MIEF2 over-expression promotes tumor growth and metastasis through reprogramming of glucose metabolism in ovarian cancer

Human ovarian cancer (OC) cell lines (A2780, SKOV3, OVCAR3, HEY and ES2) and an immortalized but non-tumorigenic ovarian epithelial cell line IOSE80 were purchased from the Cell Bank of Chinese Academy of Sciences (Shanghai, China). All cell lines were authenticated by STR profiling test to confirm their identities and cultured in Dulbecco’s Modified Eagle (DMEM) or RPMI-1640 Medium supplied with 10% fetal bovine serum, 100 U/ml penicillin and streptomycin in an incubator with 5% CO₂ at 37 °C. In addition, 152-paired tumor and surrounding non-tumor tissue samples (30 for qRT-PCR analysis; 122 for IHC staining analysis) were collected from ovarian cancer patients at the First Affiliated Hospital of the Fourth Military Medical University in Xi’an, China. The study was approved by the Ethics Committee of Fourth Military Medical University. Informed consent was obtained from all individual participants included in the study.

The transient knockdown of MIEF2 was obtained by transfection of small interference RNA (siRNA) with Lipofectamine 2000 (Invitrogen, California, USA) according to the manufacturer’s protocol. The sequence of si-MIEF2#1 was 5′- ACACCTAAGTTCAGCACTATAGCAC-3′; The sequence of si-MIEF2#2 was 5′- GCCATGCCTTGAAGATGTGAATAAA-3′. The stable knockdown of MIEF2 was obtained by transfection with the shRNA expression vector generated by a pSilencer™ 3.1-H1 puro vector (Ambion, Austin, TX, USA). For MIEF2 over-expression, the open reading frame sequence of MIEF2 was amplified and cloned into a pcDNA™3.1(C) vector (Invitrogen, V790–20). Synthetic miRNA mimics and control oligonucleotide (NC) were purchased from RiboBio Inc. (Guangzhou, China) and transfected into OC cells with Lipofectamine 2000 according to the manufacturer’s instruction.

RNA was extracted from OC cells using Trizol reagent (Invitrogen, USA). Then, reverse transcription of extracted RNA was performed using a PrimeScript® RT reagent kit (Takara, Japan) following to the manufacturer’s instructions. PCR amplification was performed using SYBR Premix Ex Taq (Takara, Japan). Relative expressions of target genes were calculated using the 2^-△△Ct method and β-actin was considered as a reference gene for normalization. The primer sequences were listed in the supplementary Table 1.

Cells were lysed with RIPA buffer containing the protease inhibitor cocktail (Sigma, USA). Proteins (35 μg) were separated in SDS-PAGE gels and transferred onto PVDF membrane (Millipore, USA). The membranes were then blocked with 5% milk and probed with primary and secondary horseradish-peroxidase-labeled antibodies. After washing three times, the signaling was detected by an enhanced chemiluminescence detection system (ECL; Amersham Pharmacia Biotech). Primary antibodies used in the present study were listed in the supplementary Table 2.

Paraffin-embedded tissue sections (4 μM) were rehydrated, blocked with 3% hydrogen peroxide and treated with hot citrate buffer. After that, primary antibodies of MIEF2 and Ki-67 were added and incubated overnight at 4 °C. The results were determined by an IHC detection kit (MXB, Fuzhou, China) according to the manufacturer’s protocol. The staining intensity was scored independently by two observers. Briefly, the scores for the proportion of positive staining (1, < 5%; 2, 5–30%; 3, 30–70%; 4, > 70%) and staining intensity (0, no staining; 1, weak; 2, moderate; 3, strong) were multiplied for each observer and then averaged.

A total of 1 × 10³ OC cells were plated into 96-well cell culture plates (020096, Xinyou Biotech, Hangzhou, China). After grown for 0, 24, 48, 72, 96 and 120 h, 20 μL MTS solution (Promega, G3581) was added to each well and incubated 2 h at 37 °C. Finally, the absorption values at 490 nm were measured with a Bio-Rad’s microplate reader to determine the relative cellular proliferation capacities.

A total of 500 OC cells were seeded into 6-well plates and cultured for 15 days. Formed colonies were fixed with 4% paraformaldehyde and stained with crystal violet for 15 min, respectively.

OC cells were washed with PBS and then analyzed with a cell cycle (F-6012, US Everbright Inc) kit or an Annexin V (FITC-conjugated) apoptosis (F-6012, US Everbright Inc) kit, according to their manufacturer’s protocols. Cell cycle distribution in each phase and percentage of apoptotic cells were determined with a flow cytometry (Beckman, Fullerton, CA).

OC cells were cultured in 6-well plates and grown to 90% confluence. Then, a plastic pipette tip was used for scratching in the bottom of the wells. After washing two times with the culture medium without fetal bovine serum, images were captured with a light Olympus microscope at 0 and 24 h. Image J software was used for the determination of relative migration in each group.

Matrigel-coated Invasion Chamber (BD Biosciences, UJ, USA) was used for assessment of cell invasion. Briefly, 2 × 10⁴ OC cells were seeded in the upper chamber of the transwell insert in serum-free culture medium and cultured for 48 h. Penetrated cells were fixed with 4% paraformaldehyde and stained with 0.5% crystal violet. The number of penetrated cells in each group was counted under a light Olympus microscope.

A total of 1 × 10⁷ OC cells with different MIEF2 level were subcutaneously injected into the flank of 4–5 weeks old female BALB/c athymic nude mice (six mice per group). Tumor volumes were measured once every week for 5 weeks. Then, tumors were removed and their sizes and weights were determined. The animal study was approved by the Institutional Animal Experiment Committee of Xijing hospital and carried out in accordance with the UK Animals (Scientific Procedures) Act, 1986.

A total of 5× 10⁶ OC cells with different MIEF2 level were intravenously injected into the tail vein of 4–5 weeks old female BALB/c athymic nude mice. After 7 weeks, the mice were sacrificed and metastatic tumors formed in their lungs were determined with hematoxylin and eosin (H&E) staining analysis.

OC cells were plated into an XF96 plate at a density of 1.0 × 10⁴ cells/well and cultured overnight. The XF96 Extracellular Flux Analyzer (Seahorse Bioscience) was used for detection of cellular oxygen consumption in OC cells, according to the manufacturer’s protocol.

A commercial kit from abcam (ab110419) was used for detection of the activities of the five mitochondrial respiratory chain complexes, following to the manufacturer’s protocol. The absorption values at 340 nm (complexes I and V), 550 nm (complexes III and IV) and 600 nm (complex II) were measured with a Bio-Rad’s microplate reader.

ATP production was determined with ATP Determination Kit (Thermo Fisher Scientific, A22066) following to the manufacturer’s instruction. Briefly, OC cells with different treatment were homogenized in the lysis buffer. The results were determined by luminescence (Promega, Glomax 20/20 luminometer) and normalized to protein concentration.

Glucose and lactate detection kits purchased from Nanjing jiancheng Bioengineering institute (Nanjing, China) were used for determination of glucose and lactate concentrations before and after 24 h cell culture in OC cells, following to their manufacturer’s protocols. In addition, a pH meter (PB-11 Basic Meter, The Netherlands) was used for the measurement of pH value in cell culture medium.

The expression of MIEF2 was firstly evaluated in tumor and corresponding peritumor tissues from 30 patients with ovarian cancer (OC) using quantitative real-time PCR (qRT-PCR) analysis. Our results showed a significantly up-regulation of MIEF2 in OC tissues when compared with peritumor tissues (Fig. 1a). Consistently, increased MIEF2 expression was also detected in five OC cell lines (A2780, SKOV3, OVCAR3, HEY and ES2) as compared with an immortalized ovarian epithelial cell line IOSE80 (Fig. 1b-c).

To explore the relationship between MIEF2 expression and survival of OC patients, immunohistochemical (IHC) analysis was applied for detection of MIEF2 protein expression level in another 122-paired OC tumor and peritumor tissues. MIEF2 was significantly higher in tumor tissues of OC compared to peritumor tissues (Fig. 1d). Kaplan-Meier survival analysis demonstrated that OC patients with higher MIEF2 expression had obvious poorer overall survival as compared to MIEF2 lower expression patients (Fig. 1e). Consistent with this, bioinformatics analysis using the KM-plotter [26] also indicated that OC patients with high MIEF2 expression had significant shorter overall (P = 0.001) and progression free survival (Fig. 1f). Taken together, MIEF2 expression was up-regulated in OC tissues/cells and associated with poor prognosis for patients with OC.

Increased MIEF2 expression implies that MIEF2 may function as an oncogene in the tumorigenesis of OC. To prove this, MIEF2 expression was knocked-down in OVCAR3 and ES2 cells (Fig. 2a and b) with relatively high MIEF2 expression as shown in Fig. 1b and Fig. 1c. Knockdown of MIEF2 significantly suppressed cell proliferation and colony formation in OVCAR3 and ES2 cells (Fig. 2c and d), as determined by MTS cell viability and colony formation assays. To characterize the mechanism by which MIEF2 knockdown suppressed OC cell growth, the effects of MIEF2 knockdown on cell proliferation and apoptosis were determined by EdU (5-ethynyl-2′-deoxyuridine) incorporation assay, as well as flow cytometry cell cycle distribution and apoptosis assays. As shown in Fig. 2e-g, knockdown of MIEF2 in OVCAR3 and ES2 cells resulted in significant lower percentage of proliferating cells (Fig. 2e) and cell cycle arrest at G1 phase (Fig. 2f), while a significant increase of cell apoptosis (Fig. 2g), suggesting that MIEF2 knockdown suppressed OC cell growth through induction of G1-S cell cycle arrest and cell apoptosis.

The effects of MIEF2 knockdown on cell migration and invasion of OC cells were also explored. Knockdown of MIEF2 significantly suppressed the migration abilities of OVCAR3 and ES2 cells when compared with control cells (Fig. 3a), as evidenced by wound healing assay. In addition, MIEF2 knockdown also inhibited the invasion abilities of OVCAR3 and ES2 cells, as shown by transwell matrigel invasion assay (Fig. 3b). Previous studies have shown that epithelial-mesenchymal-transition (EMT) plays crucial roles during cancer metastasis through decreasing cell-cell contact and increasing cell migration and invasion [27]. To investigate how MIEF2 controls OC migration and invasion, the expressions of principal epithelial and mesenchymal regulators were determined by qRT-PCR and Western blot analyses. MIEF2 knockdown significantly increased the levels of epithelial regulators of E-cadherin and ZO-1, while decreased the levels of mesenchymal regulators of N-cadherin and Vimentin (Fig. 3c and d), indicating that MIEF2 knockdown suppressed the migration and invasion of OC cells through inhibiting EMT.

We then explored the in vivo tumor-promoting effects of MIEF2 in OC. Stably MIEF2 knockdown (shMIEF1) and control (shCtrl) OVCAR3 cells (Fig. S1A and S1B) were injected into the flanks of nude mice to construct xenograft models. MIEF2 knockdown significantly inhibited the growth of tumors (Fig. 4a) and decreased their weights (Fig. 4b). Immunochemistry (IHC) staining showed significantly decreased MIEF2 expression in shMIEF2 tumor tissues compared to shCtrl (Fig. 4c), implying that the tumor growth inhibiting effect was exerted by MIEF2 knockdown. In addition, in line with the in vitro results, significantly fewer proliferating and more apoptotic cells were detected in xenografts from shMIEF2 group compared to those from shCtrl group, as determined by Ki-67 and TUNEL staining assays, respectively (Fig. 4d and e). Moreover, MIEF2 knockdown also significantly suppressed lung metastasis of OVCAR3 cells in nude mice (Fig. 4f).

To provide further support for the promoting effects of MIEF2 on cell growth and metastasis in OC, MIEF2 was over-expressed in SKOV3 and HEY cells with relatively low MIEF2 expression shown in Fig. 1b and Fig. 1c. Over-expression of MIEF2 expression (Fig. 5a and b) markedly increased the proliferation and colony formation capacities of SKOV3 and HEY cells (Fig. 5c and d). In addition, forced expression of MIEF2 also obviously enhanced the migration and invasion abilities of SKOV3 and HEY cells (Fig. 5e and f).

MicroRNAs (miRNAs) are important post-transcriptional regulators of gene expression. To identify potential miRNAs contribute to MIEF2 over-expression in OC, target prediction was applied using microRNA Data Integration Portal (mirDIP) [28]. Among the top five predicted miRNAs targeting MIEF2 (Fig. S2), only miR-424-5p transfection decreased MIEF2 expression in SKOV3 and HEY cells (Fig. 6a and b). In addition, a significant negative correlation was observed between the levels of miR-424-5p and MIEF2 in tumor tissues from 30 OC patients (Fig. 6c). As expected, a significantly down-regulation of miR-424-5p was observed in tumor tissues of OC as compared to their normal counterparts from 30 OC patients (Fig. 6d), indicating that MIEF2 over-expression is mainly mediated by the down-regulation of miR-424-5p in OC. Furthermore, we found that forced expression of miR-424-5p significantly attenuated the promoting effects of MIEF2 over-expression on OC growth and metastasis in SKOV3 and HEY cells (Fig. 6e-i).

Mitochondrial plays important roles in the regulation of cellular metabolism. Considering that MIEF2 is a crucial regulator of mitochondrial fission and morphology, we thus hypothesized that MIEF2 over-expression may contribute to the reprogramming of metabolism in OC cells. To define the metabolic alterations induced by MIEF2, we first examined the effects of MIEF2 knockdown and over-expression on mitochondrial oxygen consumption rate (OCR), oxidative phosphorylation (OXPHOS) activity and ATP production. Our results showed that knockdown of MIEF2 in OVCAR3 cells significantly increased the rate of oxygen consumption (Fig. 7a), activities of respiratory chain complexes I-V (Fig. 7b) and ATP production (Fig. 7c), while forced MIEF2 expression exhibited the opposite effects in SKOV3 cells (Fig. 7a-c). Electron microscopy showed that MIEF2 significantly induced mitochondrial fragmentation with increased cristae width (Fig. 7d), a phenotype consistent with mitochondrial OXPHOS defects [29]. These results suggest that MIEF2 suppressed mitochondrial respiration in OC cells mainly through mitochondrial fragmentation-suppressed cristae formation. Considering that impaired mitochondrial OXPHOS is often accompanied by increased glycolysis, which has been well-known as the “Warburg effect”, we accordingly assessed the potential role of MIEF2 in the glycolysis of OC cell. Glucose consumption and lactate production assays revealed that MIEF2 knockdown significantly suppressed glucose consumption and lactate production, whilst pH value in the culture medium was significantly increased. In contrast, MIEF2 over-expression exhibited the opposite effects (Fig. 7e-g). To further corroborate these results, cellular metabolites were relatively quantified by gas chromatography-mass spectrometry (GC-MS) analysis. We found that MIEF2 knockdown resulted in a significant decrease in intracellular concentrations of glycolytic intermediates (glucose 6-phosphate (G6P), fructose 6-phosphate (F6P), glyceraldehyde 3-phosphate (GA3P), 3-phosphoglycerate (3PG) and lactate), while a significant increase in TCA cycle metabolites (citrate, aconitate, α-ketoglutarate, fumarate, malate) in OVCAR3 cells. In contrast, over-expression of MIEF2 was associated with increased glycolytic intermediates, while decreased TCA cycle metabolites in SKOV3 cells (Fig. 7h). These results indicate that MIEF2 switched the glucose metabolism from oxidative phosphorylation to glycolysis in OC cells.

Increased aerobic glycolysis has been coupled with various malignant phenotypes of cancer cells, including tumor growth and metastasis [7, 30]. To test whether the promoting effects of MIEF2 on OC cell growth and metastasis were dependent on increased aerobic glycolysis, glucose in cell culture medium was replaced by galactose (cannot be fermented), which induced cells to rely on mitochondrial metabolism to generate sufficient ATP for survival. As shown in Fig. 8a-d, inhibition of glycolysis by galactose significantly attenuated the growth and metastasis promoted by MIEF2 over-expression in SKOV3 and HEY cells, as determined by MTS cell viability, colony formation, wound healing migration and transwell matrigel invasion assays. These results imply that MIEF2 may exert its oncogenic functions in OC cells through activating aerobic glycolysis.