MiR-152 Regulates Apoptosis and Triglyceride Production in MECs via Targeting ACAA2 and HSD17B12 Genes

Abstract

Mammary epithelial cells (MECs) affect milk production capacity during lactation and are critical for the maintenance of tissue homeostasis. Our previous studies have revealed that the expression of miR-152 was increased significantly in MECs of cows with high milk production. In the present study, bioinformatics analysis identified ACAA2 and HSD17B12 as the potential targets of miR-152, which were further validated by dual-luciferase repoter assay. In addition, the expressions of miR-152 was shown to be negatively correlated with levels of mRNA and protein of ACAA2, HSD17B12 genes by qPCR and western bot analysis. Furthermore, transfection with miR-152 significantly up-regulated triglyceride production, promoted proliferation and inhibited apoptosis in MECs. Furthermore, overexpression of ACAA2 and HSD17B12 could inhibit triglyceride production, cells proliferation and induce apoptosis; but sh234-ACAA2-181/sh234-HSD17B12-474 could reverse the trend. These findings suggested that miR-152 could significantly influence triglyceride production and suppress apoptosis, possibly via the expression of target genes ACAA2 and HSD17B12.

Figure 1

Target sites of miR-152 and luciferase assay. (A) The pathways of ACAA2 and HSD17B12 were analyzed using the DAVID system. (B) Binding sites of miR-152 on the 3′UTR of ACAA2 and HSD17B12. (C) Luciferase activities were detected in MECs co-transfected with miR-152 and pmiR-RB-REPORT-ACAA2-mut/WT/NC vector or pmiR-RB-REPORT- HSD17B12-mut/WT/NC vector. (**p < 0.01).

Figure 2

MEC transfection efficiency. Green fluorescence could be observed under a fluorescence microscope 24 h after the transfection. The expression rate of green fluorescence in mammary epithelial cells of dairy cow cells was 60%. (A) Cells transfected with miR-152 mimics; (B) Cells transfected with miR-152 inhibitor; (C) Cells transfected with miR-shNC.

Figure 3

The expressions of miR-152b and target genes. (A) qPCR analysis of the expressions of miR-152 in MECs transfected with miR-152 mimics, miR-152 inhibitor and miR-shNC. (B) Western blot analysis of the protein levels of ACAA2 and HSD17B12 genes in MECs transfected with miR-152 mimics, miR-152 inhibitor and miR-shNC. (C) qPCR analysis for the mRNA levels of ACAA2 and HSD17B12 genes in MECs transfected with miR-152 mimics, miR-152 inhibitor and miR-shNC. (D) Relative levels of ACAA2 and HSD17B12 protein in MECs transfected with miR-152 mimics, miR-152 inhibitor and miR-shNC. (**p < 0.01, *p < 0.05).

Figure 4

miR-152 effect on cell apoptosis, cell proliferation and triglycerides production in MECs. (A) Apoptosis ratio of MECs transfected with miR-152 mimics, miR-152 inhibitor and miR-shNC. (B) MECs proliferation determined by the MTT assay in MECs transfected with miR-152 mimics, miR-152 inhibitor and miR-shNC. (C) Triglycerides level in MECs transfected with miR-152 mimics, miR-152 inhibitor and miR-shNC. (*p < 0.05).

Figure 5

ACAA2 gene effect on cell apoptosis, proliferation and triglyceride production. (A) PBI-CMV3-ACAA2 and sh234-ACAA2-181 plasmids were respectively transfected into cells. Then qPCR analysis of the expressions of ACAA2. (B) Proteins of ACAA2 in cells after transfection. (C) MECs proliferation were analyzed after PBI-CMV3-ACAA2 and sh234-ACAA2-181 plasmids treatment. (D) triglyceride production in MECs. (E) Apoptosis rate was detected in MECs transfected with PBI-CMV3-ACAA2 and sh234-ACAA2-181.

Figure 6

HSD17B12 gene effect on cell apoptosis, proliferation and triglyceride production. (A) HSD17B12 mRNA detected in MECs that transfected PBI-CMV3-HSD17B12 and sh234-HSD17B12-474 plasmids. (B) Proteins of HSD17B12 were analyzed by western blot in MECs. (C) MTT was used to assay the effect of HSD17B12 on MECs proliferation ability. (D) Influence of HSD17B12 on triglyceride was assessed. (E) Apoptosis rate were analyzed after plasmids transfection.