MicroRNAs synergistically regulate milk fat synthesis in mammary gland epithelial cells of dairy goats

Abstract

Synergistic regulation among microRNAs (miRNAs) is important to understand the mechanisms underlying the complex molecular regulatory networks in goats. Goat milk fat synthesis is driven by a gene network that involves many biological processes in the mammary gland. These biological processes are affected by several miRNAs rather than a single miRNA. Therefore, identifying synergistic miRNAs is necessary to further understand the functions of miRNAs and the metabolism of goat milk fat synthesis. Using qRT-PCR, we assessed the expression of 11 miRNAs that have the potential to regulate milk fat synthesis in the goat mammary gland. Six of these miRNAs exhibited expression during the lactation cycle. Additionally, we also found that prolactin, the key hormone that regulates lactation, promotes the expression of four miRNAs (miR-23a, miR-27b, miR-103, and miR-200a). Further functional analysis showed that overexpression of all four miRNAs by using recombinant adenovirus in goat mammary gland epithelial cells can affect gene mRNA expression associated with milk fat synthesis. Specifically, elevated miR-200a expression suppressed the mRNA expression of genes involved in fat droplet formation. To analyze the synergistic regulation among these four miRNAs (miR-23a, miR-27b, miR-103, and miR-200a), we used the Pearson correlation coefficient to evaluate the correlation between their expression levels in 30 lactating goats. As a result, we found a strong correlation and mutual regulation between three miRNA pairs (miR-23a and miR-27b, miR-103 and miR-200a, miR-27b and miR-200a). This study provides the first experimental evidence that miRNA expression is synergistically regulated in the goat mammary gland and has identified the potential biological role of miRNAs in goat milk fat synthesis. The identification of synergistic miRNAs is a crucial step for further understanding the molecular network of milk fat synthesis at a system-wide level.

Figure 1

miRNA expression correlates with lactation stage and prolactin concentration. (A) MiR-23a, miR-27b, miR-103, and miR-200a in goat mammary gland are differentially regulated during early (30 days after parturition) and midlactation (120 days after parturition). The relative expression levels of miR-23a, miR-27b, miR-103, and miR-200a were calculated by the 2^−ΔΔCT method, normalized with U6, and plotted relative to their respective expression in early lactation. Data are presented as the mean ± SD (n = 10). (B) MiR-23a, miR-27b, miR-103, and miR-200a positively respond to prolactin concentration in goat mammary gland epithelial cells. MiRNA expression was assessed by qRT-PCR, normalized to U6 internal control, and plotted relative to the expression at a concentration of 0 µg/ml. All data are presented as the mean ± SD (n = 9). *p < 0.05, **p < 0.01.

Figure 2

Overexpression of miRNA regulates gene mRNA expression associated with milk fat synthesis in epithelial cells. (A) miRNAs are overexpressed by using their respective Ad-miRNA in mammary gland epithelial cells. miRNA expression was assessed at 0, 24, 48, and 72 h postinfection. The expression levels of miR-23a, miR-27b, miR-103, and miR-200a were measured by qRT-PCR, normalized with internal control U2, and plotted relative to level of Ad-infected cells (control). Data are expressed as the mean ± SD (n = 10). *p < 0.05. (B) Overexpression of miR-23a promotes mRNA expression of FASN, ADRP, CD36, and TIP47. (C) Overexpression of miR-27b increases mRNA levels of SCD, but decreases mRNA levels of FASN, ADRP, and BTN1A1. (D) Overexpression of miR-103 upregulates mRNA levels of ACACA, FASN, ADRP, and LPL. (E) Overexpression of miR-200a promotes mRNA expression of SLC27A6 and CD36 but suppresses mRNA expression of GPR41 and TIP47. mRNA expression (B–E) was assessed 0, 24, 48, and 72 h after goat mammary gland epithelial cells were infected with their respective Ad-miRNA. The mRNA levels were measured by qRT-PCR, normalized with GAPDH, and presented relative to the mRNA amounts of Ad-infected cells (control). All data are expressed as the mean ± SD (n = 18). *p < 0.05.

Figure 3

Correlation between miRNA expression in mammary gland of lactating goats. The expression levels of miR-23a, miR-27b, miR-103, and miR-200a were measured in each of the 30 goats (midlactation). Pearson analysis using the SPSS software was performed to identify the correlation coefficient. Intensity scatter plot shows comparison of two miRNA profiles in mammary gland. The expression of miRNAs was normalized to U6 (n = 30).

Figure 4

The location of pre-miRNAs in genome.

Figure 5

miR-27b and miR-23a regulate the mRNA level of PPARg in goat mammary gland epithelial cells. (A) Overexpression of miR-27b decreases the mRNA level of PPARg. (B) Overexpression of miR-23a increases the mRNA level of PPARg. mRNA levels of PPARγ (A, B) were determined in epithelial cells 0, 24, 48, and 72 h after infecting with Ad-miRNAs or Ad (control). The mRNA levels were measured by qRT-PCR, normalized with GAPDH, and expressed relative to the mRNA amounts of Ad-infected cells (control). All data are presented as the mean ± SD (n = 18). *p < 0.05.