Cell Culture
Human gastric cancer cell line HGC-27 (CL-0107, Procell, Wuhan, China) was cultured in HGC-27 cell specific medium (CM-0107, 125mL, Procell, Wuhan, China), and human gastric cancer cell line MGC-803 (iCell-h141, icellbioscience, Shanghai, China) was cultured in MGC-803 cell specific medium (iCell-h141-011b, 500mL, icellbiosciencel, Shanghai, China). Both cell lines were cultured in 5% CO2 and 37℃ incubator at constant temperature.
Sirna Transfection
The siRNA lyophilized powder was prepared into a 20µM stock with RNase-free Water, and 10µL of 20µM siRNA-OCT4 stock was diluted with 120µL of 1× riboFect™ CP Buffer (C10511-05, Ruibo, Guangzhou, China), and the siRNA-OCT4 was diluted by gently mixing. Transfection complexes were prepared by adding 12µL riboFect™ CP Reagent, and incubating at room temperature for 15min. The riboFect™ CP mixture was added to 1858µL of cell culture medium and gently mixed. The prepared transfection reagents were added to the corresponding 6-well plates at 2mL/ well and cultured in an incubator at 37℃.
γ-h2ax Immunofluorescence Assay
DNA damage was detected using a DNA Damage Assay Kit by γ-H2AX Immunofluorescence (C2035S, Beyotime, Shanghai, China). Cell samples in 6-well plates were added to 1ml of fixative for 5–15 min. The fixative solution was removed by suction and washed three times. 1ml of immunostaining blocking solution was added and blocked for 10 to 20 min at room temperature. The immunostaining blocking solution was removed by aspiration, and 50µl of rabbit γ-H2AX mab (C2035S-4, Beyotime, Shanghai, China) was added and incubated overnight at 4ºC. After washing, 1ml of anti-rabbit 488 was added and incubated for 1 h at room temperature. After washing, DAPI staining solution was added and stained at room temperature for about 5 minutes. DNA damage was observed by fluorescence microscopy after nuclear staining solution was removed and washed three times.
Western Blot
Total cellular protein was obtained by lysing GC cells in RIPA buffer (Pierce; servicebio, Wuhan, China). Protein concentrations were detected using a BCA Protein Assay Kit (Beyotime, Shanghai, China). Samples containing 30 µg of protein were separated by 10% SDS-PAGE, and the protein bands were transferred onto PVDF membranes (Invitrogen; Sigmaaldrich, Shanghai, China). The membranes were then blocked with 5% skim milk and subsequently incubated overnight with primary antibodies against OCT4 and DNMT1 (ab200834, ab188453, dilution: 1:2000; Abcam, Shanghai, China), 4°C overnight. After washing the membrane 3 times with Phosphate-Buffered Saline, horseradish peroxidase (HRP)-conjugated secondary antibody (ab6721, dilution: 1:5000; Abcam, Shanghai, China) was added and incubated with the membrane for 60 min at room temperature. After washing, the chemiluminescence solution was added, and it was incubated in the dark for 2–3 min. Then, the membrane was exposed and developed.
Cell Cycle Was Detected By Cck8 Assay
HGC-27 and MGC-803 cells in the logarithmic growth phase were washed with PBS and collected after trypsin digestion. The cells were centrifuged at 250g for 5min. The supernatant was removed, and an appropriate amount of medium was added to make the single cell suspension. The cell density was adjusted to 4×104/mL, and 100µL/ well were seeded in 96-well plates (the edge Wells were filled with sterile PBS) and incubated at 37 ℃ with 5%CO2. Group administration: After cell adhesion, set group: Experiment 1: Control + CDDP induction, siRNA-NC + CDDP induction, siRNA-OCT4 + CDDP induction; Experiment 2: siRNA-NC + OV-NC + CDDP induction, siRNA-NC + OV-DNMT1 + CDDP induction, siRNA-OCT4 + OV-NC + CDDP induction, siRNA-OCT4 + OV-DNMT1 + CDDP induction. The CDDP induced concentrations were 0µM, 2.5µM, 5µM, 10µM, 20µM, respectively, four replicates in each group. After 12h of cell transfection, the supernatant was discarded by aspiration. The CCK8 reagent (BS350A, Biosharp, Anhui, China) was diluted 1:10 in serum-free medium, and the diluted CCK8 working solution 110µL/ well was added. The plates were shaken gently for several times, and then incubated at 37℃ and 5%CO2 for 2 hours. The absorbance values of each well were measured at 450nm wavelength using a microplate reader.
Colony-forming Unit Assays
The HGC-27 and MGC-803 ingle cell suspension were seeded in 35-mm Petri dishes and incubated at 37℃ with 5%CO2. Experimental group 1: siRNA-NC group, siRNA-NC + CDDP induction group, siRNA-OCT4 group, siRNA-OCT4 + CDDP induction group; Experiment 2: siRNA-NC + OV-NC + CDDP induction group, siRNA-NC + OV-DNMT1 + CDDP induction group, siRNA-OCT4 + OV-NC + CDDP induction group, and siRNA-OCT4 + OV-DNMT1 + CDDP induction group. After the cells adhered to the wall, drugs with corresponding concentration gradients were added. After 12h, the cells were washed with PBS, digested with trypsin, centrifuged, and cultured as single cell suspension. After dilution, the cells were seeded in 6-well plates containing 2mL preheated culture medium at 37℃ at a gradient density of 800 cells per well, and incubated at 37℃ with 5%CO2. Cultures were terminated when a single cell grew to more than 50 cell clones under the microscope and macroscopic clones appeared in 6-well plates. The supernatant was discarded and washed twice with PBS. After 30 min of ethanol fixation, the cells were washed once with PBS, the supernatant was removed by aspiration, stained with 0.1% crystal violet for 15min, washed with PBS, dried in air, and collected and photographed for image acquisition. Clone count: The well plates were inverted and clones were directly counted by naked eye.
Cell Cycle Was Detected By Flow Cytometry
HGC-27 and MGC-803 cells were cultured and adhered to the wall, and the experimental group was set as above, with 3 replicates in each group, added with corresponding concentration gradient of drugs, and the supernatant was sucked. The supernatant was discarded after washing with PBS. After the cytoplasm retracted, the cells were no longer connected to each other. The digestion was terminated by adding the collected cell supernatant, and the supernatant was centrifuged at 250g for 5min. The supernatant was absorbed and discarded, and the cell precipitation was obtained by adding an appropriate amount of PBS to wash and centrifuge. 1mL of 75% precooled ethanol was added to the cell precipitate and the cells were resuspended. After fixation overnight at 4 ℃, the cells were centrifuged at 250g for 5min, the supernatant was aspirated and discarded, and the cell precipitate was collected after washing with PBS. The cells were resuspended in 500µL PI/RNase A staining solution. After 30min at room temperature and dark, the cells were detected and analyzed by flow cytometry analyzer (cytoflex, Beckman, America).
Cell Apoptosis Was Detected By Flow Cytometry
Annexin V-APC/PI double staining apoptosis detection kit was used to detect cell apoptosis (KGA1030, 100T, Kaiji, Jiangsu, China). HGC-27 and MGC-803 cells were cultured and adherent, and grouped as above. Cells obtained from the above experiments were precipitated. After the cells were resuspended in 500µL Binding Buffer, 5µL Annexin V was added to blow gently, and 5µL PI was added to mix. The reaction time was 15min at room temperature in the dark, and the staining was performed.
Statistical analysis
All data were analyzed using SPSS 26 (IBM, USA) statistical analysis software, and shown as mean ± standard deviation (SD). A two-tailed student's t-test, one-way or two-way analysis of variance (ANOVA) was applied to analyze the significant differences between groups, P < 0.05 was considered as a statistically significant difference.
Effect Of Cisplatin On Dna Damage And Oct4 Expression In Gastric Cancer Cells
In order to determine the difference in OCT4 expression between GC cells with CDDP induced, in the present study, the expression levels of OCT4 in HGC-27 and MGC-803 cells were detected by western bolt (Fig. 1A). Compared with the control group (0µm), the expression of OCT4 in the cells induced by CDDP was significantly increased, with statistically significant differences, and 20µm CDDP induced was the best. Then the γH2AX immunofluorescence assay was used to detect DNA damage (Fig. 1B). After CDDP treatment, the cells were observed under fluorescence microscope, CDDP induced cells to exhibit Green Fluorescent Protein (GFP), proving that γH2AX fluorescence was enhanced after CDDP induction, indicating that CDDP caused DNA damage. These results reveal that OCT4 plays a certain role in the function of cisplatin sensitivity of GC cells.
Figure 1. Effect of cisplatin on DNA damage and OCT4 expression in gastric cancer cells (20x). (A) Western blot analysis of OCT4 expression in HGC-27 and MGC-803 cells; a: Control; b: 2.5µm CDDP; c: 5µm CDDP; d: 10µm CDDP; e: 20µm CDDP. (B) DNA damage in HGC-27 cells and MGC-803 were detected by γH2AX immunofluorescence assay. Data were expressed as mean ± standard deviation. Compared with Control (0µm CDDP) group, **P < 0.01, *P < 0.05.
Effect Of Oct4 Expression On Cisplatin Resistance Of Gastric Cancer Cells
To investigate the biological function of OCT4 in HGC-27 and MGC-803 cells, a transient transfection plasmid si-RNA and its negative control si-NC were designed by GenePharma (Shanghai, China). si-OCT4 or si-NC was transfected into HGC-27 and MGC-803 cells, respectively. After resistance screening, the expression level of OCT4 in HGC-27 and MGC-803 cells was detected by western blot to determine the difference of OCT4 expression in CDDP induced GC cells (Fig. 2A). Compared with control and siRNA-NC, the expression of OCT4 in siRNA-OCT4 group was significantly decreased (P < 0.01). To explore the effect of OCT4 on the IC50 of HGC-27 and MGC-803 cell proliferation induced by CDDP, CCK8 was used to detect the cell viability induced by different concentrations of CDDP (Fig. 2B). After interfering with OCT4, the proliferation ability of HGC-27 and MGC-803 cells was significantly decreased. The IC50 of cell viability in HGC-27 decreased from 21.09 mM to 15.178 mM, and the IC50 in MGC-803 decreased from 24.388 mM to 17.109 mM (P < 0.01). This indicated that the cell model with high OCT4 expression was successfully established.
Figure 2. Effect of OCT4 expression on cisplatin resistance of gastric cancer cells. (A) Western blot analysis of OCT4 expression in HGC-27 and MGC-803 cells. a: Control; b: siRNA-NC; c: siRNA-OCT4. (B) Cell viability was detected by CCK8 assay. Data were expressed as mean ± standard deviation. Compared with Control (0µM CDDP) group, **P < 0.01, *P < 0.05.
Effects Of Oct4 Expression On Proliferation, Apoptosis, And Dna Damage Of Gastric Cancer Cells
To investigate the effect of OCT4 on the proliferation of GC cells lines HGC-27 and MGC-803 after CDDP induction, si-OCT4 and si-NC were transfected into HGC-27 and MGC-803 cells, respectively. After CDDP induction, resistance screening was performed, and DNA damage was detected by γH2AX immunofluorescence assay (Fig. 3A). After transfection, CDDP-induced DNA damage in gastric cancer cells increased with a statistically significant difference (P < 0.05). Meanwhile, the results of cell cloning assay showed that siRNA-OCT4 group reduced the colony formation rate of HGC-27 and MGC-803 cells after CDDP induction (Fig. 3B) (P < 0.01), indicating that OCT4 could promote the proliferation of gastric cancer cells, and cisplatin could effectively inhibit the proliferation of gastric cancer cell lines HGC-27 and MGC-803. To confirm the effect of interfering OCT4 on CDDP resistance of GC cells, cell cycle analysis was performed by flow cytometry (Fig. 3C). The results showed that in siRNA-OCT4 + CDDP group of HGC-27 cells G1 phase (31.58 ± 0.59%) was higher than that of the siRNA-OCT4 group (13.88 ± 0.11%) (P < 0.01), G2 phase (30.79 ± 0.04%) was lower than that of the siRNA-OCT4 group (32.18 ± 0.41%) (P < 0.01), and S phase (37.63 ± 0.57%) was higher than that of the siRNA-OCT4 group (53.93 ± 0.36%) (P < 0.01); and in siRNA-OCT4 + CDDP group of MGC-803 cells G1 phase(60.39 ± 0.18%) was higher than that of the siRNA-OCT4 group (31.39 ± 0.52%) (P < 0.01), G2 phase(12.29 ± 0.73%) was lower than that of the siRNA-OCT4 group (22.38 ± 0.06%) (P < 0.01), and S phase (27.32 ± 0.70%) was lower than that of the siRNA-OCT4 group(46.22 ± 0.50%) (P < 0.01). The G2 and S phase of siRNA-OCT4 group cells was decreased after CDDP induction in both cell lines (P < 0.01). Annexin V/PI double staining flow cytometry was used to analyze the effect of OCT4 interference on apoptosis of MGC-803 and HGC-27 cells (Fig. 3D). The apoptosis rate in siRNA-OCT4 + CDDP group of HGC-27 cells (37.68 ± 0.44%) was higher than that of the siRNA-NC group (20.30 ± 0.76%) (P < 0.01). And siRNA-OCT4 + CDDP group of MGC-27 cells apoptosis rate (29.30 ± 0.41%) is higher than siRNA-NC group (22.18 ± 1.52%).
Figure 3. Effects of OCT4 expression on proliferation, apoptosis, and DNA damage of gastric cancer cells. (A) DNA damage in HGC-27 cells and MGC-803 were detected by γH2AX immunofluorescence assay. (B) Cell cloning assay was used to detect cell proliferation. (C) Flow cytometry was used to detect cell cycle. (D) Cell apoptosis was detected by Annexin V/PI. Data were expressed as mean ± standard deviation. Compared with siRNA-NC, **P < 0.01; *P < 0.05. Compared with siRNA-NC + CDDP, ++P < 0.01; +P < 0.05. Compared with siRNA-OCT4, ##P < 0.01; #P < 0.05.
Regulation Of Dnmt1 Expression By Oct4 During Cisplatin Induction In Gastric Cancer Cells
The relationship between DNMT1 expression and the development of drug resistance in gastric cancer cells has not been studied. Here, we first describe the relative expression of DNMT1 in CDDP-resistant gastric cancer cells. WB was used to detect the effect of CDDP on the expression of DNMT1 in HGC-27 and MGC-803 cells. Compared with the control group, the expression level of DNMT1 gradually increased after 5µM (Fig. 4A) (P < 0.05). Then OCT4 was silenced to observe the expression level of DNMT1 in HGC-27 and MGC-803 cells, the expression of DNMT1 was decreased in siRNA-OCT4 group (Fig. 4B) (P < 0.05).
Figure 4. Regulation of DNMT1 expression by OCT4 during cisplatin induction in gastric cancer cells. (A) Western blot was used to detect the effect of cisplatin on the expression of DNMT1 in HGC-27 and MGC-803 cells. a: Control; b: 2.5µm CDDP; c: 5µm CDDP; d: 10µm CDDP; e: 20µm CDDP. (B) Effect of silencing OCT4 on DNMT1 expression in HGC-27 and MGC-803 cells. f: Control; g: siRNA-NC; h: siRNA-OCT4. Data were expressed as mean ± standard deviation. Compared with control (0µm CDDP), **P < 0.01; *P < 0.05.
Overexpression of DNMT1 reversed the ameliorative effect of OCT4 silencing on drug resistance of gastric cancer cells
In order to determine the effects of OCT4 up-regulation of DNMT1 on drug resistance of gastric cancer cells. As shown in Fig. 5A and 5B, OCT4 and DNMT1 expression was significantly lower in the siRNA-OCT4 of HGC-27 and MGC-803 cells than that control and siRNA-NC (P < 0.01). In order to explore the effect of OCT4 on the IC50 of HGC-27 and MGC-803 cells proliferation after CDDP induction, CCK8 was used to detect the cell viability after CDDP induction with different concentrations (Fig. 5C), as shown in Fig. 1B. After OCT4 disturb, the cell proliferation of HGC-27 and MGC-803 cells decreased significantly. After OCT4 disturb, the IC50 of cell proliferation in HGC-27 decreased from 21.09 mM to 15.178 mM, and the IC50 of cell proliferation in MGC-803 decreased from 24.388 mM to 17.109 mM. These results reveal that OCT4 plays a certain role in the function of cisplatin sensitivity of GC cell.
Overexpression of DNMT1 reversed the effects of OCT4 silencing on proliferation, apoptosis and DNA damage of GC cells
To investigate the effect of OCT4 upregulating DNMT1 on the proliferation of GC cells lines HGC-27 and MGC-803 after CDDP induction, si-OCT4 and si-NC were stably transfected into HGC-27 and MGC-803 cells, respectively. After CDDP induction, resistance screening was performed, and DNA damage was detected by γH2AX immunofluorescence assay (Fig. 6A). Compared with the siRNA-NC + OV-DNMT1 + CDDP group, the siRNA-OCT4 + OV-DNMT1 + CDDP group had increased DNA damage (P < 0.01). Meanwhile, the results of cell cloning assay showed that siRNA-OCT4 + OV-DNMT1 + CDDP group reduced the colony formation rate of HGC-27 and MGC-803 cells (Fig. 6B, P < 0.05), indicating that OCT4 could promote the proliferation of gastric cancer cells, and cisplatin could effectively inhibit the proliferation of gastric cancer cell lines HGC-27 and MGC-803. To confirm the effect of OCT4 upregulating DNMT1 on CDDP resistance of GC cells, cell cycle analysis was performed by flow cytometry (Fig. 6C). The results showed that in siRNA-OCT4 + OV-DNMT1 + CDDP group of HGC-27 cells G1 phase (17.44 ± 0.07%) was lower than that of the siRNA-OCT4 + OV-NC + CDDP group (31.92 ± 0.53%) (P < 0.01), G2 phase(33.59 ± 1.00%) was higher than that of the siRNA-OCT4 + OV-NC + CDDP group (31.66 ± 0.70%) (P < 0.05), and S phase (48.91 ± 1.07%) was higher than that of the siRNA-OCT4 + OV-NC + CDDP group(36.42 ± 1.20%) (P < 0.01); and in siRNA-OCT4 + OV-DNMT1 + CDDP group of MGC-803 cells G1 phase(41.18 ± 0.33%) was lower than that of the siRNA-OCT4 + OV-NC + CDDP group (59.27 ± 0.48%) (P < 0.01), G2 phase (19.37 ± 0.13%) was higher than that of the siRNA-OCT4 + OV-NC + CDDP group (13.58 ± 0.10%) (P < 0.01), and S phase (39.44 ± 0.44%) was higher than that of the siRNA-OCT4 + OV-NC + CDDP group (27.15 ± 0.40%) (P < 0.01). Annexin V/PI double staining flow cytometry was used to analyze the effect of OCT4 upregulating DNMT1 on apoptosis of MGC-803 and HGC-27 cells (Fig. 6D). The apoptosis rate in siRNA-OCT4 + OV-DNMT1 + CDDP group of HGC-27 cells apoptosis rate (38.63 ± 0.79%) is lower than siRNA-OCT4 + OV-NC + CDDP group (46.74 ± 0.81%) (P < 0.01). And siRNA-OCT4 + OV-DNMT1 + CDDP group of MGC-803 cells (30.64 ± 1.28%) was lower than that of the siRNA-OCT4 + OV-NC + CDDP group (32.91 ± 1.26%) (P < 0.05).