Comprehensive MicroRNA Expression Profile of the Mammary Gland in Lactating Dairy Cows With Extremely Different Milk Protein and Fat Percentages

Abstract

A total of 31 differentially expressed genes in the mammary glands were identified in our previous study using RNA sequencing (RNA-Seq), for lactating cows with extremely high and low milk protein and fat percentages. To determine the regulation of milk composition traits, we herein investigated the expression profiles of microRNA (miRNA) using small RNA sequencing based on the same samples as in the previous RNA-Seq experiment. A total of 497 known miRNAs (miRBase, release 22.1) and 49 novel miRNAs among the reads were identified. Among these miRNAs, 71 were found differentially expressed between the high and low groups (p < 0.05, q < 0.05). Furthermore, 21 of the differentially expressed genes reported in our previous RNA-Seq study were predicted as target genes for some of the 71 miRNAs. Gene ontology and KEGG pathway analyses showed that these targets were enriched for functions such as metabolism of protein and fat, and development of mammary gland, which indicating the critical role of these miRNAs in regulating the formation of milk protein and fat. With dual luciferase report assay, we further validated the regulatory role of 7 differentially expressed miRNAs through interaction with the specific sequences in 3'UTR of the targets. In conclusion, the current study investigated the complexity of the mammary gland transcriptome in dairy cattle using small RNA-seq. Comprehensive analysis of differential miRNAs expression and the data from previous study RNA-seq provided the opportunity to identify the key candidate genes for milk composition traits.

Keywords: RNA-seq; dairy cattle; mRNA; mammary gland; miRNA.

FIGURE 1

The pmirGLO vectors with the predicted 3’UTR target sequences of the 4 differentially expressed genes (A) pmirGLO-TRIB3-3′UTR; (B) pmirGLO- M-SAA3.2-3′UTR; (C) pmirGLO-PTHLH-3′UTR; (D) pmirGLO-VEGFA-3′UTR.

FIGURE 2

Domain structures of the 4 differentially expressed genes showing the locations of the seed sequence of the miRNAs within the 3’UTR of theirs.

FIGURE 3

Locations and sequences of the miRNAs target sites in the 3’UTR of the 4 differentially expressed genes. The sequences of the miRNAs are indicated, along with mutations introduced in the target sites (underlined nucleotides) for generating the mutated reporter constructs.

FIGURE 4

mRNA expression levels of the 15 randomly selected miRNAs validated with qRT-PCR. *indicates p < 0.05. Blue columns represent the relative miRNA expression levels by qRT-PCR normalized by U6 in the high group and red columns represent the relative miRNA expression levels by qRT-PCR normalized by U6 in the low group.

FIGURE 5

MicroRNAs represses the expression of TRIB3 via binding the 3’UTR target sequence. Luciferase activity in HEK293 cells co-transfected with miRNA mimic, miRNA inhibitor, miRNA control and empty vector for the TRIB3 3’UTR. Luciferase activity was assayed 24 h after transfection. All luciferase values were normalized to Renilla luciferase. Blue columns represent the luciferase activity co-transfected with miRNA mimic control; Red columns represent the luciferase activity co-transfected with miRNA inhibitor control; Green columns represent the luciferase activity co-transfected with miRNA mimic; Pink columns represent the luciferase activity co-transfected with miRNA inhibitor. (A) Represents the luciferase activity of TRIB3 after over- or down-expressed miR-2904 compared with controls. (B) Represents the luciferase activity of TRIB3 after transfecting mutant vector of miR-2904 compared with control. *Significant difference between the control and the treatment; **Very significant difference between the control and the treatment.

FIGURE 6

MicroRNAs represses the expression of M-SAA3.2 via binding the 3’UTR target sequence. Luciferase activity in HEK293 cells co-transfected with miRNA mimic, miRNA inhibitor, miRNA control and empty vector for the M-SAA3.2 3’UTR. Luciferase activity was assayed 24 h after transfection. All luciferase values were normalized to Renilla luciferase. The meanings of different colors are consistent with Figure 5. (A,C,E) Represents the luciferase activity of M-SAA3.2 after over- or down-expressed miR-146b, miR-339a and miR-339b compared with controls, respectively. (B,D,F) Represents the luciferase activity of M-SAA3.2 after transfecting mutant vector of miR-146b, miR-339a and miR-339b compared with control, respectively. **Very significant difference between the control and the treatment.

FIGURE 7

MicroRNAs represses the expression of PTHLH via binding the 3’UTR target sequence. Luciferase activity in HEK293 cells co-transfected with miRNA mimic, miRNA inhibitor, miRNA control and empty vector for the PTHLH 3’UTR. Luciferase activity was assayed 24 h after transfection. All luciferase values were normalized to Renilla luciferase. The meanings of different colors are consistent with Figure 5. (A,C,E) Represents the luciferase activity of PTHLH after over- or down-expressed miR-29c, miR-106b and miR-190a compared with controls, respectively. (B,D,F) Represents the luciferase activity of PTHLH after transfecting mutant vector of miR-29c, miR-106b and miR-190a compared with control, respectively. **Very significant difference between the control and the treatment.

FIGURE 8

MicroRNAs did not repress the expression of VEGFA via binding the 3’UTR target sequence. Luciferase activity in HEK293 cells co-transfected with miRNA mimic, miRNA inhibitor, miRNA control and empty vector for the VEGFA 3’UTR. Luciferase activity was assayed 24 h after transfection. All luciferase values were normalized to Renilla luciferase. The meanings of different colors are consistent with Figure 5. (A,B,C) Represents the luciferase activity of VEGFA after over- or down-expressed miR-2904, miR-106b and miR-21-3p compared with controls, respectively.