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Introduction

Ovarian cancer is one of the most common gynecological tumors (1). Although surgical resection combined with platinum and taxane-based chemotherapy inhibits the development of tumors and improves short and medium-term survival, the 5-year survival rate remains <30% due to drug resistance and recrudescence worldwide (2). Therefore, exploring the underlying mechanisms of ovarian cancer development and metastasis may aid the identification of a novel therapeutic target.

N6-methyladenosine (m6A) is a highly conserved functional modification of RNA widely distributed in all eukaryotic cells (3). The m6A modification functions in a number of biological processes, such as mRNA post-transcriptional processing, location and translation; m6A is important in human diseases, including obesity and liver cancer development (4,5). Catalyzation of the m6A modification is mediated by a methyltransferase complex consisting of three proteins: Methyltransferase-like 3 (METTL3), methyltransferase-like 14 and Wilms tumor 1-associating protein (6). Functional studies have demonstrated that METTL3 is closely associated with the development of various tumors, and its functions (pro or anti-tumor) vary between different tumor types. For example, Lin et al (7) have reported that METTL3 functions as a translational promotor for multiple oncogenes, including epidermal growth factor receptor (EGFR) and tafazzin (TAZ), in lung cancer, contributing to tumor proliferation, survival and invasion. Reduced METTL3 expression levels in human myeloid leukemia cell lines induces cell differentiation and apoptosis, delaying leukemia progression in recipient mice in vivo (8). Li et al (9) have demonstrated that METTL3 functions as a tumor suppressor in the development of renal cell carcinoma by inhibiting proliferation and invasion. However, the function of METTL3 in human ovarian cancer remains unclear.

The present study aimed to investigate METTL3 expression levels in different ovarian cancer tissue histotypes and analyze the functional effect of METTL3 knockdown in the human ovarian cancer cell lines SKOV3 and OVCAR3.

Materials and methods

Patient tissue samples

The present study was approved by The Ethics Committee in Shandong Provincial Hospital (Jinan, China) and all patients provided informed written consent. A total of 52 ovarian cancer tissue and adjacent normal tissue specimens were collected from patients diagnosed with ovarian cancer and treated at Shandong Provincial Hospital between February 2018 and March 2019. All patients were aged between 35 and 67 years and had only undergone surgery, receiving no other treatment. The clinicopathological data of patients obtained included age, tumor size, tumor site, lymph node metastasis and clinical stages (Table I).

Table I. METTL3 expression levels associated with the clinicopathological parameters in ovarian cancer tissues.

Table I.

METTL3 expression levels associated with the clinicopathological parameters in ovarian cancer tissues.

		METTL3 expression level
Clinicopathological parameters	n	High, n (%)	Low, n (%)	P-value
Age, years
<50	38	28 (73.7)	10 (26.3)	0.863
≥50	14	10 (71.4)	4 (28.6)
Tumor diameter, cm
<3	23	13 (56.5)	10 (43.5)	0.019a
≥3	29	25 (86.2)	4 (13.8)
Lymph node metastasis
Yes	34	29 (85.3)	5 (14.7)	0.016a
No	18	9 (50)	9 (50)
Pathological grading
I–II	13	6 (12.9)	7 (87.1)	0.030a
III–IV	39	32 (56.5)	7 (43.5)
Histotype
Serous adenocarcinoma	17	14 (82.4)	3 (17.6)	0.613
Mucinous adenocarcinoma	16	11 (68.8)	5 (31.2)
Endometrioid adenocarcinoma	12	8 (66.7)	4 (33.3)
Clear cell carcinoma	7	5 (71.43)	2 (28.57)

a P<0.05. The pathological grade was staged according to the International Union against Cancer/American Joint Committee on Cancer system (11). METTL3, methyltransferase-like 3.

Immunohistochemistry

The ovarian cancer tissues and adjacent paracancer tissues were stained using an EliVision™ Plus kit (Fuzhou Maixin Biotech Co., Ltd.) according to the manufacturers protocol. Images were obtained using an upright light microscope system (Nikon Corporation; magnifications, ×100 and ×400). The METTL3 immunostaining score was the sum of the staining intensity score and the positive staining cell rate score. The staining intensity was scored as follows: no staining, 0; weak staining, 1; moderate staining, 2; and strong staining, 3. The positive staining cell rate was scored as follows: 0 to 5%, 0; 5 to 25%, 1; 26 to 50%, 2; 51 to 75%, 3; and >75%, 4. A score <2 points was considered low METTL3 expression, whereas >3 points was considered high METTL3 expression.

Ovarian cancer cell culture and transfection

Human ovarian cancer cell lines SKOV3 and OVCAR3 were purchased from The Cell Bank of Type Culture Collection of the Chinese Academy of Sciences. The cells were cultured in Dulbeccos Modified Eagles Medium (DMEM; HyClone; GE Healthcare Life Sciences) supplemented with 10% FBS (Gibco; Thermo Fisher Scientific, Inc.) at 37°C in 5% CO2. Short hairpin (sh)RNAs (pSUPER) shRNA1, shRNA2 and shRNA3 targeting METTL3 and a control shRNA were designed and synthesized by Shanghai GenePharma Co., Ltd. Ovarian cancer cells were seeded into a 6-well plate and cultured to logarithmic phase for 24 h at 37°C. shRNA transfection was performed using Lipofectamine® 2000 (Invitrogen; Thermo Fisher Scientific, Inc.). Following transfection for 48 h, METTL3 mRNA expression levels were determined using reverse transcription-quantitative (RT-q) PCR.

RT-qPCR

Total RNA of human ovarian cancer cells was prepared using an Ultrapure RNA Extraction kit (CWBio) according to the manufacturers protocol. A total of 1 µg RNA was reverse-transcribed into cDNA with random primers using a HiFiScript cDNA Synthesis kit (CWBio) according to the manufacturers protocol. The reverse transcription reaction conditions were as follows: incubation at 42°C for 50 min, followed by incubation at 85°C for 5 min to terminate the reaction. METTL3 mRNA expression levels were determined by fluorescence qPCR using UltraSYBR mixture (CoWin Biosciences) according to the manufacturers protocol and an Applied Biosystems 7500 FAST Real-time PCR system (Applied Biosystems; Thermo Fisher Scientific, Inc.). The following thermocycling conditions were used for qPCR: Initial denaturation at 95°C for 30 sec; 40 cycles of 95°C for 5 sec, 60°C for 30 sec and one cycle of melting curve at 95°C for 15 sec, 60°C for 1 min, 95°C for 15 sec and 50°C for 30 sec. Quantification of METTL3 mRNA expression levels was performed using the 2−ΔΔCt method (10) with β-actin as an internal control. The sequences of primers were as follows: METTL3 forward, 5′-ACCCTGACAGATGATGAGATGC-3′ and reverse, 5′-CGTTCATACCCCCAGAGGTTTAG-3′; β-actin forward, 5′-TCCTCCCTGGAGAAGAGCTAC-3′ and reverse, 5′-TCCTGCTTGCTGATCCACAT-3′.

CCK-8 assay

Cells were seeded into a 96-well plate (3,000 cells/well) and cultured in DMEM at 37°C for 0, 24, 48 or 72 h, and 10 µl CCK-8 solution (Beyotime Institute of Biotechnology) was added into each well according to the manufacturers protocols. After incubation for 2 h at 37°C and 5% CO2, the absorbance of cells at 450 nm was detected using a microplate reader.

Colony formation assay

SKOV3 and OVCAR3 cells were seeded in a 6-cm petri dish at the density of 500 cells/well and normally cultured in DMEM at 37°C for 14 days. Subsequently, the colonies were fixed with 4% methanol and stained with 0.1% crystal violet at 37°C for 30 min (Sigma-Aldrich; Merck KGaA). Images of visible colonies were captured with a HP Scanjet G4010 scanner and counted manually.

Flow cytometry detection of cell apoptosis

Transfected SKOV3 and OVCAR3 cells were stained using an Annexin V-FITC Apoptosis Detection kit I (BD Biosciences) according to the manufacturers protocols. Cells were analyzed using the BD FACS Canto II system (BD Biosciences) and analyzed using FlowJo software version 4.5 (Tree Star, Inc.). Viable cells were negative for both PI and Annexin V, while apoptotic cells were positive for Annexin V and negative for PI. Late apoptotic dead cells showed both Annexin V and PI positivity.

Transwell invasion assay

Cell invasion was evaluated using Matrigel-coated Transwell chambers at 37°C for 24 h (BD Biosciences). A total of 1×105 ovarian cancer cells in 200 µl serum-free DMEM were added into the upper chamber. A total of 500 µl DMEM with 10% FBS was added to the lower chamber, and the cells were incubated for 24 h. The non-invasive cells remaining in the upper chamber of the Transwell plate were scraped off with a cotton swab. Invaded cells on the lower surface of the chamber were stained with 0.1% crystal violet at 25°C for 10 min. The cell number was counted as the average of five random fields under a light microscope (Nikon TE2000; Nikon Corporation).

Western blotting

Following transfection for 48 h, cell lysates were prepared using RIPA lysis buffer and protease cocktail inhibitor I (Merck KGaA). The protein was separated using 10% SDS-PAGE and subsequently transferred to a PVDF membrane. After blocking in a 5% non-fat milk for 1 h, the membrane was washed with TBS + 0.1% Tween-20. Membranes were incubated with primary antibodies against METTL3 (1:1,000; cat. no. GTX105037; GeneTex, Inc.), Bcl-2 (1:2,000; cat. no. 60178-1-Ig; ProteinTech Group, Inc.); Bax (1:1,000; cat. no. 50599-2-Ig; ProteinTech Group, Inc.); active caspase3 (1:1,000; rabbit polyclonal antibody; cat. no. 19677-1-AP; ProteinTech Group, Inc.); p-AKT (1:1,000; cat. no. 66444-1-Ig; ProteinTech Group, Inc.); AKT (1:500; cat. no. 9272; Cell Signaling Technology, Inc.); p70S6K (1:1,000; cat. no. GTX107562; GeneTex, Inc.); Cyclin D1 (1:1,000; cat. no. GTX108624; GeneTex, Inc.) and tubulin (1:1,000; cat. no. GTX76511; GeneTex, Inc.) at 4°C overnight, followed by incubation with anti-rabbit IgG (1:2,000; cat. no. GTX300119; GeneTex, Inc.) or anti-mouse IgG (1:2,000; cat. no. GTX300120; GeneTex, Inc.) secondary antibodies for 1 h at room temperature. Protein bands were visualized using Pierce ECL Western Blotting Substrate (Thermo Fisher Scientific, Inc.). Protein band intensity was analyzed using Image J software, v1.41 (National Institutes of Health).

Statistical analysis

Data analysis was performed using GraphPad Prism 7 (GraphPad Software, Inc.), and all experiments were performed in triplicate. Differences between two groups were evaluated using Students t-test; one-way ANOVA was used to analyze multiple groups, followed by Tukeys post-hoc test. P<0.05 was considered to indicate a statistically significant difference.

Results

METTL3 expression levels are associated with clinical parameters in patients with ovarian cancer

METTL3 expression levels were significantly highly in serous adenocarcinoma, mucinous adenocarcinoma, endometrioid adenocarcinoma and clear cell carcinoma tissues compared with corresponding paracancerous tissues. METTL3 expression levels were not associated with different histotypes of ovarian cancer tissues. High METTL3 expression levels were associated with large tumor size (P=0.0188), lymph node metastasis (P=0.0163) and high pathological grade (P=0.0303). The pathological grade was staged according to the International Union against Cancer/American Joint Committee on Cancer system (11). These data indicated that METTL3 expression levels may be associated with the tumor growth and metastasis of ovarian cancer (Fig. 1 and Table II).

Figure 1. METTL3 expression levels are high ovarian cancer tissues. Areas indicated by arrows indicate sites of high METTL3 expression levels. (A-D) Immunohistochemical staining of METTL3 in ovarian cancer tissues and para-carcinoma in cases of: (A) serous adenocarcinoma, (B) mucinous adenocarcinoma, (C) endometrioid adenocarcinoma and (D) clear cell carcinoma. Magnifications, ×100 and ×400. Scale bars, 100 and 400 µm. METTL3, methyltransferase-like 3.

Table II. METTL3 expression levels in ovarian cancer and para-carcinoma tissue.

Table II.

METTL3 expression levels in ovarian cancer and para-carcinoma tissue.

		METTL3 expression level
Tissue	n	Low, n (%)	High, n (%)	P-value
Ovarian cancer	52	14 (18.3)	38 (81.7)	<0.001a
Para-carcinoma	52	40 (87.1)	12 (12.9)	<0.001a

a P<0.05. METTL3, methyltransferase-like 3.

Downregulation of METTL3 inhibits proliferation and colony formation in human ovarian cancer tissues

To investigate the biological function of METTL3 in human ovarian cancer, loss-of-function assays were performed. The interference efficiency results demonstrated that shRNA1, shRNA2 and shRNA3 targeting METTL3 significantly downregulated METTL3 mRNA expression levels in SKOV3 and OVCAR3 cells compared with negative control group (P<0.05; Fig. 2A and B). shRNA3 in SKOV3 cells and shRNA1 in OVCAR3 cells (shMETTL3) were selected for further experiments due to the interference efficiencies, and knockdown efficacy was validated at the protein level (Fig. 2C and D). The cell proliferation assay results indicated that METTL3 knockdown significantly inhibited the proliferation of SKOV3 and OVCAR3 cells (Fig. 3A and B; P<0.05). In addition, cell colony number was also significantly decreased when METTL3 expression was silenced (Fig. 3C and D).

Figure 2. METTL3 knockdown in SKOV3 and OVCAR3 ovarian cancer cells mediated by shRNA transfection. All experiments were performed in triplicate. shRNA1, shRNA2, shRNA3, shMETTL3 or a negative control shRNA was introduced into SKOV3 and OVCAR3 cells. METLL3 mRNA expression levels in (A) SKOV3 and (B) OVCAR3 cells treated with shRNA1, shRNA2, shRNA3 or NC. (C) METTL3 protein expression levels in SKOV3 and OCVAR3 cells treated with shRNA3 and shRNA1, respectively. (D) Quantified METTL3 protein expression levels using ImageJ software in SKOV3 and OVCAR3 cells. *P<0.05 vs. NC. METTL3, methyltransferase-like 3; sh, short hairpin; NC, negative control; shMETTL3, shRNA3 in SHOV3 cells or shRNA1 in OVCAR3 cells.

Figure 3. Downregulation of METTL3 expression inhibits proliferation and colony formation of human ovarian cancer cells. (A) Proliferation of SKOV3 cells treated with shMETTL3 or NC at different time points. (B) Proliferation of OVCAR3 cells treated with shMETTL3 or NC at different time points. (C and D) Colony formation of SKOV3 and OVCAR3 cells treated with shMETTL3 or NC. All experiments were performed in triplicate. *P<0.05 vs. NC. METTL3, methyltransferase-like 3; sh, short hairpin; NC, negative control.

METTL3 knockdown induces apoptosis and may lead to the activation of the mitochondrial apoptosis pathway in ovarian cancer cells

Apoptosis analysis results suggested that the percentage of SKOV3 and OVCAR3 cells undergoing apoptosis was significantly increased when METTL3 expression levels were knocked down compared with the control at 48 h and 72 h (Fig. 4A-D). METTL3 knockdown also led to a significant upregulation of the pro-apoptotic protein Bax and downstream effector Caspase 3 expression levels, whereas the expression of the anti-apoptotic protein Bcl2 was downregulated (Fig. 4E and F). These data suggested that METTL3 knockdown activated the mitochondrial apoptosis pathway in SKOC3 and OVCAR3 cells.

Figure 4. Downregulation of METTL3 expression increases apoptosis in human ovarian cancer cells. (A and B) Quantification of Annexin V/PI staining in SKOV3 cells transfected with shMETTL3 or NC. (C and D) Quantification of Annexin V/PI staining in OVCAR3 cells transfected with shMETTL3 or NC. The data were analyzed by using FlowJo version 10 software. (E and F) Bcl2, Bax and Caspase 3 protein expression levels in SKOV3 and OVCAR3 cells with treated with shMETTL3 or NC. All experiments were performed in triplicate. *P<0.05 vs. NC. METTL3, methyltransferase-like 3; sh, short hairpin; NC, negative control; PI, propidium iodide.

METTL3 knockdown inhibits cell invasion and may reduce the activation of the AKT signaling pathway

The effect of METTL3 knockdown on the invasive ability of SKOV3 and OVCAR3 cells was investigated using Transwell assays. The invasive ability of SKOV3 and OVCAR3 cells was significantly reduced when METTL3 expression levels were knocked down compared with the control (P<0.05; Fig. 5A and B). It was hypothesized that thr AKT signaling pathway may be associated with the biological function of METTL3. METTL3 knockdown led to decreased expression levels of phosphorylated AKT and its downstream effectors p70S6K and Cyclin D1 (Fig. 5C and D). These results suggested that knockdown of METTL3 expression led to reduced activation of the AKT signaling pathway in ovarian cancer cells.

Figure 5. METTL3 knockdown inhibits cell invasion and may reduce the activation of the AKT signaling pathway. (A and B) Quantified cell migration and invasion of SKOV3 and OVCAR3 cells treated with shMETTL3 or NC. (C and D) Expression of the components of the AKT signaling pathway, including AKT, p-AKT, p7026K and Cyclin D1, in SKOV3 and OVCR3 cells treated with shMETTL3 or NC. All experiments were performed in triplicate. Scale bar, 100 µm. *P<0.05 vs. NC. METTL3, methyltransferase-like 3; sh, short hairpin; NC, negative control; p-, phosphorylated.

Discussion

In the present study, METTL3 expression levels in ovarian cancer tissues were investigated; the results demonstrated that knockdown of METTL3 significantly inhibited proliferation, colony formation and invasion of ovarian cancer cells. In addition, the apoptotic rate was increased when METTL3 expression levels were knocked down. Downregulation of METTL3 increased the expression levels of the pro-apoptotic Bax and Caspase 3, whereas the expression levels of the anti-apoptotic Bcl2 were decreased. During apoptosis, Bax binds to the mitochondrial outer membrane and promotes its permeability, causing the release of cytochrome c into the cytoplasm, which induces the activation of Caspase 3 (12). By contrast, Bcl2 functions as an apoptosis inhibitor and blocks the promotion of mitochondrial permeability (13). To the best of our knowledge, the present study demonstrated the oncogenic function of METTL3 in the biological process of ovarian cancer cells for the first time.

An increasing number of studies have demonstrated the function of METTL3 in tumor formation and progression. For example, Lin et al (7) have reported that METTL3 promotes the translation of epidermal growth factor receptor and the Hippo pathway effector TAZ in human lung cancer cells. In addition, reduced METTL3 expression levels inhibit tumor growth and metastasis to the lung of hepatocellular carcinoma (HCC) in vitro and in vivo (14). Du et al (15) have demonstrated that microRNA-33a functions as a tumor suppressor in non-small-cell lung carcinoma cells, suppressing the translation of METTL3 mRNA. However, another study identified METTL3 as a tumor suppressor in renal cell carcinoma, inhibiting tumor proliferation, migration and cell cycle progression (9). These results from previous studies suggest that the functions of METTL3 can vary between different tumor types, and a possible explanation for this may be the high degree of tumor heterogeneity or gene mutation-induced function alteration.

The modes of METTL3 action can be divided into two types: m6A-dependent and m6A-independent (14,16). For example, METTL3 depletion can promote suppressor of cytokine signaling 2 expression by decreasing m6A methylation-mediated degradation, thus blocking the progression of HCC (14). Cai et al (16) demonstrated that METTL3-induced m6A modification increased the expression levels of hepatitis B X-interacting protein, thus promoting the proliferation of breast cancer cells. METTL3 has also been identified to elevate oncoprotein expression levels in tumor cells by functioning as a transcription enhancer factor or promoting the assembly of mRNA translation machinery (7). In the present study, the mechanisms underlying the oncogenic function of METTL3 in human ovarian cancer were investigated. A previous study reported that the AKT signaling pathway regulates numerous biological processes, such as promoting cell proliferation and survival (17). The present study demonstrated that METTL3 downregulation decreased the expression levels of phosphorylated AKT and its downstream effectors, including p70S6K and Cyclin D1, indicating reduced activation of the AKT pathway. However, the present study was unable to identify the direct target of METTL3 function in human ovarian cancer cell lines due to the experimental conditions.

In conclusion, METTL3 knockdown inhibits tumorigenesis and tumor progression of human ovarian cancer cells in vitro, which may be mediated by reduced activation of the AKT signaling pathway. These results may provide novel insight into the potential targeting of METTL3 in ovarian cancer treatment.

Acknowledgements

Not applicable.

Funding

The present study was supported by The National Natural Science Foundation of China (grant no. 81503298) and Natural Science Foundation of Shandong Province (grant no. ZR2014HM008).

Availability of data and materials

The datasets used and/or analyzed during the present study are available from the corresponding author upon reasonable request.

Authors contributions

SL designed the study. SL, HG, XL, NL, FG and JL performed experiments and analyzed data. SL wrote the manuscript. All authors read and approved the final manuscript.

Ethical approval and consent to participate

The present study was approved by The Ethics Committee of Shandong Provincial Hospital (China). All patients provided written informed consent.

Patient consent for publication

Not applicable.

Competing interests

The authors declare that they have no competing interests.

Glossary

Abbreviations

Abbreviations:

References