Functional analysis of the PIP5K1A gene in Liaoning Cashmere goats: an investigation based on bioinformatics, tissue localization, and biological functions.

Abstract

Liaoning cashmere goat is an outstanding breed in China primarily for cashmere production, with strict controls against genetic outflow. Melatonin(MT) is a key factor affecting cashmere growth, and preliminary transcriptome sequencing indicated that melatonin upregulates the expression of the PIP5K1A gene in skin fibroblasts. To predict the physicochemical properties of PIP5K1A in Liaoning cashmere goats, ascertain the tissue localization of PIP5K1A in their skin, and explore the role and mechanism of PIP5K1A in the proliferation of skin fibroblasts. This aims to provide new insights into improving the yield and quality of cashmere. Bioinformatics software was used to predict the physicochemical properties, hydrophobicity, signal peptides, transmembrane regions, secondary structure, subcellular localization, and conserved sites of PIP5K1A, and to construct a phylogenetic tree for the PIP5K1A gene across different species. siRNA technology was employed to interfere with PIP5K1A in skin fibroblasts; lentiviral vector construction techniques were used to overexpress PIP5K1A in skin fibroblasts; Western blot analysis detected changes in protein expression in cells; RT-qPCR was used to detect changes in gene expression; cell viability was assessed using the CCK-8 method; hair follicle structure was observed by HE staining; and immunohistochemistry was used to detect the distribution of PIP5K1A in skin hair follicles. The PIP5K1A gene in Liaoning cashmere goats encodes 573 amino acids, classified as a soluble unstable protein without signal peptides or transmembrane regions; its secondary structure is primarily random coil; it contains one conserved domain - PIPKc superfamily. Phylogenetic analysis of the PIP5K1A gene from different species shows that goats have the closest kinship with sheep. Immunohistochemistry confirmed that PIP5K1A is specifically expressed in the outer root sheath of hair follicles; melatonin promotes the expression of the PIP5K1A gene and protein level; overexpression of PIP5K1A can promote cell proliferation, whereas interference with PIP5K1A significantly reverses melatonin-induced cell proliferation; after overexpressing PIP5K1A, the expression levels of chi-miR-34c-5p and chi-miR-novel-86 are downregulated. Conclusion: PIP5K1A is located in the outer root sheath of skin hair follicles in Liaoning Cashmere Goats. Overexpression of PIP5K1A can promote the proliferation of skin fibroblasts, interference with PIP5K1A can significantly reverse cell proliferation induced by melatonin, and PIP5K1A can regulate the expression of chi-miR-34c-5p and chi-miR-novel-86 in skin fibroblasts of Liaoning cashmere goats.