Transcriptional Differences of Coding and Non-Coding Genes Related to the Absence of Melanocyte in Skins of Bama Pig

Abstract

Skin is the body's largest organ, and the main function of skin is to protect underlying organs from possible external damage. Melanocytes play an important role in skin pigmentation. The Bama pig has a "two-end-black" phenotype with different coat colors across skin regions, e.g., white skin (without melanocytes) and black skin (with melanocytes), which could be a model to investigate skin-related disorders, specifically loss of melanocytes. Here, we generated expression profiles of mRNAs and long noncoding RNAs in Bama pig skins with different coat colors. In total, 14,900 mRNAs and 7549 lncRNAs were expressed. Overall, 2338 mRNAs/113 lncRNAs with FDR-adjusted p-value ≤ 0.05 were considered to be differentially expressed (DE) mRNAs/lncRNAs, with 1305 down-regulated mRNAs and 1033 up-regulated mRNAs in white skin with|log₂(fold change)| > 1. The genes down-regulated in white skin were associated with pigmentation, melanocyte-keratinocyte interaction, and keratin, while up-regulated ones were mainly associated with cellular energy metabolisms. Furthermore, those DE lncRNAs were predicted to be implicated in pigmentation, keratin synthesis and cellular energy metabolism. In general, this study provides insight into the transcriptional difference involved in melanocyte-loss-induced keratinocyte changes and promotes the Bama pig as a biomedical model in skin research.

Keywords: Bama pig; melanocyte deficiency; model; transcriptome.

Figure 1

White and black skin were separately sampled from the pigs’ back and buttocks, and the pie plot shows the comparisons of fibroblast, keratinocyte and melanocyte in two different skins after evaluation by CIBERSORT [13]. Except for fibroblast, keratinocyte and melanocyte varied significantly between the two groups using the Student’s t-test (two-tailed). (*: p < 0.05).

Figure 2

Principal component analysis of mRNA (A) and lncRNA (B).

Figure 3

Enrichment analysis of differentially expressed (DE) mRNAs. (A) Volcano plot for 2338 DE genes, of which 1305 were down-regulated in white skins, while 1033 were up-regulated. (B) Functional enrichment analysis of the KEGG pathway (genes down-regulated in white skin were marked blue and up-regulated marked red; Benjamini corrected p-value < 0.05). (C) Gene ontology (GO) enrichment analysis (genes down-regulated in white skin marked blue and up-regulated marked red p-value < 0.05). (D) Identification of genes associated with melanogenesis. The genes in bold are transcript factor genes (cluster (1)).

Figure 4

Functional enrichment analysis of DE lncRNAs. (A) DE lncRNAs between two groups (79 DE lncRNAs were up-regulated in white skin while 34 DE lncRNAs were down-regulated). (B) Functional enrichment analysis with 88 DE genes adjacent to DE lncRNAs (p-value < 0.05). (C) Genomic location and reads abundance of TCONS_00019024 and its target gene CYGB. (Pearson correlation coefficient: 0.932, p = 0.01). (D) Functional enrichment analysis of trans target genes related to DE lncRNA.

Figure 5

RT-PCR validation of the differentially expressed mRNAs and lncRNAs. (A) RT-PCR validation results of 10 coat color genes (n = 3). (B) RT-PCR validation results of melanocyte-specific expression gene (n = 3). (C) RT-PCR validation results of mRNA and lncRNA that were differentially expressed in the skin (n = 3). The lncRNA TCONS_00019024 and its target gene CYGB were marked utilizing a pink star. (D) Correlation curve of TCONS_00019024/CYGB. RT-PCR data from skins were used to create a correlation curve (n = 6). Linear correlation coefficient of TCONS_00019024/CYGB was 0.71 (R² = 0.50, p = 0.035). (E) Relative expression of TCONS_00019024 and CYGB in 8 tissues (n = 3). * p-value < 0.05. ** p-value < 0.01.