The cBioPortal for Cancer Genomics (http://www.cbioportal.org/) was used to analyze the relationship between mutation of the MTCH2 gene in OC and the overall survival time of patients with OC by whole exome sequencing (33). Using the cBioPortal database, OC data from the Pan-Cancer Atlas and the Cancer Genome Atlas were selected for analysis.

Human ovarian carcinoma SK-OV-3 cells were purchased from Shanghai Zhongqiao Xinzhou Biotechnology Co., Ltd. (cat. no. ZQ0074). Cells were cultured in RPMI-1640 medium (cat. no. C11875500BT; Gibco; Thermo Fisher Scientific, Inc.), supplemented with 15% fetal bovine serum (FBS; cat. no. G24-70500; Genial Biologicals, Inc.) and 1% penicillin/streptomycin (cat. no. CA0075, Leagene; Beijing Regen Biotechnology Co., Ltd.) at 37°C with 5% CO₂.

The pcDNA3.1 cloning vector (cloning sites, NheI and BamHI) was used to generate the MTCH2-overexpression (OE) plasmid, which encodes the MTCH2 transcript (accession no. NM_001317231.2). The plasmid and small interfering (si)RNAs were purchased from Sangon Biotech Co., Ltd. The sequences of MTCH2 and AIMP2 siRNAs used were as follows: MTCH siRNA sense (S), 5′-UCCUCCAACAAUAGGACGAAATT-3′ and anti-sense (AS) 5′-UUUCGUCCUAUUGUUGGAGGATT-3′; AIMP2 siRNA1 S, 5′-GAUGCAGACUUGGAUGUAATT-3′ and AS, 5′-UUACAUCCAAGUCUGCAUCTT-3′; AIMP2 siRNA2 S, 5′-GAUUCACUUUAAUUUGGAATT-3′ and AS, 5′-UUCCAAAUUAAAGUGAAUCTT-3′; AIMP2 siRNA3 S, 5′-AGUGCUUUGGAGAACAGAATT-3′ and AS 5′-UUCUGUUCUCCAAAGCACUTT-3′; and negative control (NC) S, 5′-UUCUCCGAACGUGUCACGUTT-3′ and AS, 5′-ACGUGACACGUUCGGAGAATT-3′. SK-OV-3 cells were seeded into 6-well cell culture plates. The OE transfection plasmid (1.25 µg/ml) and siRNAs (50 pmol/ml) were separately transfected into SK-OV-3 cells using the Lipo8000™ transfection reagent (cat. no. C0533; Beyotime Institute of Biotechnology) at 37°C for 24 h. Co-transfection with AIMP2 siRNA and MTCH2 siRNA or MTCH2-OE was performed to study the downstream mechanism of MTCH2 (34). The control group comprises untransfected cells. The efficiency of the transfection was evaluated through western blotting. After 24 h, cells were collected and used in the follow-up experiments.

Tumor tissue samples were collected from high-grade serous ovarian carcinoma (HGSOC). The samples were collected in May 2021. Tumor tissues and normal tissues adjacent to tumor tissues (≥4 cm from the tumor edge) (35) were collected from 67 patients with HGSOC at The Fourth Hospital of Hebei Medical University (Shijiazhuang, China). Patients with a history of chemotherapy or radiotherapy were excluded from the present study. Paired tumor tissues and adjacent normal tissues were collected and stored in liquid nitrogen until further use. The protocol was approved by the Ethics Committee of The Fourth Hospital of Hebei Medical University (approval no. 2020166; March 14 2021; Shijiazhuang, China). All protocols were performed in accordance with the Declaration of Helsinki.

OC and adjacent normal tissue samples were fixed using 10% formalin at room temperature for 3 h, embedded in paraffin and sectioned to a 4-µm thickness. After dewaxing with xylene and rehydrated in a decreasing concentration of ethanol, antigen retrieval was performed in a microwave (100°C for 7 min). Endogenous peroxidase activity was quenched with 3% H₂O₂ for 12 min at 37°C and sections were then blocked with 5% sheep serum (cat. no. ZLI-9056; OriGene Technologies, Inc.) at room temperature for 10 min. After which, the sections were incubated at 4°C overnight with primary antibodies against MTCH2 (1:200; cat. no. 16888-1-AP; Proteintech Group, Inc.) and AIMP2 (1:200; cat no. 10424-1-AP; Proteintech Group, Inc.). The next day, the slides were incubated with secondary antibodies (cat. no. PV-9000; OriGene Technologies, Inc.) at 37°C for 20 min. Staining was performed using 3,3-diaminobenzidine (cat. no. ZLI-9019; OriGene Technologies, Inc.) at room temperature for 5 min and sections were counterstained using hematoxylin for 3 min. A fluorescence microscope used to capture the images. Evaluation of the tissue sample staining intensity was performed as previously described by Wang et al (36). Sections were scored according to the staining intensity as follows: Negative staining, 0; weak positivity, 1; moderate positivity, 2; and strong positivity, 3 points. Further scoring was performed according to the proportion of the section with positive staining as follows: 0–10%, 1; 10–50%, 2; 51–80%, 3; and >80%, 4 points. Subsequently, the two scores were multiplied and the resulting score ranged from 0 to 12. Scores from 0 to 3 were considered negative and scores from 4 to 12 were considered positive. In addition, the mean density was calculated using the following formula: Mean density=IOD/area, where IOD indicates integrated optical density value. The protein expression levels were quantitatively analyzed using Image-Pro Plus software (version 6; Media Cybernetics, Inc.).

Initially, 300 µl Matrigel was used to coat the upper chamber of a Transwell system (24-well plate; pore size, 8.0 µm) at 37°C for 3 h. SK-OV-3 cells were first cultured in serum-free RPMI-1640 medium for 24 h. The next day, SK-OV-3 cells (1×10⁵ cells/ml) were seeded into the upper chambers with serum-free RPMI-1640 medium. RPMI-1640 medium supplemented with 15% FBS and 1% penicillin/streptomycin was added into the bottom of a 24-well plate and placed in an incubator for 48 h (37°C, 5% CO₂). Following this, 4% paraformaldehyde (cat. no. DF0135; Leagene; Beijing Regen Biotechnology Co., Ltd.) was used to fix the migrated cells for 10 min and 0.1% crystal violet (cat. no. G1063; Beijing Solarbio Science & Technology Co., Ltd.) was used to stain the cells for 20 min at room temperature. Any residual cells that remained in the upper chambers were gently removed using a cotton swab after staining. A CX31 inverted light microscope (Olympus Corporation) was used to image randomly selected fields of view to evaluate the migrated cells.

SK-OV-3 cells (3×10⁵ cells/well) were seeded in 6-well plates and transfected with MTCH2-OE and MTCH2 siRNA for 48 h according to the aforementioned method. When the cell density reached ~90%, 200 µl pipette tips were used to scratch two parallel lines in the central area where the cells grew. PBS was used to wash each well twice, after which, the cells were cultured for 48 h in serum-free RPMI-1640 medium. Images of the scratched area were captured using a CX31 inverted light microscope (Olympus Corporation) at 0 and 24 h. The images were analyzed using the Image-Pro Plus software (version 6; Media Cybernetics, Inc.). The cell migration distance was calculated as follows: (width of the wound at 0 h-width of the wound at 24 h)/width of the wound at 0 h.

ATP content was detected using an ATP fluorescent probe pCMV-AT1.03 (cat. no. D2604, Beyotime Institute of Biotechnology) which allowed ATP levels to be monitored within living single cells in real time. SK-OV-3 cells (2×10⁵ cells/well) were seeded in 12-well plates and cultured at 37°C in 5% CO₂ for 24 h. Next, 12 µl pCMV-AT1.03 was added to each well and cultured for 24 h at 37°C. The cells were then imaged at 24-h timepoints using a BX53 fluorescence microscope (Olympus Corporation) and fluorescence intensities were calculated using Image-Pro Plus software (version 6; Media Cybernetics, Inc.).

The mitochondrial network structure was evaluated using Mito-Tracker Red CMXRos (cat. no. C1049B; Beyotime Institute of Biotechnology) according to the manufacturer's instructions. SK-OV-3 cells (1×10⁵ cells/well) were transfected as aforementioned and cultured in 6-well plates for 24 h. The Mito-Tracker Red CMXRos working solution was prepared by adding 1 µl of 200 µM Mito-Tracker Red CMXRos storage solution to 1 ml of cell culture medium (final concentration, 200 nM) mixed well and the cells were then incubated with this working solution for 45 min. The staining solution was then replaced with fresh culture medium prior to imaging. Images were captured of living cells using a fluorescence microscope (BX53; Olympus Corporation) and the fluorescence intensities analyzed using Image-Pro Plus software (version 6, Media Cybernetics).

The BeyoClick™ EdU-594 Cell Proliferation Assay kit (cat. no. C0078S; Beyotime Institute of Biotechnology) was used to detect the proliferation rate of OC cells, according to the manufacturer's instructions. Briefly, SK-OV-3 cells (1×10⁴ cells) were cultured in a cell incubator at 37°C (5% CO₂) for 24 h. Cells were treated with 10 µM of prewarmed EdU working solution at 37°C for 2 h. Then, the cells were fixed with 4% paraformaldehyde for 15 min at room temperature, followed by rinsing three times with PBS solution containing 3% BSA (3 min/wash). The cells were then incubated with PBS containing 0.3%Triton X-100 for 15 min at room temperature. Next, 0.5 ml Click reaction solution was added and incubated in the dark for 30 min at room temperature. The cells were counterstained with DAPI (10 µg/ml) at room temperature for 3 min and imaged using a BX53 fluorescence microscope (Olympus Corporation). The number of positively stained proliferating cells were counted for analysis of proliferation rate.

Apoptosis was detected using Caspase-3 Activity and Apoptosis Detection kit for Live Cells (cat. no. C1077S; Beyotime Institute of Biotechnology) according to the manufacturer's instructions. SK-OV-3 cells were washed with PBS, following which 194 µl annexin V-mCherry Binding Buffer and 5 µl Annexin V-mCherry and 1 µl GreenNuc™ Caspase-3 Substrate (1 mM) were added, gently mixed and incubated at room temperature in the dark for 30 min. Images were captured using a BX53 fluorescence microscope (Olympus Corporation). Annexin V-mCherry labels apoptotic cells with red fluorescence and GreenNuc™ Caspase-3 Substrate labels the nuclei of apoptotic cells with green fluorescence representing caspase-3 activity. The number of apoptotic cells was calculated as follows=(number of apoptotic cells/number of total cells) ×100.

SK-OV-3 cells (1×10⁵ cells) were spread on slides, fixed with 3.7% formaldehyde in PBS solution for 20 min at room temperature and then washed thrice with PBS containing 0.1% Triton X-100 at room temperature (5 min/wash). Actin-Tracker Green (cat. no. C2201S; Beyotime Institute of Biotechnology) was added dropwise onto the slides and incubated in the dark at room temperature for 60 min. Next, the cells were washed three times with PBS containing 0.1% Triton X-100 at room temperature (5 min/wash), the plates were air-dried, counterstained with DAPI at room temperature for 3 min and images were captured using a BX53 fluorescence microscope (Olympus Corporation). The images were analyzed using the Image-Pro Plus software (version 6; Media Cybernetics, Inc.).

SK-OV-3 cells (2×10⁵ cells) were fixed using 4% paraformaldehyde at room temperature for 10 min, permeabilized with 0.1% Triton-X-100 in PBS and blocked with goat serum (cat. no. ZLI-9056; OriGene Technologies, Inc.). The cells were then incubated with primary antibodies against MTCH2 (1:200; cat no. 16888-1-AP; Proteintech Group, Inc.) and AIMP2 (1:100; cat no. 10424-1-AP; Proteintech Group, Inc.) at 4°C overnight. Next, using dilution buffer (cat. no. ZLI-9030; OriGene Technologies, Inc.) to dilute the antibody, cells were incubated with Alexa Fluor^® 488 goat anti-rabbit IgG (1:100; cat. no. ZF-0511; OriGene Technologies, Inc.) secondary antibodies was performed at 37°C for 1 h. PBS was used to wash the section thrice and DAPI was used to stain the nucleus at room temperature for 5 min. Images were captured using a BX53 fluorescence microscope (Olympus Corporation). The fluorescence intensities were analyzed using the Image-Pro Plus software (version 6; Media Cybernetics, Inc.).

The cells were lysed with Lysis Buffer with Protease Inhibitor Cocktail from the IP kit (cat. no. P2179M; Beyotime Institute of Biotechnology). Lysed cells were centrifuged at 13,000 × g for 20 min at 4°C and the supernatant was collected, of which 50 µl was taken for use as the positive control input group. From each sample, 2 µl was used to determine the concentration and the remaining supernatant was stored for subsequent use. The antibodies used for IP were MTCH2 (3 µg; cat no. 16456-1-AP; Proteintech Group, Inc.) and AIMP2 (3 µg; cat. no. 10424-1-AP; Proteintech Group, Inc.) and 3 µg of normal IgG was used as a negative control group. Next, 100 µl Protein A+G magnetic beads (cat. no. P2179M; Beyotime Institute of Biotechnology) were added and incubated with antibodies and IgG on a shaker at 4°C for 2 h. The precipitate was collected by centrifugation at 1,000 × g for 5 min at 4°C. The 500-µl cell lysates were added to protein A+G magnetic beads conjugated with antibody or normal IgG on a shaker at 4°C overnight to bind the proteins to the antibodies. The next day, the samples were centrifuged at 1,000 × g for 5 min at 4°C and the supernatant discarded. The precipitate was washed four times with PBS using 1 ml each time. The precipitate and the input (50 µl of the supernatant after cell lysis) were boiled at 95°C for 5 min with 1× loading buffer (cat. no. ZS306; Beijing Zoman Biotechnology Co., Ltd.). The samples were centrifuged at 1,000 × g for 5 min and the supernatant was used as samples for MTCH2-IP and AIMP2-IP experimental groups for western blotting.

Total protein was extracted from cells using a Total Protein Extraction kit (cat. no. KGP2100; Nanjing KeyGen Biotech Co., Ltd.). The BCA protein quantification kit (cat. no. PT0001, Leagene; Beijing Regen Biotechnology Co., Ltd.) was used to quantify the concentrations of extracted total proteins. The proteins were then separated by SDS-PAGE on a 9% gel and transferred on to PVDF membranes. The membranes were blocked with 5% milk/TBST (0.05% Tween) for 1 h at room temperature to prevent non-specific binding. The membranes were incubated with MTCH2 (1:1,000; cat. no. 16888-1-AP; Proteintech Group, Inc.), claudin-3 (1:1,000; cat. no. AF0129; Affinity Biosciences), AIMP2 (1:1,000; cat. no. 10424-1-AP; Proteintech Group, Inc.) and β-actin (1:5000, cat. no. AF7018; Affinity Biosciences) overnight at 4°C. Following primary antibody incubation, the membranes were incubated with horseradish peroxidase-conjugated secondary antibodies (cat. no. ZB-2301; OriGene Technologies, Inc.) at room temperature for 1 h. Protein bands were visualized using electrochemiluminescence solution (cat. no. P1050; Applygen Technologies, Inc.). ImageJ software (version 2.14.0; National Institutes of Health) was used for data analysis.

Statistical analysis was conducted using SPSS (version 27; IBM Corp.). Data were presented as mean ± standard deviation. Paired Student's t-test was used to compare two groups. Multiple groups were compared using a one-way ANOVA followed by Bonferroni's post hoc test. Correlation analysis was performed using Pearson correlation test. A survival analysis was performed using Kapan-Meier analysis and log rank test. Contingency tables were assessed using the χ² test. Data presentation was performed using the GraphPad Prism software (version 8; Dotmatics). P<0.05 was considered to indicate a statistically significant difference.

The expression levels of MTCH2, AIMP2 and claudin-3 in OC and adjacent normal tissues were evaluated in tissue samples from 67 patients with OC (median age, 57 years; range, 24-72 years). Immunohistochemical results showed significantly increased expression levels of MTCH2, AIMP2 and claudin-3 in OC tissue samples compared with in adjacent normal tissue samples (Fig. 1A-D), and the tumor samples were scored). According to the immunohistochemical scoring criteria, in OC tissues, 46 cases were positive for MTCH2 and 21 were negative; 42 cases were positive for AIMP2 and 25 were negative; and 43 cases were positive for claudin-3 and 24 were negative (Fig. S1; Table I). Pearson correlation analysis demonstrated a significant positive correlation between MTCH2 expression levels and the expression levels of both AIMP2 and claudin-3 (Table II). Subsequently, the relationship between MTCH2 and the clinicopathological features of patients with OC were assessed, and it was demonstrated that MTCH2 expression levels were significantly associated with International Federation of Gynecology and Obstetrics stage (37) and tumor differentiation (Table III). No significant difference in overall survival time of patients with OC between the MTCH2 mutation group and the MTCH2 control group was detected (Fig. 1E).

To further explore the role of MTCH2 in OC cells, an MTCH2 overexpression plasmid and MTCH2 siRNA were designed and synthesized. Western blotting was used to confirm the transfection efficiency of the MTCH2-OE plasmid and MTCH2 siRNA in OC cells compared with the negative controls (Fig. 2A-D). The wound healing assay demonstrated that, compared with the NC-OE group, the scratch area of the MTCH2-OE group was significantly reduced at 24 h. By contrast, the scratch area of the MTCH2 siRNA group was significantly larger compared with the NC-siRNA group (Fig. 2E, F). Transwell assays demonstrated that the number of migrating cells in the MTCH2-OE group were significantly increased compared with the NC-OE group (P<0.001), whereas the number of migrating cells were significantly decreased in the MTCH2 siRNA group compared with the NC-siRNA group (Fig. 2G, H).

The ATP fluorescence probe assay demonstrated that the fluorescence intensity of the MTCH2-OE group was significantly increased compared with the NC-OE group, which represented increased ATP production in the MTCH2-OE group. By contrast, the fluorescence intensity significantly decreased in the MTCH2 siRNA group compared with the NC-siRNA group (P<0.01; Fig. 3A, B). MitoTracker Red CMXRos staining of mitochondria demonstrated decreased mitochondrial fluorescence in the MTCH2 siRNA group compared with the NC-siRNA group, which indicated mitochondrial dysfunction (Fig. 3C, D). The proliferation of OC cells was assessed through EdU staining. Significantly increased cell proliferation was observed in the MTCH2-OE group compared with the NC-OE group, whereas significantly decreased cell proliferation was demonstrated in the MTCH2 siRNA group compared with the NC-siRNA (Fig. 4A, B). The number of apoptotic cells were significantly decreased in the MTCH2-OE group compared with the NC-OE group, but were significantly increased in the MTCH2 siRNA group compared with the NC-siRNA group (P<0.001; Fig. 4C and D).

Transfection with MTCH2-OE in SK-OV-3 cells demonstrated a significant increase in the fluorescence intensity of microfilaments, filamentous feet and pseudopodia compared with the NC-OE group. By contrast, the fluorescence intensity significantly decreased in the MTCH2 siRNA group compared with the NC-siRNA group (Fig. 5A, B). Immunofluorescence and western blot analyses showed that, compared with the NC-OE group, the expression levels of claudin-3 and AIMP2 were significantly increased in the MTCH2-OE group, and were significantly reduced in the MTCH2 siRNA group compared with that in the NC-siRNA group (Fig. 5C-I).

To evaluate the pathways involved in MTCH2-mediated OC cell metastasis, a number of AIMP2 siRNA sequences were designed and synthesized. Western blotting was used to assess the transfection efficacy of AIMP2 siRNA. Compared with NC-siRNA, the transfection effects of AIMP2 siRNA2 and AIMP2 siRNA3 were notably better than AIMP2 siRNA1. The results of western blotting revealed that the inhibitory effect of AIMP2 siRNA2 on claudin-3 was markedly stronger than that of AIMP2 siRNA3 therefore, AIMP2 siRNA2 was chosen as the interference sequence for use in subsequent experiments (Figs. 6C and D and S2). The expression levels of claudin-3 were significantly decreased in the AIMP2 siRNA group compared with the NC-siRNA group. However, there was no significant regulatory effect on MTCH2 expression levels upon AIMP2 knockdown by AIMP2 siRNA. Transfection of AIMP2 siRNA with the MTCH2-OE plasmid in OC cells could not restore the expression levels of AIMP2 and claudin-3 to the NC-siRNA group levels, which indicated that knockdown AIMP2 blocked the regulatory effect of MTCH2 on these targets (Figs. 6E-H). Co-immunoprecipitation demonstrated that MTCH2 interacts with both claudin-3 and AIMP2 (Fig. 6A and B).