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

Abstract

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-β-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.

Keywords: mammary gland; microRNA; milk protein; mouse.

Figure 1

Overview of the small RNA sequencing data: (A) Distribution of total identified small RNAs’ lengths. (B) Classification of all detected small RNAs. (C) Principal component analysis of the total identified miRNAs. (D) Venn diagram illustrating the overlap of miRNAs across five different stages.

Figure 2

Validation of the expression of miRNAs using qPCR: (A) (a–h). The relative expression levels of eight selected miRNAs measured by qPCR in five stages. The expression levels were normalized against the expression level of the internal control U6 using the comparative cycle threshold (2^−ΔΔCt) method. Meanwhile, for each miRNA, the stage of lowest sequencing abundance in the five different stages was used as a reference sample for comparisons. Data are presented as the mean ± standard error of the mean (SEM) for three replicates. (B) Scatterplot between the qPCR and RNA-Seq results of the eight selected miRNAs. The y-axis corresponds to the log2 of the qPCR ratios, while the x-axis shows the log2 of the RNA-Seq ratios, for the eight selected miRNAs.

Figure 3

Expression dynamics of 12 miRNA clusters during murine mammary gland development. The purple line represents the miRNAs’ expression trend in each cluster.

Figure 4

GO and KEGG enrichment analysis of cluster 1 miRNAs’ target genes: (A) GO term enrichment analysis of cluster 1 miRNAs’ target genes, including biological process (BP), cellular component (CC), and molecular function (MF). (B) KEGG pathway analysis of cluster 1 miRNAs’ target genes. The size of the circle represents the number of genes enriched in the pathway, and the color scale from blue to red (more significant enrichment) represents the enriched significance.

Figure 5

FISH analysis of novel-mmu-miR424-5p at distinct stages of mammary gland development. In the luminal and basal epithelial cell layers, the novel-mmu-miR424-5p was abundantly expressed. The luminal cell is shown by the arrow without a tail, whereas the basal cell is indicated by the arrow with a tail. Scale bars = 20 µm.

Figure 6

Novel-mmu-miR424-5p targets the 3′ UTR of CSN2: (A) A map of pmirGLO vectors carrying the predicted WT or Mut target sequences of novel-mmu-miR424-5p in the 3′ UTR of CSN2. (B) The designed luciferase reporter sequences—WT: the WT CSN2-3′UTR sequence includes a novel-mmu-miR424-5p binding site; Mut: the sequence of CSN2-3′UTR with mutation in the novel-mmu-miR424-5p binding site. (C) Luciferase activity in 293T cells co-transfected with WT or Mut CSN2-3′UTR luciferase reporter and novel-mmu-miR424-5p mimics or negative control mimics (miR-NC). +: the vector or mimics was transfected in cells; −: the vector or mimics was not transfected in cells. The relative amounts of firefly luminescence normalized to Renilla luminescence are plotted. Data are shown as mean ± SEM values (n = 3, * p < 0.05, Student’s t-test).