Bta-miR-106b Regulates Bovine Mammary Epithelial Cell Proliferation, Cell Cycle, and Milk Protein Synthesis by Targeting the CDKN1A Gene

Abstract

Our previous studies found that bta-miR-106b and its corresponding target gene, CDKN1A, were differentially expressed between the mammary epithelium of lactating Holstein cows with extremely high and low milk protein and fat percentage, implying the potential role of bta-miR-106b in milk composition synthesis. In this study, with luciferase assay experiment, bta-miR-106b was validated to target the 3'-untranslated region (UTR) of bovine CDKN1A, thereby regulating its expression. Moreover, in bovine mammary epithelial cells (BMECs), over-expression of bta-miR-106b significantly down-regulated the CDKN1A expression at both mRNA and protein levels, and inhibitors of bta-miR-106b increased CDKN1A expression. Of note, we observed that bta-miR-106b accelerated cell proliferation and cell cycle, and changed the expressions of protein synthesis related pathways such as JAK-STAT and PI3K/AKT/mTOR through regulating CDKN1A expression. Our findings highlight the important regulatory role of bta-miR-106b in milk protein synthesis by targeting CDKN1A in dairy cattle.

Keywords: BMECs; CDKN1A; bta-miR-106b; cell proliferation; milk protein synthesis.

Figure 1

Bta-miR-106b regulated transcript activity by targeting CDKN1A 3′UTR with luciferase assays. (A) It was predicted Bta-miR-106b targeted the 3′UTR of bovine CDKN1A with TargetScan. (B) Luciferase activity was down-regulated after co-transfecting 3′UTR of CDKN1A with Bta-miR-106b mimics. (Note: * means p value < 0.05.)

Figure 2

BMECs isolation. (A) Separation and purification of the BMECs. (B) CK-18 immunofluorescence and DAPI dying results of the BMECs (200×).

Figure 3

Bta-miR-106b regulated cell proliferation by targeting CDKN1A in BMECs. (A,B) The expression levels of bta-miR-106b at 48 h after transfection with bta-miR-106b mimics and inhibitors. (C,D) The mRNA levels of CDKN1A after transfection with bta-miR-106b mimics and inhibitors with qRT-PCR. (E,F) The protein expression of CDKN1A after transfection with bta-miR-106b mimics and inhibitors with Western blot. (G,H) The cell proliferation of BMECs at 0, 24, 48, and 72 h after transfection of bta-miR-106b mimics and inhibitors. (I) The proliferating BMECs were labeled with EdU. The click-it reaction revealed the EdU staining (red). The cell nuclei were stained with DAPI (blue). (Note: * means p value < 0.05; ** means p value < 0.01.).

Figure 4

Bta-miR-106b promoted cell cycle progression from G0/G1 to the S phase in BMECs. (A) and (B) Distribution of cell cycle phases of BMECs with propidium iodide (PI) staining after transfection with bta-miR-106b mimics and bta-miR-106b inhibitors for 48 h. (C) and (D) The mRNA expression of cell cycle regulators specific to the G1/S phase in BMECs with bta-miR-106b mimics and inhibitors with qRT-PCR. (Note: * means p value < 0.05; ** means p value < 0.01.)

Figure 5

Regulation of Bta-miR-106b on the expressions of genes related to protein and fat synthesis in BMECs. (A,B) The mRNA levels of 16 genes involved in fat synthesis at 48 h after transfection with bta-miR-106b mimics and inhibitors in BMECs. (C,D) The mRNA levels of 8 genes involved in protein synthesis at 48 h after transfection with bta-miR-106b mimics and inhibitors in BMECs. (E–G) Western blot results and the protein expression levels of the genes involved in PI3K/AKT/mTOR pathway in BMECs at 48 h after transfection with bta-miR-106b mimics and inhibitors. (Note: * means p value < 0.05; ** means p value < 0.01.)

Figure 6

CDKN1A regulates BMECs cell cycle and milk protein synthesis through PI3K/AKT/mTOR pathways of BMECs. (A) Screening of the best inhibiting fragment and time of three siRNA. (B) Distribution of cell cycle phases of BMECs with PI staining after interference on CDKN1A for 48 h. (C) The mRNA expression of cell cycle regulators specific to G1/S phase in BMECs when CDKN1A was down-regulated with qRT-PCR. (D–F) The mRNA and protein expression levels of eight genes in protein synthesis after interference on CDKN1A in BMECs. (G) Distribution of cell cycle phases of BMECs with PI staining after over-expression of CDKN1A. (H) The mRNA expression of cell cycle regulators specific upon over-expression of CDKN1A. (I,J) The expression of eight genes involved in protein synthesis after up-regulation of CDKN1A. (Note: * means p value < 0.05; ** means p value < 0.01.)