Cell culture and CRISPR/Cas9 gene editing
The human ovarian cancer cell lines A2780, HO-8910, ES-2, SKOV3, and OVCAR-3, as well as the human embryonic kidney cell line 293T, were acquired from the American Type Culture Collection (ATCC, Manassas, VA, USA). The benign human ovarian epithelial cell line IOSE was generously supplied by Hengyu Fan from Zhejiang University [27]. Cells were grown in Dulbecco's modified Eagle's medium (Gibco | Thermo Fisher Scientific, Waltham, MA, USA) supplemented with 10% fetal bovine serum (Gibco | Thermo Fisher Scientific) and 1% penicillin-streptomycin (Gibco | Thermo Fisher Scientific). Cells were kept at a temperature of 37°C in a humidified environment with 95% air and 5% CO2, which are the standard culture conditions. PIF1-deficient cells were generated through the application of CRISPR/Cas9 technology. Lentivirus-mediated transfection was used to introduce plasmids into ES-2 and OVCAR-3 cells. Following 24 h of transfection, cells were treated with a concentration of 2 µg/mL puromycin for 3 days. Western blot analysis was employed to validate the partial knockout of the PIF1 gene and to assess PIF1 protein expression levels. Supplementary Table S1 provides the sequences of PIF1 sgRNAs. CRISPR/Cas9 plasmid was kindly provided by Kun-Liang Guan, West Lake University [28].
Cell proliferation and colony formation
For the examination of the cell growth curve, cells were placed in 6-well plates (Corning, New York, NY, USA) at a density of 1 × 105 cells per well. Cell counts were performed in triplicate for each well at 24, 48, and 72 h after seeding.
In the colony formation experiments, 1.5 mL of bottom agarose, consisting of 0.5% agarose (Sigma-Aldrich, St. Louis, MO, USA) in standard culture medium, was used to coat each well of a 6-well plate. Around 2,500 cells were mixed with 1.5 mL of top agarose, which included 0.35% agarose in a regular growth medium. This mixture was then applied as a layer onto each well. For cellular nourishment, a volume of 2 mL of cell culture media was applied to the topmost layer twice a week. The cells were incubated for 21 days under conventional culture conditions. After this period, they were stained with 0.5% crystal violet (Sigma-Aldrich) to quantify the colonies. There were six replicates in each experimental group.
Western blotting
Whole-cell lysates were generated with western blot and IP lysis buffer (Beyotime Biotechnology, Shanghai, China), and the protein content was determined using the bicinchoninic acid assay kit (Beyotime Biotechnology). Proteins were subjected to denaturation at a temperature of 95°C for 5 min. Subsequently, protein samples of 20-µg were isolated using SDS-PAGE. The gel was then transferred onto PVDF membranes (Merck | Millipore, Burlington, MA, USA). Membranes were obstructed with 5% milk at room temperature for 1 h, treated with primary antibodies (Table S2) against the target protein overnight at 4°C, and then exposed to horseradish peroxidase-conjugated secondary antibodies (Table S2) at room temperature for 1 h. Gels were visualized using an enhanced chemiluminescence detection kit (Merck | Millipore). Band intensity was quantified using an Imager 680 (Amersham | GE Healthcare, Chicago, IL, USA) and analyzed with ImageJ software (National Institutes of Health, Bethesda, MD, USA).
Immunohistochemistry
The tumor tissues removed from the nude mice were preserved overnight in a solution of 4% paraformaldehyde. After fixing, the tissues were dehydrated with ethanol at increasing concentrations. Then, xylene was used to replace the ethanol, and finally, the tissues were embedded in paraffin. The tissue blocks fixed in paraffin were cut into sections with a thickness of 5 µm. The deparaffinized sections were treated in 0.3% hydrogen peroxide (H2O2) for 10 min to suppress endogenous peroxidase activity. The process of antigen retrieval was performed using a 10 mM sodium citrate buffer with a pH of 6.0 for 15 min. The sections were subsequently kept at a temperature of 4°C overnight and exposed to primary antibodies targeting PIF1, p-H2AX, BrdU, and RAD51 (Table S2). Subsequently, the sections were treated with biotinylated and peroxidase-conjugated secondary antibodies (1:400; Cell Signaling Technology, Danvers, MA, USA) for 30 min. Ultimately, the sections were stained again using a Vectastain ABC kit and a 3,3'-diaminobenzidine peroxidase substrate kit (Vector Laboratories, Burlingame, CA, USA).
Ovarian tissue microarray analysis and tumor tissue biopsy
The tissue microarrays containing paraffin-embedded human ovarian cancer tissue samples were acquired from Fanpu Biotech (Guilin, China). The data on tumor type, staging, and grading are presented in Table 1. Immunohistochemical analysis was conducted on the ovarian tissue chips to assess PIF1 expression. The protein expression levels were determined by multiplying the proportion of cells that tested positive by the intensity of immunostaining. The allocation scores for percentage were as follows: 0 for non-positive cells, 1 for 1–25%, 2 for 26–50%, 3 for 51–75%, and 4 for 76–100%. Staining intensity was scored as: 0 for negative (-), 1 for weak (+), 2 for moderate (++), and 3 for strong (+++) (Fig. 1A). The final expression score was categorized as no expression (0), weak expression (1–3), moderate expression (4–6), and high expression (8–12) (Table 1). In addition, fresh ovarian tumor tissues (n = 6) and adjacent non-tumor tissues (n = 2) were obtained by biopsy from patients at the First Affiliated Hospital of Jiaxing University (Jiaxing, Zhejiang, China). All participants or their legal guardians provided informed consent, and the study protocol was approved by the institutional review board of the First Hospital of Jiaxing City (LS2020-148). The study adhered to the principles of the Declaration of Helsinki.
Table 1
Demographic and clinical characteristics of the patients with ovarian cancer (n = 99).
Parameter | | Mean (standard deviation) (range) |
Age, years | | 49.70 (13.00) (18–82) |
| | Patients, n (%) |
Pathological type | Normal Benign tumor Serous cystadenocarcinoma Endometrial Mucinous Clear cell | 2 (2.0%) 3 (3.0%) 48 (48.5%) 30 (30.3%) 15 (15.2%) 1 (1.0%) |
Pathological grading | Ⅰ Ⅰ-Ⅱ Ⅱ Ⅱ-Ⅲ Ⅲ | 19 (20.4%) 9 (9.7%) 19 (20.4%) 7 (7.5%) 39 (41.9%) |
T stage | T1 T2 T3 | 46 (48.9%) 23 (24.5%) 25 (26.6%) |
N stage | N0 N1 | 88 (93.6%) 6 (6.4%) |
M stage | M0 M1 | 72 (76.6%) 22 (23.4%) |
Ovarian cancer PIF1 IHC score | Negative (0) + (1–3) ++ (4–6) +++ (8–12) | 1 (1.0%) 15 (16.0%) 44 (46.8%) 34 (36.2%) |
Quantitative real-time polymerase chain reaction (qRT-PCR)
The TRIzol reagent (Invitrogen | Thermo Fisher Scientific) was used to extract total RNA from grown cells. The RNA that was obtained was converted into complementary DNA (cDNA) using the PrimeScript RT Reagent Kit (Takara Bio, Shiga, Japan). The TB Green Master Mix reagent kit (Takara Bio) was used for real-time PCR analysis using the Realplex2 PCR equipment (Eppendorf, Hamburg, Germany). The mRNA levels of each gene were standardized by comparing them to the levels of the housekeeping gene GAPDH. The experiment was conducted three times. The primer sequences can be found in Table S3.
RNA interference
GenePharma Co. (Suzhou, China) designed and synthesized siRNA oligonucleotides targeting the RAD51 gene (Table S4). A negative control siRNA was also provided by GenePharma Co. Ovarian cancer cells were placed into 6-well plates with a concentration of 5 × 105 cells per well. Following the manufacturer's instructions, siRNA sequences were transfected into the cells using Lipofectamine RNAiMAX reagent (Invitrogen). After 48 h of transfection (final siRNA concentration 100 nM), the cells were harvested, and the interference efficiency was analyzed by qRT-PCR or western blot.
Mice and xenograft models
Female BALB/c nude mice, free from specific pathogens, were acquired from Jiangsu Jicui Yaokang Biotechnology Co. (Nanjing, China). The mice were 6–8-weeks-old and were kept in facilities that met the standards for specific pathogen-free conditions for the whole duration of the study. The animals were cared for and used in accordance with institutional protocols and the National Institutes of Health Guide for the Care and Use of Laboratory Animals (8th edition) [29]. The study received ethical approval from the Medical College Animal Ethics Committee of Jiaxing University (Registration No. JUMC2020-069). Mice were anesthetized with diethyl ether and wild-type and PIF1-deleted cells (5 × 106 cells in phosphate buffered saline (PBS)) were subcutaneously injected into both flanks of the mice, with each group comprising 3–4 mice. Tumor volume was measured every 2 days using calipers, calculated with the formula: volume (mm³) = (width)² × height × 0.523. Mice were terminated by cervical dislocation when the diameter of the tumor exceeded 15 mm, which is the human endpoint. Tumor tissues were subsequently gathered for western blotting and immunohistochemistry investigations.
Flow cytometry
In order to perform cell cycle analysis, a total of 1 × 106 cells were gathered and treated with 70% ethanol for 24 h. Subsequently, the cells were separated by centrifugation and underwent two rounds of washing with PBS. Afterward, the cells were placed in 500 µL of PI/Rnase Staining Buffer (BD Biosciences, Franklin Lakes, NJ, USA) and incubated at 37°C in the absence of light for 30 min. They were then analyzed using flow cytometry (model flow cytometer; BD Biosciences) to quantify the cells in each phase. Data analysis was performed using ModFit software (Verity Software House, Topsham, ME, USA) with each experiment conducted in triplicate.
Detection of senescence-associated β-galactosidase activity
To analyze cell senescence, a total of 1 × 105 control cells and PIF1 partial knockout cells were collected and grown overnight in 12-wells plates. Following the washing of cells with 1 × PBS, they were then treated with 1 × fixation solution at room temperature for 15 min. This was subsequently followed by another round of washing with 1 × PBS. The process of senescence staining was performed using the senescence reagent (β-Galactosidase Staining Kit; Beyotime Biotechnology) in accordance with the instructions provided by the manufacturer. Following the staining process, the cells were left overnight to incubate at a temperature of 37°C. Subsequently, they were rinsed with a solution of 1 × PBS. The images were acquired using an inverted microscope (model CKX53, Olympus), with a minimum of three randomly selected fields. The quantity of senescent cells stained with blue dye in these regions was determined as a proportion of the overall cell count.
Immunofluorescence and co-localization
For the immunofluorescence analysis, a total of 5 × 104 grown cells were placed in 24-well plates. The cells were then rinsed with PBS and subsequently treated with 4% paraformaldehyde for 30 min to ensure fixation. Subsequently, the cells were obstructed with a 5% solution of bovine serum albumin for 1 h. Primary antibodies against p-H2AX, Ki-67, and RAD51 (Table S2) were incubated with the cells overnight at 4°C. After washing, the cells were incubated with Alexa488- or Alexa594-conjugated secondary antibodies (Abcam, Cambridge, UK) (Table S2) and stained with DAPI (Beijing Solarbio Science & Technology Co., Beijing, China) to visualize the cell nuclei. The laser scanning confocal microscope (model FV3000; Olympus) was used to obtain digital images. A minimum of three images were acquired for each group.
For immunofluorescence co-localization analysis, plasmids carrying enhanced green fluorescent protein (EGFP)-tagged PIF1 (Fenghui Biotechnology, Hunan, China) and EGFP empty vector (Fenghui Biotechnology) were separately transfected into cells. Following 36 h of incubation, the cells were fixed and stained with DAPI as described above. Digital images were collected using a laser scanning confocal microscope (model FV3000; Olympus).
Plasmids and overexpression of RAD51 in PIF1 partial knockout cells
The human PIF1 cDNA expression construct (EGFP-PIF1) was purchased from Fenghui Biotechnology (Hunan, China). The Flag empty vector was provided by Dr. Heng-Yu Fan [30]. The Flag-PIF1 construct was created by inserting the human PIF1 cDNA (cloned by Fenghui Biotechnology) into the Flag empty vector, with sequencing validation performed by Sangon Biotech (Shanghai, China). The HA empty vector was acquired from Miaoling Biotechnology (Wuhan, China). Human RAD51 cDNA, cloned from the ovarian cancer cell line ES-2 using specific primers (Table S5), was then inserted into the HA empty vector. This construct was also validated by sequencing at Sangon Biotech (Shanghai, China).
To construct the RAD51 overexpression vector, the pQCXIH retroviral vector containing the RAD51 cDNA sequence was utilized. pQCXIH plasmid was kindly provided by Kun-Liang Guan, West Lake University [31]. 293T cells were transfected with either the pQCXIH empty vector or pQCXIH-RAD51, together with Retro VSVG and Retro GPE constructs, using the PolyJet™ DNA In Vitro Transfection Reagent (Signagen Laboratories, Frederick, MD, USA), following the manufacturer's instructions. The transfection was carried out in a medium devoid of serum. After 48 h, the retroviral supernatant was collected, mixed with 5 µg/mL Polybrene (GeneChem Co., Shanghai, China), passed through a sterile 0.45-µm filter (Merck | Millipore), and employed to infect PIF1 partially deficient ES-2 cells. Following 48 h of infection, the specific cells were chosen using a concentration of 200 µg/mL puromycin in a completely supplied medium for 3 days. Stable RAD51 expression in the cells was confirmed via western blot analysis.
Co-immunoprecipitation (Co-IP)
After transfecting the plasmids into 293T cells for 36 h, the cells were lysed and centrifuged using a western blot and IP lysis buffer (Beyotime Biotechnology). The respective antibodies and protein A/G magnetic beads (MCE Magnetic, Mianyang, China) were incubated together at 4°C for 4 h, followed by washing four times with 400 µL of binding/washing buffer (1× PBS + 0.5% Tween-20 + 150 mM NaCl). Subsequently, the antibody-bead complexes and protein supernatants were incubated together overnight at 4°C. The immune complexes were washed six times with PBST buffer (1× PBS + 0.5% Tween-20 + 150 mM NaCl), resuspended in 1× loading buffer, and heated at 95°C for 5 min. Finally, the samples were analyzed by western blot.
Cell counting kit-8 (CCK8) assay
Three thousand cells were resuspended in 200 µL of medium and seeded into a 96-well plate. After cell adhesion, they were treated with increasing concentrations of cisplatin for 24 h. After the treatment period, cells were incubated with 10 µL of CCK-8 reagent (MCE Magnetic, Mianyang, China) in 100 µL of medium for 1–4 h. The Spark multimode microplate reader (Tecan, Männedorf, Switzerland) was used to measure absorbance at 450 nm, with data collected using TECAN software. Each condition was replicated four times.
Kaplan-Meier plotter database
An online database called Kaplan Meier plotter (http://kmplot.com/analysis) can be used to assess the relationship between gene mRNA expression and survival in a variety of cancer cohorts, such as patients with acute myeloid leukemia, multiple myeloma, lung, gastric, pancreatic, and breast cancers [32]. Histology, stage, grade and the use of chemotherapy were among the clinical data available for ovarian cancer patients (Table 2 and Table 3). An online analysis was conducted to examine the correlation between PIF1 mRNA expression and survival in ovarian cancer patients. The results were expressed as the hazard ratio and logarithmic rank P-value. Patients with PIF1 gene expression were grouped using the best available cut-off value [33].
Table 2
Demographic and clinical characteristics of patients with ovarian cancer (n = 655).
Parameter | | Patients, n (%) |
Pathological type | Serous cystadenocarcinoma | 523 (79.8%) |
Endometrial | 30 (4.6%) |
Other | 102 (15.6%) |
Stage | 1 | 51 (8.9%) |
2 | 32 (5.6%) |
3 | 426 (74.7%) |
4 | 61 (10.7%) |
Grade | 1 | 41 (6.7%) |
2 | 162 (26.4%) |
3 | 392 (63.9%) |
4 | 18 (2.9%) |
Treatment | Platin | 478 (73.0%) |
Taxol | 357 (54.5%) |
Platin + Taxol | 356 (54.4%) |
Other | 176 (26.9%) |
Table 3
Demographic and clinical characteristics of patients with ovarian cancer (n = 382).
Parameter | | Patients, n (%) |
Pathological type | Serous cystadenocarcinoma | 346 (90.6%) |
Endometrial | 10 (2.6%) |
Other | 26 (6.8%) |
Stage | 1 | 7 (1.8%) |
2 | 13 (3.4%) |
3 | 312 (81.9%) |
4 | 49 (12.9%) |
Grade | 1 | 9 (2.4%) |
2 | 109 (29.0%) |
3 | 240 (63.8%) |
4 | 18 (4.8%) |
Treatment | Platin | 373 (97.6%) |
Taxol | 274 (71.7%) |
Platin + Taxol | 273 (71.4%) |
Other | 8 (2.1%) |
GEPIA database
The GEPIA database is an online database developed by Zhang's Lab at Peking University in Beijing, China [34]. It may be accessed at http://gepia.cancer-pku.cn/. The association between PIF1 and ovarian cancer was analyzed with the “Boxplots” module of GEPIA. GEPIA employs the log2 (TPM + 1) method to represent data on a logarithmic scale when comparing TCGA normal and GTEx data.
UALCAN database
The UALCAN database provides a thorough and interactive analysis of bioinformatics utilizing RNA-seq and clinical data from 31 tumors in the TCGA dataset (http://ualcan.path.uab.edu/) [35]. The association between PIF1 and ovarian cancer grades was analyzed with the “tumor grade” module of UALCAN.
Statistical analysis
Data are presented as mean ± standard deviation. Non-normally distributed variables were analyzed using the Mann-Whitney U test. Unpaired parametric tests were employed for normally distributed variables. Three or more groups were compared using one-way ANOVA. Statistical analyses were conducted using GraphPad Prism software (GraphPad Software, San Diego, CA, USA). Statistical significance was set at P < 0.05. Data obtained from mice that died were excluded from the analysis.