miRNA-200a suppresses GNAI1 and PLCB4 to modulate skin pigmentation in cashmere goats

Abstract

Coat colour formation in mammals is influenced by melanogenesis and pigmentation processes regulated by miRNAs, including miRNA-200a. Although miRNA-200a is differentially expressed in the skin of cashmere goats with varying coat colours, its regulatory mechanism remains unclear. In this study, miRNA-200a target genes were predicted using miRBase and TargetScan, identifying GNAI1 and PLCB4 as the target genes through GO and KEGG analyses. Dual-luciferase assays using wild-type and mutant plasmids confirmed a direct interaction between miRNA-200a and the 3'UTR regions of these genes. RT-qPCR and Western blot analyses demonstrated that the expression levels of miRNA-200a and its target genes differed significantly between black and white goat skin. In HaCaT cells, transfection with miRNA-200a mimics or inhibitors altered GNAI1 and PLCB4 expression at both mRNA and protein levels. To validate these findings in vivo, subcutaneous injection of antagomiR-200a into BALB/c mice significantly reduced melanin content (P < 0.01) and increased the expression of GNAI1 and PLCB4. These results indicate that miRNA-200a modulates skin pigmentation by suppressing GNAI1 and PLCB4, thereby influencing coat colour in cashmere goats. This study provides a foundational understanding for leveraging genetic regulation to enhance coat colour diversity and develop naturally pigmented breeds.

Keywords: GNAI1; PLCB4; Cashmere goat; Coat colour; Melanogenesis; MiRNA-200a; Pigmentation.

Fig. 1

In vivo expression analysis of miRNA-200a and its target genes GNAI1 and PLCB4 in cashmere goat skin pigmentation. (A) KEGG pathway analysis demonstrating involvement of GNAI1 and PLCB4 (highlighted in red) in melanogenesis (pathway ID: hsa04916). Pathway map adapted from KEGG (Kanehisa et al., 2023); (B) mRNA expression levels of target genes GNAI1 and PLCB4 in black and white skin of cashmere goats; (C) Protein expression of target genes GNAI1 and PLCB4 in black and white skin of cashmere goats. M: marker; 1: black (black group); 2: white (white group). **, P < 0.01; ****, P < 0.0001.

Fig. 2

Predicted binding sites and dual-luciferase assay validation for miRNA-200a and its target genes GNAI1 and PLCB4. (A) Predicted binding sites of miRNA-200a with the target genes GNAI1 and PLCB4; (B) Validation of the targeted interactions. **, P < 0.01.

Fig. 3

Identification of in vitro expression of miRNA-200a and its target genes GNAI1 and PLCB4. (A) mRNA expression of GNAI1 in HaCaT cells transfected with miRNA-200a mimics/inhibitors/NC; (B) mRNA expression of PLCB4 in HaCaT cells post-transfection; (C) Protein expression levels of GNAI1 and PLCB4 in HaCaT cells post-transfection. 1: inhibitor group; 2: NC group; 3: mimics group. **, P < 0.01; ***, P < 0.001; ****, P < 0.0001.

Fig. 4

Measurement of melanin content and localisation of GNAI1 and PLCB4 protein expression. (A) Standard curve of melanin content and melanin levels in mouse skin; (B) Immunohistochemical results for target genes GNAI1 and PLCB4 in mouse skin. **, P < 0.01.

Fig. 5

Expression of miRNA-200a and its target genes in the skin of BALB/c mice injected with antagomiR-200a. (A) mRNA expression levels of miRNA-200a after antagomiR-200a injection; (B) mRNA expression levels of target genes GNAI1 and PLCB4 after antagomiR-200a injection; (C) Protein expression levels of GNAI1 and PLCB4 in mouse skin. 1: AntagomiRNA-200a group; 2: DEPC group. *, P < 0.05; **, P < 0.01; ***, P < 0.001; ****, P < 0.0001.