeting Foxd3 3'-untranslated (3'-UTR) region and silencing its expression in vivo would repress the transcription level of Mitf and its downstream genes (Yan et al, 2013). We have identified 164 differentially expressed miRNAs (DEMs) and 14 overlapping including miR-206 on different skin colors (black, red, and white) through Illumina sequencing in koi carp (Luo et al, 2018). MiRNAs specific regulation mechanisms related to skin color differentiation in koi carp remains unclear
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