Human ovarian cancer cell line SKOV3 and its cisplatin-resistant variant SKOV3/DDP were purchased from the cell bank of China Academy of Sciences. Normal human ovarian epithelial cell line IOSE80 is preserved in our laboratory. SKOV3 and SKOV3/DDP cells were cultured in DMEM medium (Gibco) containing 10% fetal bovine serum (FBS, Gibco) and 1% penicillin/streptomycin (P/S, Gibco). IOSE80 cells were cultured in DMEM medium (Gibco) containing 10% FBS and 1% p/s. All cells were cultured in an incubator with 37°C and 5% CO2.

Cisplatin (CDDP): purchased from Sigma-Aldrich Company; β-catenin agonist BML-284: purchased from Tocris Bioscience company; Apatinib: purchased from Jiangsu Hengrui Pharmaceutical Co., Ltd.; SIRT1 siRNA: synthesized by GenePharma Company; Lipofectamine 3000 transfection reagent: purchased from Thermo Fisher Scientific Company; CCK8 kit: purchased from Dojindo Company; Annexin V-FITC/PI Apoptosis Kit: purchased from BD Biosciences; Matrigel matrix adhesive: purchased from Corning company; Glucose uptake test kit: purchased from Abcam Company; Lactic acid detection kit: purchased from Abcam Company; ATP test kit: purchased from Beyotime company; ROS detection kit: purchased from Beyotime company; NADPH/NADP+ detection kit: purchased from Beyotime Company; Western Blotting related antibodies: antibodies such as SIRT1, β-catenin, c-myc, PKM2, HK2, LDHA, GLUT1, VEGFA, Ang-1, IL-8 and GAPDH were purchased from Cell Signaling Technology, Abcam and Santa Cruz Biotechnology Company; QRT-PCR related reagents: RNA extraction kit, reverse transcription kit and SYBR Green qPCR Mix were purchased from Takara Company; Flow cytometry: BD FACSCanto II; Enzyme marker: Bio-Rad iMark; Inverted microscope: Olympus IX71.

In this study, cells were divided into the following groups according to the experimental purpose:

SKOV3 and SKOV3/DDP cells were inoculated into a 6-well plate at a density of 5×105 cells per well, and cultured until the cell density reached 70-80%. SIRT1 siRNA was transfected into cells according to the instructions of Lipofectamine 3000 transfection reagent. After transfection for 6 hours, the fresh medium was replaced, and the follow-up experiment was carried out after 48 hours of continuous culture.

Protein was extracted from cells or tissues by RIPI lysis buffer to obtain total protein samples. SDS-PAGE electrophoresis: the concentration of protein was determined by BCA protein determination method, and electrophoresis was carried out with 10% and 12% polyacrylamide gel (SDS-PAGE), and the loading mass was 40 μ g per well; After electrophoresis at 80 V for 120 min, the protein was transferred to polyvinylidene fluoride (PVDF) membrane at 300 mA and 90 min, and sealed with 5% skim milk powder at room temperature for 1 h;; The PVDF membrane was washed with PBST for 4 times (5 min/time), and the target protein band was cut according to the position of Marker, and put into the incubator. Add the target proteins SIRT1, HXK2, PKM, LDHA, PDHA1), c-mycALDOA, GLUT1, VEGFA, Ang-1, IL-8, β-catenin, TCF1/TCF7, MMP-7, PKM2 and mouse-derived GAPDH respectively, and then wash PVDF membrane 4 with PBST. Accord/ming to the source of antibody species, the goat anti-rabbit or mouse polyclonal antibody (1:10000, China Kangwei Reagent Biotechnology Co., Ltd.) combined with horseradish peroxidase was added, and incubated for 1 h; at room temperature in a shaking table at the speed of 70 ~ 80 rpm. The PVDF film was washed with PBST for 4 times (5 min/time), and the signal of the combination of the secondary antibody and the primary antibody was detected by ECL color development. The photo was taken by chemiluminescence detection system, and the ratio of the optical density of the target strip to GAPDH was detected by Image J. Collect cells or tumor tissues, add RIPA lysate (containing protease inhibitor) and crack on ice for 30 minutes. Centrifuge at 4°C and 12000rpm for 15 minutes, and collect supernatant. The protein concentration was determined by BCA method. Take the same amount of protein for SDS-PAGE electrophoresis, and then transfer the protein to PVDF membrane. Sealed with 5% skim milk for 1 hour, and then added with primary antibody (incubated overnight at 4°C). TBST washes the membrane three times, each time for 10 minutes. Add secondary antibody (incubate at room temperature for 1 hour). TBST washes the membrane three times, each time for 10 minutes. ECL development and Image J software were used to analyze the gray value of protein bands.

Total RNA was extracted from samples (cells, tissues, etc.) with Trizol reagent, and the total RNA was quantified with Qubit fluorometer. The extracted RNA was reverse transcribed into cDNA, and 2g of total RNA was taken and reverse transcribed using a high-capacity cDNA reverse transcription kit (Applied Biosystems, USA). The reaction procedures were 25°C for 5 min, 50°C for 15 min and 85°C for 5 s;Prepare a PCR reaction system, put the reverse transcribed cDNA and primers into a PCR reaction tube, and carry out PCR reaction with ABI-7300 fluorescence quantitative PCR instrument. The reaction conditions are denaturation at 95°C for 10 s, annealing at 60°C for 20 s, extension at 72°C for 15 s, and repeat 40 cycles. The relative expression level of each RNA is determined by QCPR using SYBR GreenMaster reagent (American Applied Biological Systems Company). The gene and primer sequences are shown in Table 1. The Llevels of genes were have been quantified using the 2-ΔΔCT method., and the result was expressed as mean standard deviation.

Cells were inoculated into 96-well plates at a density of 2×103 cells/well, cultured for 24 hours, and then treated with different concentrations of cisplatin (0, 2.5, 5, 10, 20 μM). After 48 hours of continuous culture, 10 μl CCK8 reagent was added to each well and incubated for 2 hours. The absorbance value was detected at 450 nm by enzyme-labeled instrument.

Take logarithmic growth cells, blow them with trypsin, suspend them in 1640 medium containing 10% FBS and double antibody, and incubate them in an incubator for 2 to 3 weeks. Discard the supernatant and wash it carefully with PBS twice. Cells were fixed with 4% paraformaldehyde in 5mL for 15 minutes. Then remove the stationary solution, add an appropriate amount of Giemsa for 10 ~ 30 min, and then slowly rinse the dyeing solution with running water to calculate the clone formation rate.

First, mark the back of the 6-hole plate with a marker pen, compare it with a ruler, and draw a horizontal line evenly, about every 0.5~1cm, and cross the hole. Each hole passes through at least 5 lines, and about 5 X 105 cells are added in the air; The next day, compare the gun head with the ruler, and try to hang down to the horizontal scratches behind. The gun head should be vertical and not inclined; Wash the cells with PBS for three times, remove the scratched cells, add serum-free medium, and put them in an incubator for culture. Take samples and take photos at 0,24 hours.

Take out the matrix adhesive from -20°C and melt it in the refrigerator at 4°C overnight. Then, the matrix glue was diluted with serum-free medium according to a certain proportion and evenly coated on the upper layer of Transwell chamber; Add the cell suspension to the upper layer of Transwell chamber, and pay attention to control the number and volume of cells. Put the Transwell chamber into a 24-well plate, and add the lower culture medium to put the 24-well plate into a cell incubator for culture according to the experimental requirements; After the culture, the Transwell chamber was taken out, washed with PBS, and then cell fixation and staining were carried out. The cell invasion was observed under the microscope, and the cell count was carried out.

Cell samples of each group were collected, and the cells were washed twice with precooled PBS to remove the interference in the culture medium. Then, the cells were resuspended with Annexin V binding buffer, and the cell concentration was adjusted to an appropriate range. Then, according to the proportion of the kit instructions, respectively add Annexin V-FITC and PI dye solution, gently mix well, and incubate at room temperature in the dark for 15–30 minutes. Finally, add an appropriate amount of Annexin V binding buffer to stop the reaction, and use flow cytometry to detect as soon as possible (usually within 1 hour).

According to the instructions of the kit, cells were labeled with fluorescently labeled deoxyglucose analog 2-NBDG, and glucose uptake was evaluated for 30 minutes. Subsequently, the cells were obtained by flow cytometry and the data were analyzed. For lactic acid determination, lactic acid concentration in whole cell lysate was detected by lactic acid determination kit according to the instructions.

According to the instructions of the kit, the ATP level, ROS level and NADPH/NADP+ ratio of the cells were detected respectively.

Matrigel matrix glue was spread in a 96-well plate and incubated at 37°C for 30 minutes. Cells were seeded on Matrigel at a density of 2×104 cells/well. After 24 hours of culture, the tube formation was observed.

BALB/c female nude mice aged 4–6 weeks were selected and purchased from Beijing Weitong Lihua Experimental Animal Technology Co., Ltd. All animal experiments are approved by the Ethics Committee. SKOV3/DDP cells were resuspended with PBS, and the cell concentration was adjusted to 1×108 cells/ml. 100 μl cell suspension was injected subcutaneously into each mouse. On the second day after tumor implantation, mice were randomly divided into the following 7 groups (6 mice in each group):

The other two groups were not treated with cisplatin:

The tumor volume was measured once a day by three experimenters independently or showed signs of ulcer. The tumor size in current work is between 474.37-1579.67 mm³. Then, the mice were killed, the tumor tissue was taken out and weighed. Euthanasia procedures were performed in strict accordance with the guidelines of the Institutional Animal Care and Use Committee (IACUC) and followed the AVMA Guidelines for the Euthanasia of Animals (2020 edition). Fora anesthesia, mice were first anesthetized with 3-5% isoflurane in an induction chamber with oxygen flow rate of 1 L/min, anesthesia was maintained with 1.5-2% isoflurane via nose cone. Depth of anesthesia was confirmed by loss of righting reflex, absence of toe pinch response and stable respiratory rate. Euthanasia was performed by carbon dioxide asphyxiation, briefly, carbon dioxide have been filled at 20-30% chamber volume replacement per minute, followed by cervical dislocation as secondary physical method. Some tumor tissues were used for Western Blotting and qRT-PCR analysis, and some tumor tissues were used for immunofluorescence staining. The blood of mice was collected and the indexes of liver and kidney function were detected. This study is reported in accordance with ARRIVE guidelines, and the animal study was approved by ethical committee of Affiliated Hospital of North Sichuan Medical College hospital.

The tumor tissue was embedded in paraffin and sliced. Dewaxing, antigen repair. Blocked with 5% BSA for 1 hour. Add primary antibodies (SIRT1 and CD31) and incubate overnight at 4°C. Add secondary antibody and incubate at room temperature for 1 hour. DAPI staining. Observe and take photos with a fluorescent microscope.

In this study, all the experiments were repeated biologically for three times (n=3). Each experiment is carried out independently to ensure the reliability and repeatability of the results. In addition, in each experiment, the key experimental steps (such as cell proliferation, migration and invasion experiments) were also repeated to verify the consistency of the experimental results. All experimental data were statistically analyzed by SPSS 22.0 software. The measurement data is expressed as mean standard deviation (SD). T test was used for comparison between the two groups, and one-way analysis of variance (ANOVA) was used for comparison between multiple groups, and the difference was statistically significant (P < 0.05).

In order to explore the role of SIRT1 in ovarian cancer, we first detected the expression level of SIRT1 in ovarian cancer cell lines SKOV3, SKOV3/DDP and normal ovarian epithelial cell line IOSE80. Western Blotting results showed that the protein expression level of SIRT1 in SKOV3 and SKOV3/DDP cells was significantly higher than that in IOSE80 cells (Figure 1A). The results of qRT-PCR also showed that the mRNA expression level of SIRT1 in SKOV3 and SKOV3/DDP cells was significantly higher than that in IOSE80 cells (Figure 1B). In addition, the expression level of SIRT1 in cisplatin-resistant cell line SKOV3/DDP is higher than that in SKOV3 cells, suggesting that SIRT1 may be related to chemotherapy resistance of ovarian cancer.

In order to study the effect of SIRT1 on the biological behavior of ovarian cancer cells, we constructed SKOV3 and SKOV3/DDP cell lines with low expression of SIRT1. Western Blotting results showed that the expression level of SIRT1 protein in si-SIRT1 group was significantly lower than that in si-NC group (Figure 2A), which indicated that SIRT1 low-expression cell line was successfully constructed. The results of CCK8 experiment showed that knocking down SIRT1 could significantly inhibit the proliferation of SKOV3 and SKOV3/DDP cells (Figure 2B). The results of clone formation experiments show that knocking down SIRT1 can significantly reduce the number of clone formation of SKOV3 and SKOV3/DDP cells (Figure 2C). Transwell experiment and scratch experiment show that knocking down SIRT1 can significantly inhibit the invasion and migration ability of SKOV3 and SKOV3/DDP cells (Figures 2D, E). The results of flow cytometry showed that knocking down SIRT1 could significantly promote the apoptosis of SKOV3 and SKOV3/DDP cells (Figure 2F). This suggests that knocking down SIRT1 can inhibit the biological malignant behavior of ovarian cancer cells.

In order to study the effect of SIRT1 on glycolysis and angiogenesis of ovarian cancer cells, we detected the glucose uptake, lactic acid production, ATP level, ROS level and NADPH/NADP+ ratio of SKOV3 and SKOV3/DDP cells after SIRT1 was knocked down. The results showed that knocking down SIRT1 significantly reduced glucose uptake and lactic acid production of SKOV3 and SKOV3/DDP cells (Figures 3A, B), decreased ATP level (Figure 3C), increased ROS level (Figure 3D) and decreased NADPH/NADP+ ratio (Figure 3E) compared with si-NC group. The results of Western Blotting and qRT-PCR showed that knocking down SIRT1 significantly reduced the protein and mRNA expression levels of key glycolytic enzymes (HXK2, PKM, LDHA, PDHA1) and glucose metabolites (ALDOA, GLUT1) in SKOV3 and SKOV3/DDP cells compared with si-NC group (Figures 3F, G). In addition, the experimental results of tube formation showed that knocking down SIRT1 significantly inhibited the tube formation ability of SKOV3 and SKOV3/DDP cells compared with the si-NC group, and knocking down SIRT1 significantly decreased the protein and mRNA expression levels of angiogenic factors (VEGFA, Ang-1, IL-8) in SKOV3 and SKOV3/DDP cells compared with the si-NC group (Figures 4A, B).

SIRT1 regulates glycolysis and angiogenesis of ovarian cancer cells through β-catenin/c-myc/PKM2 pathway.

In order to study whether SIRT1 regulates glycolysis and angiogenesis of ovarian cancer cells through β-catenin/c-myc/PKM2 pathway, we used β-catenin agonist BML-284 to intervene. Western Blotting and PCR results showed that knocking down SIRT1 significantly decreased the protein expression levels of β-catenin, TCF1/TCF7, MMP-7, c-myc and PKM2 in SKOV3 and SKOV3/DDP cells compared with si-NC group. However, BML-284 can reverse the inhibition of SIRT1 on the expression of β-catenin, TCF1/TCF7, MMP-7, c-myc and PKM2 (Figures 5A, B). Further experimental results show that BML-284 can reverse the inhibition of SIRT1 on glucose uptake, lactic acid production, ATP level, ROS level, NADPH/NADP+ ratio, and the expression of key glycolytic enzymes and angiogenic factors (Figures 5C–I, 6A–C). This suggests that SIRT1 regulates glycolysis and angiogenesis of ovarian cancer cells through β-catenin/c-myc/PKM2 pathway.

In order to verify the effect of SIRT1 in vivo, we constructed a nude mouse model of transplanted tumor. In animal experiments, in order to evaluate the in vivo efficacy and potential toxicity of SIRT1 knock-down combined with or without BML-284 and apatinib on cisplatin-resistant ovarian cancer, we first detected the related indexes of liver and kidney function in each group of mice. The results of ELISA (Figure 7) showed that compared with the Si-NC group, the levels of ALT, AST, Cr, Urea and UA in the Si-SIRT1 group decreased significantly, indicating that knocking down SIRT1 alone can inhibit the damage of liver and kidney function in ovarian cancer. After cisplatin (CDDP) treatment, the levels of ALT, AST, Cr, Urea and UA were inhibited to some extent, but the anti-tumor effect of SIRT1 knockdown combined with cisplatin (si-SIRT1+CDDP) was significantly better than that of cisplatin alone (si-NC+CDDP), which indicated that SIRT1 knockdown enhanced the therapeutic effect of cisplatin. Apatinib combined with SIRT1 knockdown and cisplatin (si-SIRT1+APa+CDDP) showed the strongest inhibitory effect on ALT, AST, Cr, Urea and UA levels. On the contrary, activating β-catenin pathway (si-NC+BML-284+CDDP) slightly promoted the release of ALT, AST, Cr, Urea and UA, and activating β -catenin (si-SIRT1+BML-284+CDDP) on the basis of SIRT1 knockdown combined with cisplatin partially reversed SIRT1 knockdown (Si-SIR). This suggests that SIRT1 knockdown combined with apatinib and cisplatin does not cause significant hepatorenal toxicity in the treatment of tumors, suggesting that the combined treatment scheme is safe.

In order to verify the effect of SIRT1 on β-catenin/c-myc/PKM2 pathway, we used WB and PCR to detect related proteins. The experimental results showed that compared with si-NC, si-SIRT1 significantly inhibited the growth of ovarian cancer cells in vivo (Figure 8A). The results of immunofluorescence staining showed that knocking down SIRT1 significantly reduced the expression of SIRT1 and CD31 in tumor tissues compared with si-NC group (Figure 8B). Western Blotting and qRT-PCR results showed that compared with si-NC, si-SIRT1 reduced the expression levels of angiogenesis-related proteins (VEGFA, Ang-1, IL-8) and glycolytic key proteins (HXK2, PKM2, LDHA, etc.) in tumor tissues (Figures 8C, D), while inhibiting β-catenin/c-myc. Cisplatin (CDDP) treatment inhibited tumor growth to a certain extent, but the anti-tumor effect of SIRT1 knock-down combined with cisplatin (si-SIRT1+CDDP) was significantly better than that of cisplatin alone, indicating that SIRT1 knock-down enhanced the therapeutic effect of cisplatin. Furthermore, Apatinib combined with SIRT1 knockdown and cisplatin (si-SIRT1+APa+CDDP) showed the strongest tumor growth inhibition effect and the lowest levels of angiogenesis and glycolysis. On the contrary, activating β-catenin pathway (si-NC+BML-284) slightly promoted tumor growth, and activating β -catenin (Si-SIRT1+BML-284+CDDP) on the basis of SIRT1 knockdown combined with cisplatin partially reversed the antitumor effect of Sirt1 knockdown. This suggests that SIRT1 knockdown can inhibit tumor angiogenesis and glycolysis by inhibiting β-catenin/c-myc/PKM2 pathway, thus inhibiting the growth of ovarian cancer cells in vivo and enhancing the anti-tumor effect of cisplatin.