Ssc-mir-221-3p regulates melanin production in Xiang pigs melanocytes by targeting the TYRP1 gene

Abstract

Background: MicroRNAs (miRNAs) are small endogenous non-coding RNAs that regulate gene expression by down-regulating it. Several studies have suggested that miRNAs plays a crucial role in mammalian skin color production. The TYRP1 gene, a member of the tyrosine family, is an important candidate gene that affects melanogenesis. This study aimed to identify genes and miRNAs that affect melanin production in Xiang pigs by transcriptome sequencing, and to validate their targeted regulatory relationships.

Results: 17 miRNAs and 1,230 genes were significantly differentially expressed (P < 0.05) in the black and white skin tissues of Jianbai Xiang pigs. miRNA-221-3p was identified as a candidate miRNA for melanin formation and its target gene, TYRP1, was selected. The TYRP1 gene is a member of the TYR gene family, which evolved from the TYR gene through chromosome segmental duplication. The function of the gene was highly conserved throughout the evolutionary process. overexpression of TYRP1 gene significantly increased the expression of TYR, TYRP1, and DCT genes P < 0.01, which led to an increase in the relative content of melanin. Silencing of TYRP1 through the use of TYRP1-siRNA significantly reduced the expression of TYR, TYRP1, and DCT genes in Jianbai Xiang pig melanocytes P < 0.01, which in turn decreased the relative melanin content. The targeted binding relationship between ssc-miR-221-3p and TYRP1 gene was validated. After transfection of porcine melanocytes with ssc-miR-221-3p mimic, the expression of ssc-miR-221-3p was significantly up-regulated (P < 0.01). Furthermore, the mRNA and protein levels of TYR, TYRP1, and DCT genes were significantly down-regulated (P < 0.01), and melanin content in cells was significantly reduced (P < 0.01).

Conclusion: The TYRP1 gene affects melanogenesis in melanocytes of Jianbai Xiang pigs, and ssc-miR-221-3p targets the TYRP1 gene to regulate melanogenesis in melanocytes of Jianbai Xiang pigs.

Keywords: Gene family; Melanin; Melanocyte; Ssc-miR-221-3p; TYRP1; Xiang pigs.

Fig. 1

Transcriptome analysis of miRNA and mRNA in the black and white skin tissue of Jianbai Xiang pigs. A, B The differential expression volcano map for miRNA and mRNA. C, D miRNA and mRNA differential expression clustering map. Columns represent various samples, rows represent different miRNAs and mRNAs, clustered by log₁₀(TPM + 1) and log₁₀(FPKM + 0.000001) values, respectively. The color red indicates high expression miRNAs and mRNAs, while green represents low expression miRNAs and mRNAs. The skin samples collected from the black coat on the back of Jianbai Xiang pigs are labeled as S03, S04, S05, L13, L14, and L15. On the other hand, the skin samples from the white coat on the head of Jianbai Xiang pigs are labeled as S02, S06, S07, L12, L16, and L17

Fig. 2

Experiments and correlation analysis. A-D Expression of selected miRNAs and genes in black and white skin tissue of the Jianbai Xiang pig (**, P < 0.001). A is the TPM value of each miRNA obtained through transcriptome sequencing, B is the miRNA expression of each gene detected by qRT-PCR, C is the FPKM value of each gene by transcriptome sequencing, and D is the qRT-PCR detection of each gene’s mRNA expression. E Differential target genes and miRNAs enriched in melanin synthesis pathway and tyrosine metabolism notification. Triangles and solids represent miRNAs and genes, respectively, while straight lines represented their interactions

Fig. 3

Analysis of the TYR gene family. Refer to the abbreviation directory for the meanings of the abbreviations. A Venn diagram of gene families in each species, with the center region indicating gene families that are common to all six species, and the peripheral regions shows gene families that are specific to each species. B Highlights members of the TYR gene family in pigs with red labels. C A phylogenetic tree constructed from the longest protein sequences of the TYR gene family members. D Ten conserved motifs in the protein sequence. E Exons in red regions and introns with lines, and the length of each region is indicated by scale below. F The circle plot, with the outermost circos indicating chromosome length and distribution. The second blue bar represents gene density calculated in a 5 Mb sliding window, while the green line in the third circle indicates GC content. The innermost connected circle shows the region of collinearity within the pig species. G The collinearity between pig, chicken, and cattle species. The blue line indicates TYR gene family pairs between pig and chicken, the green line shows TYR gene family pairs between pig and cattle, and the grey section shows collinearity for other genes

Fig. 4

Role of TYRP1 on melanogenesis in melanocytes of Jianbai Xiang pigs (**, P < 0.001). A-C Expression of TYR, TYRP1, and DCT genes in melanocytes after transfection with pEGFP-N3-TYRP1. D, E Expression of TYR, TYRP1, and DCT genes in melanocytes after transfection with TYRP1-siRNA. G Changes in melanin content in melanocytes after transfection with pEGFP-N3-TYRP1. H Changes in melanin content in melanocytes after transfection with TYRP1-siRNA (****, P < 0.00001)

Fig. 5

The evidence that ssc-miR-221-3p targets the TYRP1 gene. A The binding site of ssc-miR-221-3p and TYRP1. B TYRP1-wt, NC co-transfection group and TYRP1-wt, ssc-miR-221-3p mimic co-transfection group, TYRP1-mut, NC co-transfection group and TYRP1-mut, ssc-miR-221-3p mimic co-transfection group (**, P < 0.001)

Fig. 6

The rusults of an experiment analyzing the impact of ssc-miR-221-3p transfection on the genes related to melanin production. A mRNA expression of TYRP1, TYR and DCT following the transfection. B Western blotting results of genes involved in melanin production. C Protein expression of TYRP1, TYR and DCT in melanocytes post-transfection with ssc-miR-221-3p. D Effect of ssc-miR-221-3p transfection on melanin content in melanocytes(**, P < 0.001)