MiR-15a decreases bovine mammary epithelial cell viability and lactation and regulates growth hormone receptor expression

Abstract

MicroRNAs (miRNAs) are a class of small non-coding RNAs that regulate the expression of target genes at the post-transcriptional level by transcript degradation or translational inhibition. The role of bta-miR-15a in bovine mammary gland hasn’t been reported. Using miRNAs prediction software, GHR gene was predicted to be a potential target of bta-miR-15a. In this study, bovine mammary epithelial cell line was used as an in vitro cell model to address the function of bta-miR-15a on bovine mammary epithelial cells. The expression changes of bta-miR-15a and Ghr after bta-miR-15a transfection were detected by qRT-PCR; the expression of GHR protein and casein was detected by western blotting. To determine whether bta-miR-15a can affect cell viability, cells were examined using an electronic Coulter counter (CASY-TT). In conclusion, bta-miR-15a inhibited the expression of casein of bovine mammary epithelial cells, and cell number and viability were reduced by bta-miR-15a expression. Bta-miR-15a inhibited the viability of mammary epithelial cells as well as the expression of GHR mRNA and protein level, therefore suggesting that bta-miR-15a may play an important role in mammary gland physiology.

Figure 1

Bta-miR-15a mimics-FAM transfect into bovine mammary gland epithelial cells. (A) Non-transfected mammary gland epithelial cells, under fluorescent microscope (100×); (B) Mammary gland epithelial cells transfected with bta-miR-15a mimic-FAM, under fluorescent microscope (100×); (C) Mammary gland epithelial cells transfected with bta-miR-15a mimic-FAM, under light microscope (100×).

Figure 2

miR-15a targets 3'UTR of Ghr mRNA.

Figure 3

Expression of bta-miR-15a and Ghr mRNA detected by quantitative real-time PCR in bovine mammary epithelial cells transfected with bta-miR-15a mimics (A) or bta-miR-15a inhibitor (B). (A) An Endogenous miR-15a was raised in the cells with overexpress-miR-15a. ** p < 0.01. Expression of Ghr mRNA in bovine mammary epithelial cells after miR-15a mimics transfect was decreased. ** p < 0.01. (B) Endogenous miR-15a was decressed and expression of Ghr mRNA was raised after miR-15a inhibitor transfect. * p < 0.05. Values are means ± SEM (n = 3 per group). * and ** indicate significant differences from values obtained in the control group at levels of p < 0.05 and p < 0.01, respectively.

Figure 4

Protein grayscale scanning and statistical results of GHR and casein by Western blotting. (A) Immunoblot of GHR and cansein in mammary epithelialcells the 2 days after transfect with miR-15a mimics, negative control, and non-transfected group. The mediated expression of miR-15a mimics down-regulated GHR protein and casein transcribed from the endogenous Ghr transcript, compared to control and non-transfected group. 1–3 indicate bta-miR-15a mimics group, negative control group and non-treated group respectively; (B) Immunoblot of GHR and cansein in mammary epithelialcells the 2 days after transfect with miR-15a inhibitor, negative control, and non-transfected group. The mediated expression of miR-15a inhibitor up-regulated GHR protein and casein transcribed from the endogenous Ghr transcript, compared to control and non-transfected group. 1–3 indicate bta-miR-15a inhibitor group, negative control group and non-treated group respectively; C, D respectively shows expression protein relative levels of GHR and casein by transfect bta-miR-15a mimics and bta-miR-15a inhibitor. Protein expression of GHR and casein normalize by GAPDH. Values are means ± SEM (n = 3 per group). * indicate significant differences from values obtained in the control group at levels of p < 0.05.