Chi-miR-3031 regulates beta-casein via the PI3K/AKT-mTOR signaling pathway in goat mammary epithelial cells (GMECs)

Abstract

Background: MicroRNAs can regulate gene expression at the posttranscriptional level through translational repression or target degradation. Our previous investigations examined the differential expression levels of chi-miR-3031 in caprine mammary gland tissues in colostrum and common milk stages.

Results: The present study detected the role of chi-miR-3031 in the lactation mechanisms of GMECs. High-throughput sequencing was used to analyze transcriptomic landscapes of GMECs transfected with chi-miR-3031 mimics (MC) and a mimic negative control (NC). In the MC and NC groups, we acquired 39,793,503 and 36,531,517 uniquely mapped reads, respectively, accounting for 85.85 and 81.66% of total reads. In the MC group, 180 differentially expressed unigenes were downregulated, whereas 157 unigenes were upregulated. KEGG pathway analyses showed that the prolactin, TNF and ErbB signaling pathways, including TGFα, PIK3R3, IGF2, ELF5, IGFBP5 and LHβ genes, played important roles in mammary development and milk secretion. Results from transcriptome sequencing, real-time PCR and western blotting showed that chi-miR-3031 suppressed the expression of IGFBP5 mRNA and protein. The expression levels of β-casein significantly increased in the MC and siRNA-IGFBP5 groups. We observed that the down-regulation of IGFBP5 activated mTOR at the Ser2448 site in GMECs transfected with MC and siRNA-IGFBP5. Previous findings and our results showed that chi-miR-3031 activated the PI3K-AKT-mTOR pathway and increased β-casein expression by down-regulating IGFBP5.

Conclusions: These findings will afford valuable information for improving milk quality and contribute the development of potential methods for amending lactation performance.

Keywords: Dairy goats; Gene expression; Milk components; Transcriptome; miRNAs.

Fig. 1

Expression levels of chi-miR-3031 in GMECs transfected with MC and NC. **P < 0.01. NC: Negative control

Fig. 2

Increasing chi-miR-3031 and siRNA-IGFBP5 levels notably decreased IGFBP5 mRNA expression. a: Effect of chi-miR-3031 on IGFBP5 mRNA. b: Effect of siRNA-IGFBP5 on IGFBP5 mRNA. P < 0.01 is shown as **. MC: Chi-miR-3031 mimic. NC: Negative control

Fig. 3

Effects of chi-miR-3031 and siRNA-IGFBP5 on IGFBP5 protein levels. a: Western blot analysis results. b: Densitometric quantification of western blot results. Protein levels were normalized to β-actin. P < 0.05 is shown as *, and P < 0.01 is shown as **. MC: Chi-miR-3031 mimic. NC: Negative control

Fig. 4

Protein expression levels of κ-casein (a) and β-casein (b) in GMECs transfected with MC, NC and siRNA-IGFBP5. P < 0.05 is shown as *, and P < 0.01 is shown as **. MC: Chi-miR-3031 mimic. NC: Negative control

Fig. 5

Effects of chi-miR-3031 and siRNA-IGFBP5 on p-mTOR protein levels. a: Western blot analysis results. b: Densitometric quantification of western blot results. P < 0.05 is shown as *, and P < 0.01 is shown as **. MC: Chi-miR-3031 mimic. NC: Negative control