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

Abstract

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 β-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.

Keywords: AKT3; MAPK; circRNA; miRNA; transcriptome.

Figure 1

Transcriptome sequencing. (a) The distribution of reads in the reference genome in different areas. (b) Histogram presentation of the top 30 gene ontology (GO) functional annotations for the differentially expressed genes (DEGs). The x‐axis indicates the number of DEGs, the y‐axis indicates the GO terms. (c) The scatter plot of the Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment of differential genes. The x‐axis indicates the rich factor; the y‐axis indicates the KEGG pathway. The size of the dot indicates the number of differentially expressed genes in the pathway, and the color of the dot corresponds to the different Q value range

Figure 2

miR‐574‐5p regulated the proliferation of goat mammary epithelial cells (GMECs) via mitogen‐activated protein kinase kinase kinase 9 (MAP3K9). The target site for miR‐574‐5p in the MAP3K9 3′‐untranslated region (3′‐UTR) and the construction of the luciferase expression vector (Luc) fused with the MAP3K9 3′‐UTR (a). wt represents the Luc reporter vector with the wt MAP3K9 3′‐UTR 10714–10720); mut represents the Luc reporter vector with the mutation at the miR‐574‐5p site in MAP3K9 3′‐UTR. After 24 hr, the luciferase activities were measured. (b) Messenger RNA and protein expressions of MAP3K9 in GMECs transfected with miR‐574‐5p mimics or inhibitors for 24 and 48 hr. Expression was quantified by quantitative real‐time polymerase chain reaction and western blot, β‐actin was used as an internal control. (c) Cell viability was determined using the CCK‐8 assay for 24, 48, 72, and 96 hr. GMECs were transfected with miR‐574‐5p mimics, inhibitors, siRNA‐MAP3K9 or pcDNA3.1‐MAP3K9 expression vectors or cotransfected MAP3K9 with miR‐574‐5p. (d, e) EdU staining assay. After 24 hr transfection, GMECs in the S phase were stained with EdU in red, while cell nuclei were dyed with DAPI in blue. *p < .05; **p < .01

Figure 3

miR‐574‐5p regulated apoptosis and mitogen‐activated protein kinase pathways via MAP3K9 in goat mammary epithelial cells (GMECs). (a) The apoptosis of GMECs transfected with miR‐574‐5p mimics, inhibitors, si‐MAP3K9 or pcDNA3.1‐MAP3K9 expression vectors or cotransfected MAP3K9 with miR‐574‐5p for 24 hr. The histogram showed the apoptotic cell percentage detected by flow cytometry method, and error bars denote mean ± SD. After 48 hr transfection, (b) western blot analysis was used to detect the expression of Bcl‐2 and Bax in GMECs. (c, d) Cytosolic proteins were analyzed by western blot for extracellular‐signal‐regulated kinase, p38, c‐Jun N‐terminal kinase, MAP2K4, MAP2K7, c‐Jun, RSK1, Bad, and cyclin D1. (e) GMECs were treated with 5 μM anisomycin (AN) and honokiol (HO) in 24 and 48 hr. The expression of Bcl‐2 and Bax was analyzed by western blot. β‐Actin was detected as a loading control. *p < .05; **p < .01

Figure 4

miR‐574‐5p regulated milk synthesis via mitogen‐activated protein kinase kinase kinase 9 (MAP3K9) in goat mammary epithelial cells (GMECs). GMECs were posttransfected with miR‐574‐5p mimics, si‐MAP3K9, pcDNA3.1‐MAP3K9 expression vectors or cotransfected MAP3K9 with miR‐574‐5p for 48 hr. (a) The secretion of β‐casein in the cell‐free supernatants in GMECs was measured by enzyme‐linked immunosorbent assay kit. (b) The secretion of triglycerides in GMECs was measured by the detection kit. (c) Cytosolic proteins and related phosphorylation levels were analyzed by western blot for protein kinase B (AKT), phosphoinositide 3 kinase (PI3K), mammalian target of rapamycin (mTOR), S6K1, 4EBP1, RPS6, and EIF4B. (d) GMECs were treated with 1 and 5 μM anisomycin in 24 and 48 hr. The expression of AKT, PI3K, mTOR, S6K1, 4EBP1, RPS6, and EIF4B was analyzed by western blot. β‐Actin was detected as a loading control. *p < .05; **p < .01

Figure 5

miR‐574‐5p regulated milk synthesis via AKT3 in goat mammary epithelial cells (GMECs). (a) The target site for miR‐574‐5p in the AKT3 3′‐untranslated region (3′‐UTR) and the construction of the luciferase expression vector (Luc) fused with the AKT3 3′‐UTR. After 24 hr, the luciferase activities were measured. (B) Messenger RNA and protein expressions of AKT3 in GMECs transfected with miR‐574‐5p mimics or inhibitors for 24 and 48 hr. Expression was quantified by quantitative real‐time polymerase chain reaction and western blot, β‐actin was used as an internal control. (c) GMECs were posttransfected with miR‐574‐5p mimics, si‐AKT3 or pcDNA3.1‐AKT3 expression vectors or cotransfected AKT3 with miR‐574‐5p for 48 hr. The secretion of β‐casein in the cell‐free supernatants and triglycerides in GMECs was measured by enzyme‐linked immunosorbent assay kit and detection kit. (d) Cytosolic proteins and related phosphorylation levels were analyzed by western blot for mammalian target of rapamycin, S6K1, 4EBP1, RPS6, and EIF4B. β‐Actin was detected as a loading control. *p < .05; **p < .01

Figure 6

circ‐016910 acted as a sponge for miR‐574‐5p. (a) We performed a luciferase vector assay to confirm the direct binding between circ‐016910 and miR‐574‐5p in terms of their complementary sequences. (b) After cotransfecting wt‐ or mut‐ plasmids with miRNA mimics in goat mammary epithelial cells (GMECs) for 24 hr, luciferase activities were measured. After transfected si‐circ‐016910, miR‐574‐5p mimics, pc‐circ‐016910 or cotransfected circ‐016910 with miR‐574‐5p, (c) messenger RNA expressions, and (d) cytosolic proteins expressions of miR‐574‐5p, MAP3K9, and AKT3 in GMECs were quantified by quantitative real‐time polymerase chain reaction for 24 hr and western blot for 48 hr, respectively. U6 and β‐actin were used as an internal control. *p < .05; **p < .01

Figure 7

The regulation of circ‐016910 via miR‐574‐5p in goat mammary epithelial cells (GMECs). (a) Cell viability was determined using the cell counting kit‐8 assay after GMECs were posttransfected for 24, 48, 72, and 96 hr. After 24 hr transfection, GMECs in the S phase were stained with EdU in red, while cell nuclei were dyed with DAPI in blue. (b) The apoptosis of GMECs was measured 24 hr posttransfection. The histogram showed the apoptotic cell percentage detected by flow cytometry method, and error bars denote mean ± standard deviation. (c) Western blot analysis was used to detect the expression of Bcl‐2 and Bax in GMECs for 48 hr. (d) The secretion of β‐casein in the cell‐free supernatants and triglycerides in GMECs was measured by enzyme‐linked immunosorbent assay kit and detection kit. (e) After 48 hr treatment, cytosolic proteins and related phosphorylation levels were analyzed by western blot for c‐Jun N‐terminal kinase, MAP2K4/7, c‐Jun, cyclin D1, and extracellular‐signal‐regulated kinase, RSK1, Bad, and (f) protein kinase B, mammalian target of rapamycin, S6K1, RPS6, and EIF4B. (g) Histograms represented the analysis of the data. β‐Actin was detected as a loading control. *p < .05; **p < .01

Figure 8

A proposed model linking the miR‐574‐5p locus to the biology of goat mammary epithelial cells (GMECs). miR‐574‐5p promotes cell apoptosis, reduces cell proliferation and milk synthesis in GMECs. miR‐574‐5p targets mitogen‐activated protein kinase kinase kinase 9 (MAP3K9) and AKT3 directly and MAP3K9 stimulates different signal pathways. MAP3K9 actives JNK‐c‐Jun‐cyclin D1 pathway resulting in enhanced cell proliferation, JNK/ERK‐Bcl‐2/Bax pathway and ERK‐RSK1‐Bad pathway accompanied by weakening cell apoptosis. phosphoinositide 3 kinase/protein kinase B–mammalian target of rapamycin pathway is also stimulated by MAP3K9, further accelerates milk synthesis. Meanwhile, circ‐016910 acts as a sponge for miR‐574‐5p and shows a circRNA–miRNA–mRNA network to undertake biological effects