Metformin inhibits the proliferation and invasion of ovarian cancer cells by suppressing tripartite motif-containing 37-induced tumor necrosis factor receptor-associated factor 2 ubiquitination

2.1 Cell lines and reagents

Human immortal ovarian epithelial cell line HOSEpiC and human ovarian cancer cell lines including A2780, SKOV3, OVCAR3, OVCAR5, and OVCAR433 were purchased from ATCC and cultured in RPMI-1640 medium supplemented with 10% FBS (Gibco, Life Technologies), 1% penicillin, 1% streptomycin, and 1% amphotericin B at 37°C with 5% CO₂. Metformin were purchased from Sigma-Aldrich and dissolved in PBS. Both PDTC (50 μM) and TNF-α were purchased from MedChemExpress Corporation. Lipopolysaccharide (100 ng/ml) was purchased from Beyotime Biotechnology.

2.2 Patients and samples

A total of 30 control samples of normal ovarian epithelial tissues and 60 cases of epithelial ovarian cancer tissues used in the study were applied from the tissue bank of the Obstetrics and Gynecology Hospital of Fudan University and were approved by the ethics committee of our hospital. All patients signed informed consent. Patients in the ovarian cancer group were pathologically diagnosed as epithelial ovarian cancer and treated, and were without other malignant tumors. For the control group, the tissues were taken from the patients who underwent adnexectomy for hysteromyoma, and other ovarian lesions were excluded.

2.3 Plasmids and lentivirus transfection

The interference and overexpression lentiviral vectors of TRIM37 were constructed by Shanghai Genechem Chemical Technology Co., Ltd. The TRAF2 plasmid (pCDH-CMV-Human TRAF2 [NM_021138]-C-Myc-EF1-Puro) was constructed by Lnc Biotechnology Co., Ltd. According to the TRIM37 sequence, the company designed four pairs of interference sequences. The target information is shown in Table 1, and the sequence information is shown in Table 2. 293T cells were seeded into 6-well plates, and the density was approximately 70% before transfection the next day. Transfection system A, including 125 μl (Opti-MEM, Thermo Fisher scitific, USA) 2.5 μg DNA, and 5 μl (p3000 reagent, Thermo Fisher scitific, USA) (2 μl/μg DNA) was prepared and vortexed for 3 s. Transfection B system including 125 μl Opti-MEM and 7.5 μl (Lipofectamine 3000, Thermo Fisher scitific, USA) was prepared and vortexed for 3 s. Solution A was mixed with solution B and incubated at room temperature for 10 min, added to a 6-well plate, and supplemented with 2 ml RPMI-1640 medium. After 8 h, the RPMI-1640 medium was replaced with fresh medium containing 10% FBS. Western blot analysis was used to verify the transfection efficiency of four pairs of sequences, and the most effective pair was selected for virus packaging. Puromycin was used to select the stable cell lines.

2.4 Colony formation test

Cells in logarithmic growth phase were resuspended into single cell suspension with RPMI-1640 medium containing 10% FBS, and counted with a cell counting plate. Each 6-well plate was inoculated with 400 cells, gently shaken and mixed, and then put into a cell incubator for culture. After 2 weeks, the colonies could be observed by the naked eye. The culture medium was discarded. The plates were washed twice with PBS, fixed with the 4% polyformaldehyde for 15 min, and dyed with crystal violet for 10 min. After the plates were dried, photographs were taken and analyzed.

2.5 Real-time cellular analysis proliferation test

The transfected cells were digested and resuspended into single cell suspension with RPMI-1640 containing 10% FBS. The cells were counted by a cell counting plate and the cell concentration was adjusted to 5 × 10⁴ cells/ml. The cell suspension was gently mixed then 5000 cells were implanted into each hole of an E-16 plate. After standing at room temperature for 30 min, the E-16 plate was placed into the RTCA instrument. When the proliferation curve reached the platform period, the test was terminated and data could be derived.

2.6 Immunohistochemistry

The fixed tissue was embedded in paraffin and sliced into 5 μm-thick sections. After deparaffinization and dehydration, the slices underwent antigen retrieval at 95°C for 30 min and were blocked with goat serum for 1 h. The slices were then incubated with primary Abs at 4°C overnight and with HRP-conjugated secondary Abs at 37°C for 45 min. 3,3′-Diaminobenzidine (Jiehao Biotechnology) was used for color development. The slides were examined under the microscope, and representative images were captured. For each slide, five visual fields were randomly selected at 400×. The anti-TRIM37, anti-Ki-67, and anti-MMP-9 Abs were purchased from Abcam. Image-Pro Plus 6.0 (Media Cybernetics) was used to analyze the captured images. Average staining score of five fields was calculated by the software.

2.7 Western blot analysis

Cells were harvested and lysed with RIPA (Beyotime) supplemented with PMSF (Beyotime) and phosphatase inhibitors. The protein concentration was quantified with a BCA kit (Beyotime), and 30 μg protein was loaded onto SDS-PAGE gels for electrophoresis and transferred to PVDF membranes. The membranes were blocked in 5% fat-free milk for 1 h at room temperature and incubated in primary Abs at 4°C overnight. The membranes were then incubated with HRP-conjugated secondary Abs (1:4000) (Cell Signaling Technology) for 1 h at room temperature and detected with Immobilon Western substrate (Millipore). The anti-TRIM37, anti-MMP-9, anti-N-cadherin, anti-Bcl-xl, anti-Bax, anti-Bcl-2, anti-cleaved caspase-3, and anti-cleaved PARP Abs were purchased from Abcam. The anti-TRAF2, anti-P-IKB, anti-IKB, anti-P-IKK, anti-IKK Abs were purchased from Cell Signaling Technology. All primary Abs were monoclonal and applied at a dilution of 1:1000. The relative protein expression levels were normalized to the GAPDH (Abcam) level. All experiments were repeated three times.

2.8 Transwell assay

Cell migration and invasion were detected by Transwell assay. Briefly speaking, in the migration assay, transfected cells were resuspended in 200 μl FBS-free medium and were implanted into the upper chambers (purchased from BD Biosciences) of 24-well plates at a density of 10⁵ cells. For the invasion assay, the upper chambers were embedded with 50 μl Matrigel (BD Biosciences). The lower chambers were loaded with RPMI-1640 with 20% FBS. Forty-eight hours later, the cells were fixed with 4% paraformaldehyde, stained with 1% crystal violet, and rinsed in PBS three times. The migrated or invaded cells were counted under an inverted microscope. Five visual fields were randomly selected for each chamber. All experiments were repeated in triplicate.

2.9 Immunoprecipitation

The transfected cells were lysed with lysis buffer for 20 min and placed into the ultrasonic crusher. The cell lysate was centrifuged at 12,000 g for 30 min at 4°C. Cell lysate (500 μl) and 5 μg Ab of target proteins were added into a new Eppendorf tube. At the same time, nonspecific homologous Ab was set as the control group. The Eppendorf tubes were mixed gently and incubated at 4°C overnight. Protein A and Protein G (5 μl each) were added into tubes and mixed gently for 3 h. After centrifugation, the supernatant was discarded and the precipitate was retained and washed with Wash Buffer. Lysate was resuspended with 20–40 μl loading buffer and heated at 95°C for 5 min. Western blot analysis was undertaken to detect the protein.

2.10 Ubiquitination detection

Tumor necrosis factor-α (10 ng/ml) was added to transfected cells. The TRAF2 plasmid and pCMV-HA-Ub plasmid were cotransfected into cells. After 24 h, proteasome inhibitor MG-132 (20 μmol/L) was added. The protein was collected and immunoprecipitated with anti-TRAF2 Ab. The level of ubiquitination was detected by western blot.

2.11 Statistical analysis

SPSS 16.0 (IBM) was used to analyze the data. The quantitative data were expressed as mean ± SD. Comparisons between multiple groups were undertaken by one-way ANOVA. Comparisons between two groups were undertaken by two-tailed Student’s t-test, and comparisons of two constituent ratios in the classification data was carried out by χ²-test. p < 0.05 was considered statistically significant.

3.1 Expression level of TRIM37 was significantly higher in epithelial ovarian cancer than in normal ovarian tissues

To compare the expression level of TRIM37 between epithelial ovarian cancer tissues and normal ovarian tissues, we collected 60 cases of epithelial ovarian cancer tissues and 30 samples of normal ovarian tissues in our hospital. Immunohistochemistry was used to detect the expression level of TRIM37. We found that the expression level of TRIM37 was significantly higher in epithelial ovarian cancer tissues than in normal ovarian tissues (p < 0.001) (Figure 1).

3.2 High level of TRIM37 correlated with high level of Ki-67 and MMP-9

To investigate whether the level of TRIM37 was correlated with tumor proliferation marker Ki-67 and invasion marker MMP-9, we detected the expression of Ki-67 and MMP-9 simultaneously in continuous ovarian cancer tissues of patients. Data showed that the expression of TRIM37 was positively with the level of Ki-67 (p = 0.002) and MMP-9 (p = 0.000) (Figure 2).

3.3 Overexpression of TRIM37 promoted proliferation of epithelial ovarian cancer cell lines

Five common ovarian cancer cell lines, A2780, OVCAR433, SKOV3, OVCAR3, and OVCAR5, were selected to detect the expression of TRIM37 by western blot analysis. As indicated in Figure 3, TRIM37 expression is lowest in A2780 cells and highest in SKOV3. To verify the biological role of TRIM37 in epithelial ovarian cancer, we constructed a TRIM37-overexpressing A2780 cell line and TRIM37-knockdown epithelial ovarian SKOV3 cancer cell line. Colony formation test (Figure 3D) and RTCA proliferation test (Figure 3E) showed that overexpression of TRIM37 promoted the proliferation of epithelial ovarian cancer cell lines while the knockdown of TRIM37 inhibited that compared with respective controls (p < 0.05) (Figure 3).

3.4 Overexpression of TRIM37 promoted migration and invasion of epithelial ovarian cancer cell lines

Transwell assay was undertaken to clarify the influence of the expression of TRIM37 on the migration and invasion of epithelial ovarian cancer cell lines. Overexpression of TRIM37 obviously stimulated the migration and invasion of ovarian cancer cell lines, whereas the knockdown of TRIM37 inhibited migration and invasion (p < 0.05) (Figure 4A,B). Next, we detected apoptosis- and invasion-associated proteins in TRIM37-overexpressing or TRIM37-knockdown epithelial ovarian cancer cell lines. Results from western blot analysis showed that the expression of MMP-9 and N-cadherin were upregulated after TRIM37 was overexpressed. Additionally, the antiapoptosis proteins Bcl-xl and Bcl-2 were upregulated in TRIM37-overexpression cells while cleaved caspase-3 and cleaved PARP were downregulated (Figure 4C). To sum up, TRIM37 played an initial role in the proliferation and metastasis of epithelial ovarian cancer. In our previous study, we found that metformin inhibited the proliferation, migration, and invasion.^{4, 17} We wondered whether the antitumor effect of metformin might partially depend on the regulation of TRIM37.

3.5 Nuclear factor-κB signaling pathway activated by TRIM37 in epithelial ovarian cancer

It was reported that TRIM37 promoted the progression of lung cancer by activating the NF-κB signaling pathway. In this study, we found that overexpression of TRIM37 stimulated the NF-κB signaling pathway (Figure 5A). Furthermore, PDTC (50 μM), an inhibitor of the NF-κB signaling pathway, reversed the upregulated N-cadherin, MMP-9, Bcl-xl, and Bcl-2 and downregulated cleaved PARP (Figure 5B). In contrast, LPS (100 ng/ml), an activator of the NF-κB signaling pathway, played opposite roles (Figure 5C). We drew a preliminary conclusion that TRIM37 can activate the NF-κB pathway in epithelial ovarian cancer, and promote the malignant biological behavior of epithelial ovarian cancer partly depending on this pathway.

3.6 Metformin inhibited the NF-κB signaling pathway by downregulating TRIM37

To explore whether metformin could regulate TRIM37, western blot analysis was undertaken and we found that metformin (20 mM) downregulated TRIM37 significantly (Figure 6A). Furthermore, results showed that metformin inhibited NF-κB signaling pathway in SKOV3, OVCAR3, and OVCAR5 ovarian cancer cell lines (Figure 6B). Next, metformin was added to TRIM37-overexpressing cells and we found that metformin reversed the NF-κB signaling pathway, MMP-9, and N-cadherin, which were upregulated by TRIM37 (Figure 6C).

3.7 Metformin inhibited ubiquitination of TRAF2 induced by TRIM37 overexpression

It was reported that TRIM37 contains TRAF fragments, which can be combined with the same fragment on homologous or heterogenous protein.¹⁴ An important protein in the NF-κB pathway activated by TNF-α, TRAF2 contains a fragment of TRAF. Immunoprecipitation assay was used to detect the binding of TRIM37 and TRAF2 in epithelial ovarian cancer cell lines. The results showed that TRAF2 was detected in the pull-down proteins of TRIM37-overexpression cells; TRIM37 was also detected in the pull-down proteins of TRAF2-overexpression cells (Figure 7A). Therefore, we concluded that TRIM37 bound to TRAF2 in epithelial ovarian cancer cell lines.

Tumor necrosis factor receptor-associated factor 2 and RIP could be rapidly recruited to form a complex and labeled by ubiquitin when TNF-α stimulates the NF-κB pathway, which is a key step in the activation of NF-κB pathway. Tripartite motif-containing 37 is a member of the ubiquitin ligase family and can bind TRAF2. Therefore, we speculated whether TRIM37 is involved in the ubiquitination of TRAF2. To verify this idea, we cotransfected pCMV-HA-Ub plasmid and TRAF2-overexpression plasmid into TRIM37-overexpression and TRIM37-knockdown epithelial ovarian cancer cell lines, respectively. Tumor necrosis factor-α was added to stimulate the NF-κB pathway and MG132 was added to inhibit protein degradation. Results showed that after overexpression of TRIM37, the ubiquitination level of TRAF2 increased. On the contrary, after interfering with TRIM37, the ubiquitination level of TRAF2 decreased (Figure 7B). Can metformin affect the ubiquitination of TRIM37 on TRAF2? Metformin was added to the pCMV-HA-Ub and TRAF2-overexpression plasmid cotransfection system of TRIM37-overexpression cell lines. We found that metformin significantly inhibited TRAF2 ubiquitination induced by TRIM37 (Figure 7C). Through the above experiments, we concluded that metformin inhibited the ubiquitination degradation of TRAF2 induced by TRIM37, and then inhibited epithelial ovarian cancer.