miR-574-5p impaired DGKI directly and DGKH indirectly in GMECs
To authenticate the novel underlying molecular mechanism of miR-574-5p affecting GMECs, we detected miR-574-5p putative targets via Target Scan (http://www. targetscan.org/). Among them, DGKI drew our attention due to following reasons: (1) TargetScan forecasted that there was one miR-574-5p target site at nucleotides 11361 to 11369 of DGKI-3’UTR; (2) DGKI was a differentially expressed unigene in terms of transcriptome sequencing and (3) DGKI was regarded as a momentous regulator for diversiform metabolic processes, which upon activation converted DAG into PA [9, 10] To further validate that DGKI is transcriptionally mediated by miR-574-5p, two-tier luciferase reporters linked the psiCHECK-2 (psC) vector with the wild-type (wt)-DGKI-3’UTR or mutant (mut)-DGKI-3’UTR were used (Figure 1A). Indeed, miR-574-5p mimics or inhibitors attenuated or facilitated relative luciferase activities significantly, nevertheless reporters mutated in DGKI-3’UTR failed to react to miR-574-5p expression (Figure 1B). Then RT-qPCR and western blot were used to screen the mRNA and protein expressions of DGKI. Compliance with above-mentioned observation, DGKI mRNA level was apparently depressed by re-expression of miR-574-5p, whereas it improved when miR-574-5p interference in antisense molecules (Figure 1C). Cells post-transfected with miR-574-5p mimics or inhibitors emerged a remarkable decrease or increase of relative protein expression of DGKI in comparison with contrasts (Figure 1D). All the evidence indicated that miR-574-5p served as a demotivated mediator of DGKI via bonding to the 3’-UTR directly in GMECs.
As RNA-seq results indicated that the expression of DGKH was reduced in miR-574-5p-induced cells, we wanted to affirm their connections. RT-qPCR and western blot results showed that miR-574-5p inhibited the mRNA and protein expressions of DGKH, coinciding with those of transcriptome sequencing (Figure 1C, D). It suggested that miR-574-5p took negative regulatory impacts on DGKH expression.
miR-574-5p regulated milk synthesis via DGKI and DGKH in GMECs
We next screened for DGKI and DGKH that were involved in regulating milk sythesis. Since DGKs could convert DAG to PA, we identified the secretion of diglycerides and phosphatidic acid in the cell-free supernatants using detection kits. As excepted, overexpression of DGKI and DGKH both inhibited diglycerides production and promoted PA production (Figure 2A, B), while depressed DGKI and DGKH exhibited reverse secretion (Figure 2C), notably. We also found DGKI and DGKH attracted the assuasive effects of miR-574-5p on the content of diglycerides and PA (Figure 2A, B). Moreover, DGKI and DGKH showed induced production of triglycerides whether GMECs treated with overexpression plasmids or siRNAs (Figure 2D, E, F). The negative effects of miR-574-5p on triglycerides content were counteracted by DGKI or DGKH (Figure 2D, E). ELISA results revealed that DGKI acted as a promoter of secretion of β-casein and alleviated that negative impact of miR-574-5p in GMECs (Figure 2D, F). However, there was no change in β-casein secretion after GMECs treated with various expressions of DGKH (data not shown). All clues made certain that miR-574-5p regulated the production of diglycerides, PA and triglycerides via DGKI and DGKH and blocked β-casein secretion via DGKI in GMECs.
miR-574-5p depressed the proliferation and improved the apoptosis of GMECs via DGKI
In our previous work, we made certain that miR-574-5p depressed the proliferation and improved the apoptosis of GMECs. Thus we next sought to determine whether miR-574-5p were inclusive of modulating survival capabilities of GMECs via its negativity to DGKI or DGKH. SiRNA-DGKI/DGKH and vectors carrying DGKI/DGKH coding sequences (CDS), were introduced into GMECs for the blockage and motivation of DGKI or DGKH. CCK8 assay after cells cultivated for 24, 48 and 72h exhibited that inducement of DGKI promoted the cell viability (Figure 3A), while interference of DGKI using siRNA restrained the cell viability compared to contrasts (Figure 3C). Figure 3B, C showed that DGKH appeared a negative role on GMECs viability. Subsequently, the EdU staining assay was conducted to investigate the proliferation of GMECs. We observed a prominent increase in cellular proliferation following DGKI overexpression (Figure 3D, G), which was conspicuously impaired by si-DGKI in comparison with negative control (Figure 3F, G). Expectably, DGKI moderated miR-574-5p-weakened effects on GMECs proliferation via decreasing cell viability and EdU positive cells (Figure 3A, D, G), clarifying that miR-574-5p suppressed cell proliferation through DGKI in GMECs. However, DGKH functioned as an oppressive regulator on the proliferation of GMECs when we treated cells with distinct expressions of DGKH (Figure 3E, F, G), which meant miR-574-5p didn’t regulate cellular proliferation by DGKH.
On behalf of affirming the impact of DGKI and DGKH on GMECs apoptosis, the extents of apoptosis were measured utilizing Annexin V-FITC/PI staining and relative expression of decisive apoptotic genes. Flow cytometry showed that 24 h following transfection of GMECs, the apoptotic rates were higher in si-DGKI group and lower after DGKI overexpressed, significantly (Figure 4A, C, S1A, C). Since incremental documents revealed that Bcl2 and Bax were diverse valid apoptotic genes [26, 27], we implemented western blot analysis and DGKI inhibited pro-apoptotic Bax protein expression and improved anti-apoptotic Bcl-2 protein expression, separately (Figure 4D, E). DGKI topically rescued the stimulative impact of miR-574-5p on GMECs apoptosis (Figure 4A, E, S1A). All these indicated that miR-574-5p answered for facilitating cell apoptosis via its mediation of DGKI. Compared with respective control groups, interference of DGKH diminished the cells apoptotic rates (Figure 4C, S1C), and on the contrast, inducing DGKH elevated count of apoptotic cells (Figure 4B, S1B). Loss of DGKH enhanced the protein level of Bcl-2 cooperated with down-regulated Bax expression (Figure 4D), while motivated DGKH exhibited the opposite results (Figure 4F). All the data affirmed that DGKH promoted the apoptosis of GMECs.
DGKI and DGKH effected AKT-mTOR signaling pathways in GMECs
Searching for the mechanism by which DGKI and DGKH affected milk sythesis, we turned to AKT-mTOR signalling, a potent classical pathway that modulated mammary development [28, 29]. DGKI overexpression clearly increased the activation of PI3K, AKT, mTOR, S6K1, RPS6, EIF4B and 4EBP1, meanwhile, neutralized the passive influence of miR-574-5p (Figure 5A). Oppositely, cells treated with miR-574-5p mimics and si-DGKI reduced these phosphorylation levels (Figure 5C), which meant miR-574-5p suppressed the expression of milk protein synthesis-relevant proteins via down-regulating DGKI in GMECs. Afterwards, we inquired whether DGKH could affect AKT-mTOR pathway. Deficiency of DGKH triggered activation of AKT, mTOR, S6K1, RPS6, EIF4B and 4EBP1, which are components of downstream targets of mTOR (Figure 5C). Interestingly, PI3K was inactivated in si-DGKH treated cells (Figure 5C). The diagrams exhibited opposite corresponding protein levels after cells transfected with pcDNA3.1-DGKH vectors (Figure 5B). We hence suggested that DGKH could attenuate AKT-mTOR signalling in GMECs.
DGKI and DGKH effected ERK and NFKB signaling pathways in GMECs
It’s well known that NFKBs, MAPKs, incorporating ERK, p38 MAPK and JNK, are potent signaling molecules partook in cellular processes and modulate cell proliferation and cell apoptosis [30-33]. To elucidate the mechanism by which DGKs effected cellular activities, the crucial members activation in Raf-ERK signaling pathway in DGKs-induced or -silenced GMECs was subsequently measured. The key members’ activation was evaluated by their phosphorylation via western blot. In cells overexpressing DGKI, phosphorylation of Raf-1, MAP2K1&2, ERK, RSK1 and Bad were greater than that seen in control cells (Figure 6A). DGKI also partially abrogated the negative effects of miR-574-5p on aforesaid key proteins motivation (Figure 6A). Consistent with our prior observation that DGKI promoted ERK signaling, we found that DGKI depletion significantly restrained the increase in p-Raf-1, p-MAP2K1&2, p-ERK, p-RSK1 and p-Bad (Figure 6C). To further pursue the impacts of DGKI on GMECs, we assessed the activation in IKK-NFKB signaling. Astonishingly, Figure 6D showed that induced DGKI inhibited the expression of p-IKKα/β, p-IKBα/β and NFKB1. Cells treated with si-DGKI and miR-574-5p uncovered reverse results (Figure 6F), indicating that DGKI and miR-574-5p both arrested IKK-NFKB signaling pathway. Thus we confirmed that miR-574-5p was capable of regulating cell survival capabilities via the DGKI effects on Raf-ERK signaling, but not IKK-NFKB signaling in GMECs. With regard to DGKH functions, we compared ERK signaling in GMECs transfected with si-DGKH or pcDNA3.1-DGKH plasmids with controls. Blockage of DGKH caused up-regulation of phosphorylation of Raf-1, MAP2K1&2, ERK, RSK1 and Bad (Figure 6C), whereas contrary results were exhibited after DGKH enhanced (Figure 6B), supporting a critical role of DGKHin the blockage of the Raf-ERK signaling cascade. We also measured that the expression of p-IKKα/β, p-IKBα/β and NFKB1 was reduced or improved when DGKH overexpressed or knocked-down (Figure 6E, F). All clues affirmed that DGKH acted on a catalyzer in cell apoptosis and promoted cell proliferation via impairing ERK pathway and NFKB pathway.
Crosstalk between mTOR and ERK pathways
Enhancive documents reveal about novel signaling connections between mTOR and ERK pathways [34, 35]. To suppress the ERK pathway, we used either pharmacological UO126, an effective inhibitor of MAP2K1&2 or siRNAs of MAP2K1 and MAP2K2. As excepted, the phosphorylation of ERK was diminished under these conditions (Figure 7A, B). UO126 with different concentrations in 24 h triggered inactivation of mTOR, S6K1 and 4EBP1 in GMECs, intriguingly, we observed a notable increase in phosphorylation of AKT (Figure 7A). Cells transfected with siRNA-MAP2K1&2 also showed the same results compared with NC (Figure 7B). Afterwards, we examined the effects of the mTOR inhibitor, rapamycin, and the mTOR catalyzer, MHY1485 on GMECs in 1, 5 and 10 μM. The results exhibited that the expression of p-S6K1 and p-4EBP1 was down-regulated via rapamycin accompanied by induced phosphorylation of AKT, MAP2K1&2 and ERK (Figure 7C). Significantly, MHY1485 treatment gave rise to positive phosphorylation of S6K1 and 4EBP1, while AKT, MAP2K1&2 and ERK was inactived (Figure 7D). These data supported a role for ERK motivation of mTOR-S6K1/4EBP1 pathway and ERK signaling might lie upstream mTOR signaling. Since S6K1 is one of the downstream targets of mTOR, we introduced siRNA targeted against S6K1 and found higher levels of p-AKT and p-ERK in GMECs (Figure 7F, G), which meant S6K1 arrested both ERK and AKT pathways. Then we suspected whether AKT motivation via ERK inhibition was reliant on mTOR-S6K1. Western blot showed that UO126 didn’t further improved p-AKT from enhanced phosphorylation of AKT levels by rapamycin-blocked mTOR or knockdown of S6K1 (Figure 7E, F). It suggested that mTOR-S6K1 signaling was essential for ERK inhibition-induced AKT motivation in GMECs. At the same time, rapamycin-induced ERK motivation was dependent on S6K1 (Figure 7G), providing evidence that S6K1 was a central medium of ERK inhibition-enhanced AKT activation and mTOR inhibition-enhanced ERK activation.