MiR-139 suppresses β-casein synthesis and proliferation in bovine mammary epithelial cells by targeting the GHR and IGF1R signaling pathways

Abstract

Background: MicroRNAs have important roles in many biological processes. However, the role of miR-139 in healthy mammary gland remains unclear. The objective of this study was to investigate the effects of miR-139 on lactation in dairy cows.

Results: Here, we found that miR-139 was down-regulated in mid-lactation dairy cow mammary tissues compared with mid-pregnancy tissues. Then, we prioritized two of potential target genes of miR-139 in cow, growth hormone receptor (GHR) and type I insulin-like growth factor receptor (IGF1R) for further functional studies based on their roles in lactation processes. Dual luciferase reporter assays validated direct binding of miR-139 to the 3'- untranslated region (UTR) of GHR and IGF1R. Moreover, over-expression or silencing of miR-139 affected mRNA levels of GHR and IGF1R in cultured bovine mammary epithelial cells (BMECs). Furthermore, over-expression of miR-139 decreased protein levels of β-casein, proliferation in mammary epithelial cell, and the protein levels of IGF1R and key members of the GHR or IGF1R pathways as well, whereas silencing miR-139 produced the opposite result. Among these signal molecules, signal transducer and activator of transcription-5 (STAT5), protein kinase B (also known as AKT1), mammalian target of rapamycin (mTOR), and p70-S6 Kinase (p70S6K) are involed in β-casein synthesis, and Cyclin D1 is involved in cell proliferation. In addition, silencing GHR decreased protein levels of β-casein, IGF1R, and key members of the IGF1R pathway, whereas co-silencing miR-139 and GHR rescued the expression of GHR and reversed GHR silencing effects.

Conclusions: Our results demonstrate that GHR and IGF1R are target genes of miR-139 in dairy cow. MiR-139 suppresses β-casein synthesis and proliferation in BMECs by targeting the GHR and IGF1R signaling pathways.

Keywords: Growth hormone receptor; Mammary gland; Type I insulin-like growth factor receptor; miR-139; β-casein.

Fig. 1

Expression of miR-139 in Holstein cow mammary gland. Mammary tissues were from mid-pregnancy (P) (n = 3) or mid-lactation (L) (n = 3) cows. MiR-139 expression was normalized to U6. Results are shown as means ± SEM from three independent experiments. Statistical analysis was conducted using the t test.“*”, statistical significance was considered at P<0.05

Fig. 2

Identifying target genes of miR-139 via the dual luciferase reporter assay. a Bta-miR-139 binding sites in the 3′-UTR of GHR. Ten nucleotides (underlined) were mutated in luciferase reporter plasmids carrying GHR 3′-UTR. b Bta-miR-139 binding sites in the 3′-UTR of IGF1R. Eight nucleotides (underlined) were mutated in the luciferase reporter plasmids carrying IGF1R 3′-UTR. c Luciferase activity of reporter plasmids carrying wild-type or mutant GHR 3′-UTR in HeLa cells, in response to co-transfection with miR-139. d Luciferase activity of reporter plasmids carrying wild-type or mutant IGF1R 3′-UTR in HeLa cells, in response to co-transfection with miR-139. Results are shown as means ± SEM from three independent experiments. Statistical analysis was conducted using one-way ANOVA.“*”, statistical significance was considered at P < 0.05

Fig. 3

GHR and IGF1R are target genes of miR-139. BMECs were transfected with negative control of miR-139 mimic (Mimic NC), miR-139 mimic, negative control of miR-139 inhibitor (Inhibitor NC) or miR-139 inhibitor using Lipofectamine 2000. The Control group was only transfected with Lipofectamine 2000. MRNA levels of miR-139 (a, b), GHR (c, d) and IGF1R (e, f) were examined by qPCR at 24 h post-transfection. MiR-139 expression was normalized to U6. GHR and IGF1R expression was normalized to β-actin. Results are shown as means ± SEM from three independent experiments. Statistical analysis was conducted using one-way ANOVA.“*”, statistical significance was considered at P<0.05

Fig. 4

MiR-139 inhibits protein levels of β-casein in BMECs. a BMECs were transfected with Lipofectamine 2000 (Control), negative control of miR-139 mimic (Mimic NC) or miR-139 mimic. Western blotting analysis showed the protein levels of β-casein and β-actin at 48 h post-transfection. b BMECs were transfected with Lipofectamine 2000 (Control), negative control of miR-139 inhibitor (Inhibitor NC) or miR-139 inhibitor. Western blotting analysis showed the protein levels of β-casein and β-actin at 48 h post-transfection. c Relative protein levels were quantified by analyzing scanned blots in panel a using Bandscan5.0 software. β-actin was used as a loading control. d Relative protein levels were quantified by analyzing scanned blots in panel b using Bandscan5.0 software. β-actin was used as a loading control. For c and d, results are shown as means ± SEM from three independent experiments. Statistical analysis was conducted using one-way ANOVA. “*”, statistical significance was considered at P<0.05

Fig. 5

MiR-139 inhibits proliferation of BMECs. a BMECs were transfected with Lipofectamine 2000 (Control), negative control of miR-139 mimic (Mimic NC) or miR-139 mimic. Cell proliferation was measured by the MTT assay at 24, 48, 72, or 96 h post-transfection. b BMECs were transfected with Lipofectamine 2000 (Control), negative control of miR-139 inhibitor (Inhibitor NC) or miR-139 inhibitor. Cell proliferation was measured by the MTT assay at 24, 48, 72, or 96 h post-transfection. Results are shown as means ± SEM from three independent experiments. Statistical analysis was conducted using one-way ANOVA.“*”, statistical significance was considered at P<0.05

Fig. 6

MiR-139 reduces protein levels of major downstream members of GHR and IGF1R signaling in BMECs. a BMECs were transfected with Lipofectamine 2000 (Control), negative control of miR-139 mimic (Mimic NC) or miR-139 mimic. Western blotting analysis showed the protein levels of p-STAT5, STAT5, IGF1R, p-AKT1, AKT1, Cyclin D1, p-mTOR, mTOR, p-p70S6K, p70S6K and β-actin at 48 h post-transfection. b BMECs were transfected with Lipofectamine 2000 (Control), negative control of miR-139 inhibitor (Inhibitor NC) or miR-139 inhibitor. Western blotting analysis showed the protein levels of p-STAT5, STAT5, IGF1R, p-AKT1, AKT1, Cyclin D1, p-mTOR, mTOR, p-p70S6K, p70S6K and β-actin at 48 h post-transfection. c Relative protein levels were quantified by analyzing scanned blots in panel a using Bandscan5.0 software. β-actin was used as a loading control. d Relative protein levels were quantified by analyzing scanned blots in panel b using Bandscan5.0 software. β-actin was used as a loading control. For c and d, results are shown as means ± SEM from three independent experiments. Statistical analysis was conducted using one-way ANOVA. “*”, statistical significance was considered at P<0.05

Fig. 7

MiR-139 also regulates synthesis of β-casein and IGF1R signaling by influencing GHR. BMECs were transfected with (without) GHR siRNA and (or) miR-139 inhibitor. a GHR mRNA levels were examined by qPCR at 24 h post-transfection in cultured BMECs. GHR expression was normalized to β-actin. b Western blotting analysis showed the levels of IGF1R, p-AKT1, AKT1, β-casein, and β-actin at 48 h post-transfection in cultured BMECs. c Relative protein levels were quantified by analyzing scanned blots in panel b using Bandscan5.0 software. β-actin was used as a loading control. For a and c, results are shown as means ± SEM from three independent experiments. Statistical analysis was conducted using one-way ANOVA. Bars with different lowercase letters are significantly different (P < 0.05)