circ-016910 sponges miR-574-5p to regulate cell physiology and milk synthesis via MAPK and PI3K/AKT-mTOR pathways in GMECs

Incremental proofs demonstrate that miRNAs, the essential regulators of gene expression, are implicated in various biological procedures, including mammary development and milk synthesis. Here, the role of miR-574-5p in milk synthesis, apoptosis, and proliferation of goat mammary epithelial cells (GMECs) are explored without precedent, and the molecular mechanisms for the impacts are elucidated. Small RNA libraries were constructed using GMECs transfected with miR-574-5p mimics and negative control followed by sequencing via Solexa technology. Overall, 332 genes were distinguishingly expressed entre two libraries, with 74 genes upregulated and 258 genes downregulated. This approach revealed mitogen-activated protein kinase kinase kinase 9 (MAP3K9), an upstream activator of MAPK signaling, as a differentially expressed unigene. miR-574-5p targeted seed sequences of the MAP3K9 3 '-untranslated region and suppressed its messenger RNA (mRNA) and protein levels, correspondingly. GMECs with miR-574-5p overexpression and MAP3K9 inhibition showed increased cell apoptosis and decreased cell proliferation resulting from sustained suppression of MAPK pathways, while MAP3K9 elevation manifested the opposite results. miR-574-5p repressed the phosphorylation of members of protein kinase B (AKT)-mammalian target of rapamycin pathway via downregulating MAP3K9 and AKT3, resulting in reducing the secretion of beta-casein and triglycerides in GMECs. Finally, according to the constructed circular RNA (circRNA) libraries and bioinformatics prediction approach, we selected circ-016910 and found it acted as a sponge for miR-574-5p and blocked its relevant behaviors to undertake biological effects in GMECs. The circRNA-miRNA-mRNA network facilitates further probes on the function of miR-574-5p in mammary development and milk synthesis.