Proteomics Reveals the Role of PLIN2 in Regulating the Secondary Hair Follicle Cycle in Cashmere Goats

Abstract

Based on comprehensive proteomic analysis conducted across various stages of secondary hair follicles (SHFs), the growth and development regulatory mechanisms of SHFs in Jiangnan cashmere goats were studied. Proteomic analysis of skin tissue from the SHF anagen (An), catagen (Cn), and telogen (Tn) revealed 145 differentially expressed proteins (DEPs) between the An and Tn, 53 DEPs between the Cn and An, and 168 DEPs between the Cn and Tn. Gene Ontology (GO) annotations indicated that the DEPs were predominantly involved in keratin filament formation (KRTAP3-1, KRT1, KRT8), intermediate filament formation (KRT26, KRT35, KRT19, etc.), and lipid metabolism (FA2H, CERS6, ECH1, TECR, etc.). Furthermore, Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analysis identified significant enrichment of DEPs in pathways related to hair follicle growth and development. Notably, these included the PPAR signaling pathway (PLIN2, PLIN4, ACSL5, etc.), the IL-17 signaling pathway (S100A7A, LOC108633164), and the estrogen signaling pathway (KRT26, KRT35, LOC102176457.). Western blotting (WB) experiments were then performed on five DEPs (KRT28, FA2H, PLIN2, FABP7, and VNN1) to validate the consistency of the WB results with the proteomic data. Overexpression and siRNA interference of PLIN2 in dermal papilla cells (DPCs) were followed by CCK8 and flow cytometry assays, revealing that PLIN2 knockdown significantly decreased DPC proliferation while inducing apoptosis, compared to controls. These findings suggest that the PLIN2 gene plays a crucial role in modulating SHF growth cycles in cashmere goats by influencing DPC proliferation. These results provide novel insights that could inform the development of breeding strategies aimed at enhancing the cashmere yield in such goats.

Keywords: Jiangnan cashmere goats; PLIN2; proteomics; secondary hair follicle cycle.

Figure 1

Protein identification in skin tissue of Jiangnan cashmere goats. (A). Hierarchical filtering process utilized for protein identification, with distinct colors representing various levels of filtering; (B). PCA plot of the proteome, where different colors and shapes signify skin samples from varying stages of secondary follicle development. PC1 and PC2 correspond to the horizontal and vertical axes, respectively.

Figure 2

Differential expression analysis of proteins. (A–C) illustrate the volcano plot, Venn diagrams, and expression trend maps of differentially expressed proteins across various comparison groups. The figure presents Venn diagrams illustrating the up and down regulated differential proteins across various comparison groups. Proteins associated with hair follicle growth and development are indicated in red font. The color gradient from green to blue indicates the density of protein expression patterns across different stages (Cn, Tn, An) in the trend maps.

Figure 3

The potential roles of DEPs in regulating the hair follicle cycle. (A), GO analysis of DEPs across the three developmental stages. (B), KEGG analysis of DEPs across the three developmental stages. (A,B) figure: Red Font: Biological processes and pathways potentially associated with the hair follicle cycle. Red Box: Key pathways for further analysis. (C), PPI network analysis of selected DEPs. This network underscores the potential interactions among DEPs implicated in the PPAR signaling pathway, intermediate filament organization, and keratin filament dynamics. (D), Schematic representation delineating the effects of adipocyte differentiation and growth on the hair follicle cycle and the PPAR signaling pathway. The diagram illustrates the interactions between DEPs within the PPAR signaling pathway and those involved in other pathways, as well as the expression trends of DEPs during the transitions from Tn to An to Cn. The colors assigned to the DEPs correspond to the colors of their respective expression trend lines.

Figure 4

Western blot analysis, PLIN2 gene interference screening, and impact of PLIN2 on DPCs viability, apoptosis, and growth cycle regulation in Jiangnan cashmere goats. (A), Western blot was performed to assess the expression of FA2H, PLIN2, VNN1, KRT28, and FABP7, with GAPDH serving as a reference protein. Cn-P1, Cn-P2, and Cn-P3 correspond to Cn skin samples, Tn-P1, Tn-P2, and Tn-P3 correspond to Tn skin samples, and An-P1, An-P2, and An-P3 correspond to An skin samples. (B), Screening of PLIN2 interference fragments via mRNA and protein levels. (C), Effects of PLIN2 gene on DPC viability, apoptosis, and cell cycle. Data are presented as mean ± SEM. Statistical analysis of experimental results was conducted using the t-test; different lowercase letters indicate significant differences (p < 0.05), while identical lowercase letters denote insignificant differences (p > 0.05).