Dynamic miRNA Landscape Links Mammary Gland Development to the Regulation of Milk Protein Expression in Mice

Simple Summary Milk synthesis is vital for maintaining the normal growth of newborn animals. Abnormal mammary gland development leads to a decrease in female productivity and the overall productivity of animal husbandry. This study characterized the dynamic miRNA expression profile during the process of mammary gland development, and identified a novel miRNA regulating expression of beta-casein-an important milk protein. The results are valuable for studying mammary gland development, increasing milk production, improving the survival rate of pups, and promoting the development of animal husbandry. Mammary gland morphology varies considerably between pregnancy and lactation status, e.g., virgin to pregnant and lactation to weaning. Throughout these critical developmental phases, the mammary glands undergo remodeling to accommodate changes in milk production capacity, which is positively correlated with milk protein expression. The purpose of this study was to investigate the microRNA (miRNA) expression profiles in female ICR mice's mammary glands at the virgin stage (V), day 16 of pregnancy (P16d), day 12 of lactation (L12d), day 1 of forced weaning (FW1d), and day 3 of forced weaning (FW3d), and to identify the miRNAs regulating milk protein gene expression. During the five stages of testing, 852 known miRNAs and 179 novel miRNAs were identified in the mammary glands. Based on their expression patterns, the identified miRNAs were grouped into 12 clusters. The expression pattern of cluster 1 miRNAs was opposite to that of milk protein genes in mammary glands in all five different stages. Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) analyses revealed that the predicted target genes of cluster 1 miRNAs were related to murine mammary gland development and lactation. Furthermore, fluorescence in situ hybridization (FISH) analysis revealed that the novel-mmu-miR424-5p, which belongs to the cluster 1 miRNAs, was expressed in murine mammary epithelial cells. The dual-luciferase reporter assay revealed that an important milk protein gene-beta-casein (CSN2)-was regarded as one of the likely targets for the novel-mmu-miR424-5p. This study analyzed the expression patterns of miRNAs in murine mammary glands throughout five critical developmental stages, and discovered a novel miRNA involved in regulating the expression of CSN2. These findings contribute to an enhanced understanding of the developmental biology of mammary glands, providing guidelines for increasing lactation efficiency and milk quality.