miR-454 suppresses the proliferation and invasion of ovarian cancer by targeting E2F6

OVCAR3 and SKOV3 cells were obtained from Cell Bank of Chinese Academy of Sciences (Shanghai, China) and maintained in RPMI-1640 medium (HyClone, USA) supplemented with 10% FBS at 37 °C with 5% CO₂. Cells were transfected with pCMV-MIR-miR-454 (5 μg; Ribobio, Guangzhou, China) using Lipofectamine 2000 (Invitrogen, CA, USA) according to the instructions, pCMV-MIR vector (5 μg; Ribobio) was used as negative control (NC). The E2F6 cDNA sequences were cloned into pcDNA3.1 vector and the pcDNA3.1-E2F6 (5 μg; Ribobio) was transfected into cells using Lipofectamine 2000.

Seventy-five cases of ovarian cancer tissues and 15 cases of tumor-adjacent tissues were obtained from Beijing Anzhen Hospital, Capital Medical University. All patients gave informed consent in written, and this study was approved by the ethics committee of Beijing Anzhen Hospital, Capital Medical University. Venous blood was collected from 13 patients with ovarian cancer and 6 normal female subjects from Beijing Anzhen Hospital, Capital Medical University. The patient samples and normal subjects age was matched. All blood samples were taken before patient treatment.

Total RNA extracted from OVCAR3 and SKOV3 cells or serum samples using TRIzol Reagent. The SYBR PrimeScript miRNA RT PCR kit (Takara, Shiga, Japan) was performed for RT-PCR reaction to examine the expression of miR-454. For detection of E2F6 mRNA expression, a HiFiScript cDNA Synthesis Kit (CWBIO, Beijing, China) was used to reverse transcribe RNA to cDNA, and a SYBR Premix Ex Taq II kit (Takara) was performed for RT-PCR reaction. Reaction conditions were as follows: 95 °C for 10 min, followed by 40 cycles of 95 °C for 5 s and 60 °C for 30 s. U6 or GAPDH was used as reference genes. Primers of miR-454 and E2F6 were obtained from Ribobio. The relative expression of miR-454 or E2F6 mRNA was calculated using the ^2−ΔΔCT method and normalized to NC group.

Cells were seeded in a 96-well plate at a density of 1000 cells/well and cultured at 37 °C with 5% CO₂ for 0, 24, 48, and 72 h, respectively. Then, 10 μL of CCK8 reagent (Beijing Solarbio Science & Technology, Beijing, China) was added into each well. Following incubation at 37 °C for 1.5 h, the absorbance was detected at 450 nm.

Cells were cultured in 60-mm dishes (500 cells/dish) and incubated at 37 °C with 5% CO₂ for 1–2 weeks. After that, the colonies were fixed with 4% paraformaldehyde for 30 min at room temperature and dyed with 0.1% crystal violet for another 30 min at room temperature. Finally, the number of cell colonies was counted.

Transwell chambers (Millipore, MA, USA) were used to measure cell migration and invasion. About 1 × 10⁵ cells were seeded in the upper chamber and the lower chamber was filled with RPMI-1640 medium containing 20% FBS. Following incubation at 37 °C of 12 h, the migrated or invaded cells were fixed with 4% paraformaldehyde for 30 min at room temperature, and stained with 0.1% crystal violet for 20 min at room temperature. In invasion experiment, the upper chamber was coated with Matrigel (BD Bioscience, CA, USA) before seeding cells.

Cells transfected with plasmids of 24 h were cultured in serum-free medium at 37 °C for 24 h, trypsinized by trypsin (0.25%) without EDTA and centrifuged at 1000 rpm for 5 min. After washing with PBS twice, cells were re-suspended in loading buffer to prepare 1 × 10⁶ cells/mL suspension. 100 μL cell suspension were incubated with 5 μL of Annexin V/FITC (BioVision, USA) for 5 min in the dark at room temperature, then stained with 10 μL propidium iodide (PI). The apoptotic rate was examined by a flow cytometer (BD FACSC anto II, BD Biosciences, USA) and analyzed using FlowJo v7.6.5 software (Tree Star, Inc., Ashland, OR, USA).

Total proteins were extracted from transfected cells by RIPA lysis, and quantitated by a BCA kit (CWBIO). Proteins (20 μg) were subjected to 10% SDS-PAGE and transferred onto the PVDF membranes (Bio-Rad, Hercules, CA, USA). After blocked with 5% non-fat milk solution for 1 h, the membranes were probed with primary antibodies overnight at 4 °C. After washing with TBST, the blots were incubated with horseradish peroxidase (HRP)-conjugated secondary antibodies (1:5000 dilution, SA00001-1, SA00001-2, Proteintech Group, USA) for 1 h at room temperature, and developed using the Enhanced Chemiluminescence kit (CWBIO). The band intensity was quantified using Image J software (NIH, Bethesda, USA). The primary antibodies used in this study were anti-Bcl-2 (1:1000 dilution, 12789-1-AP, Proteintech Group), anti-Bax (1:1000 dilution, 50599-2-Ig, Proteintech Group), anti-cleaved Caspase 9 (1:1000 dilution, ab2324, Abcam, USA), anti-cleaved Caspase 3 (1:1000 dilution, ab2302, Abcam), anti-Akt (1:1000 dilution, 10176-2-AP, Proteintech Group), anti-Akt-Phospho-S473 (1:1000 dilution, 66444-1-Ig, Proteintech Group), anti-mTOR (1:1000 dilution, 20657-1-AP, Proteintech Group), anti-mTOR (phosphor S2448) (1:1000 dilution, ab109268, Abcam), anti-Cyclin D1 (1:1000 dilution, 26939-1-AP, Proteintech Group), anti-p70s6k (1:1000 dilution, 14485-1-AP, Proteintech Group), anti-Phospho-p70s6k (1:1000 dilution, 9204, Cell Signaling Technology, USA), anti-Wnt3 (1:1000 dilution, 67452-1-Ig, Proteintech Group), anti-β-catenin (1:1000 dilution, 17565-1-AP, Proteintech Group), anti-E-cadherin (1:1000 dilution, 20874-1-AP, Proteintech Group), and anti-GAPDH (1:1000 dilution, 10494-1-AP, Proteintech Group).

TargetScan (http://​www.​targetscan.​org/​vert_​72/​) [22] was used to explore the bindingship between miR-454 and E2F6. The wild-type of E2F6 3′-UTR (E2F6-wt) containing the predicated miR-454-bingding site or mutant type of E2F6 3′-UTR (E2F6-mut) was cloned into pmir-GLO reporter vector (Promega, WI, USA). 293T cells were co-transfected with luciferase reporter plasmids (wt or mut; 1 ng/μL) and 5 μg either pCMV-MIR-miR-454 or pCMV-MIR using Lipofectamine 2000. Following transfection of 48 h, cells were collected and lysed, the luciferase activity of each group was measured by the dual-luciferase assay system kit (Promega).

IHC was performed to evaluate the expression of E2F6 protein (1:50; Abcam) as described previously [23]. E2F6 expression levels were scored by staining intensity and percentage of positive stained cells [24]. The staining intensity was graded as follows: 0, no staining; 1, pale yellow staining; 2, buffy staining; 3, intense brown staining. The percentage of positive stained cells was scored as follows: 0 = 0–10%, 1 = 11–25%, 2 = 26–50%, 3 = 51–75%, 4 => 75%. Samples were scored by multiplying synchronically the two sections, and score > 6 as E2F6 high expression level. Ovary tissue was used as positive control, and PBS was used as the negative control instead of primary antibody.

Data was presented as Mean ± SD and statistical analyses were conducted using GraphPad Prism 7.0 software (GraphPad, CA, USA). The Chi-square test was performed for continuous or discrete data analysis, and the Student’s t test or one-way ANOVA analysis was used for comparisons between groups. Differences were considered significant when P value was less than 0.05.

To investigate whether miR-454 is correlated to the progression of ovarian cancer, we first examined the expression of miR-454 in ovarian cancer. We found that miR-454 expression was obviously down-regulated by hypoxia (after 6 or 24 h in 1% O₂) in OVCAR3 and SKOV3 cells compared with corresponding normoxic condition (Fig. 1a). These data indicated the specificity of miR-454 in hypoxic induction in ovarian cancer. Next, we examined the expression of miR-454 in the serum of ovarian cancer patients (n = 13) and normal human serum (n = 6). The expression of miR-454 was found to be significantly elevated in the serum of ovarian cancer patients compared to normal samples (Fig. 1b). Additionally, our data demonstrated that the expression of miR-454 was correlated with the clinicopathological stages of ovarian cancer (P = 0.016), the expression of miR-454 in the serum of patients in stage I–II was significantly higher than that in stage III–IV (Table 1). But the expression of miR-454 was not found to be correlated with age and lymph node metastasis of ovarian cancer patients (Table 1). Therefore, miR-454 may be involved in the progression of ovarian cancer.

To elucidate the functional role of miR-454 in ovarian cancer, pCMV-MIR-miR-454 was transfected into ovarian cancer cell lines OVCAR3 and SKOV3, pCMV-MIR plasmid was used as negative control (NC). RT-PCR analysis showed that miR-454 expression was markedly up-regulated in OVCAR3 and SKOV3 cells transfected with pCMV-MIR-miR-454 (Fig. 1c). As shown in Fig. 1d, e, miR-454 overexpression significantly reduced the proliferation of OVCAR3 and SKOV3 cells compared with corresponding control group. Consistently, OVCAR3 cells transfected with pCMV-MIR-miR-454 formed fewer colonies than control group (Fig. 1f). Similar results were also observed in SKOV3 cells, up-regulation of miR-454 inhibited colony formation ability of SKOV3 cells (Fig. 1f). Moreover, transwell assay showed that up-regulation of miR-454 dramatically inhibited the migration and invasion abilities of both OVCAR3 and SKOV3 cells with a contrast to that of the NC group (Fig. 1g, h). These results suggest that miR-454 suppresses the growth and metastasis of ovarian cancer cells, exerting a tumor suppressor effect.

As indicated by flow cytometry assay, miR-454 overexpression significantly enhanced the apoptosis rate of both OVCAR3 and SKOV3 cells with a contrast to NC group (Fig. 2a). Then, the expression of apoptosis-related proteins was detected by western blot analysis to investigate the underlying mechanism of induced apoptosis by miR-454. As shown in Fig. 2b–d, the expression of anti-apoptotic protein Bcl-2 was significantly down-regulated by miR-454 in both OVCAR3 and SKOV3 cells, while the expression levels of pro-oncogenic proteins Bax, cleaved Caspase 9 and cleaved Caspase 3 were up-regulated by miR-454. The above data indicate that miR-454 may induce apoptosis in ovarian cancer cells by regulating the Bcl-2/Bax axis and Caspase cascade.

It is well known that the Akt/mTOR and Wnt/β-catenin signaling pathways play critical roles in tumorigenesis and development. To further investigate the role of miR-454 in ovarian cancer, we examined the effect of miR-454 on activation of the Akt/mTOR and Wnt/β-catenin signaling pathways in ovarian cancer cells. As indicated by western blot analysis, the phosphorylation levels of Akt (p-Akt) and mTOR (p-mTOR) were reduced in miR-454 group compared with NC group (Fig. 2e–g). Additionally, the expression of downstream proteins Cyclin D1 and p70s6k was inhibited correspondingly in miR-454 group, as well as the level of phosphorylation form of p70s6k (p-p70s6k) (Fig. 2e–g). Similarly, the expression levels of Wnt3 and β-catenin were also down-regulated by miR-454 overexpression in OVCAR3 and SKOV3 cells, the expression of downstream protein E-cadherin was up-regulated correspondingly (Fig. 2h–j).

In order to elucidate the molecular mechanism of miR-454, we identified the potential target of miR-454. Bioinformatics analysis (TargetScan) suggested E2F6 mRNA had a possible binding site for miR-454 in its 3′-UTR (Fig. 3a). To confirm that, the wild-type (wt) or mutant-type (mut) E2F6 3′-UTR was cloned into a luciferase reporter vector, which was co-transfected with pCMV-MIR-miR-454 into 293T cells. As shown in Fig. 3b, the relative luciferase activity of E2F6-wt was markedly decreased in the presence of miR-454, while the luciferase activity of E2F6-mut was not impaired by miR-454. Moreover, the expression of E2F6 protein was significantly inhibited in OVCAR3 and SKOV3 cells transfected with pCMV-MIR-miR-454 (Fig. 3c). Therefore, E2F6 was a direct target of miR-454 in ovarian cancer cells.

The IHC analysis was performed to determine the expression of E2F6 protein in 75 cases of ovarian cancer tissues and 15 cases of tumor-adjacent tissues. As shown in Fig. 4a–d, the positive signal of E2F6 was found to mainly located in nucleoplasm. The rate of E2F6-high expression in ovarian cancer tissues was 57.3% (43/75), which was 20% (3/15) in tumor-adjacent tissues (P < 0.05). Additionally, we observed that E2F6 protein was highly expressed in ovarian cancer tissues compared with tumor-adjacent tissues (Fig. 4a–d).

To investigate the role of E2F6 in miR-454-mediated progression of ovarian cancer cells, pcDNA3.1-E2F6 plasmid was constructed and transfected separately or co-transfected with pCMV-MIR-miR-454 into OVCAR3 and SKOV3 cells (Fig. 5a). As shown in Fig. 5b, c, E2F6 overexpression significantly enhanced the proliferation ability of OVCAR3 and SKOV3 cells compared with NC group, while miR-454 overexpression attenuated the promoting effect of E2F6 on cell proliferation. Moreover, the migration of OVCAR3 and SKOV3 cells was also promoted by E2F6 overexpression, which was also abolished by miR-454 overexpression (Fig. 5d). Collectively, E2F6 acts as a functional target of miR-454 in ovarian cancer.