Ishikawa and RL95–2 cells were generously donated by the Gynecologic Oncology Laboratory at Qilu Hospital in Shandong Province, China. RL95–2 cells were maintained in Dulbecco’s modified Eagle’s medium/F-12 media (HyClone, Biological Industries, Israel) with 10% fetal bovine serum (FBS, Invitrogen, USA) with antibiotics, whereas Ishikawa cells were maintained in Roswell Park Memorial Institute (RPMI) modified medium (HyClone, Biological Industries, Israel) supplemented with 10% FBS (Invitrogen, USA) with antibiotics. All cell lines were cultured in a humidified atmosphere of 5% CO2 at 37 °C. Half of the medium was replaced with fresh medium at 3-day intervals until the attached cells reached 70–80% confluence in our experiments.
All experimental procedures were approved by the Laboratory Animal Ethics Committee of Qilu Hospital, Shandong University. The principles outlined in the ARRIVE (Animal Research: Reporting of In Vivo Experiments) guidelines and the Basel declaration (including the 3 R concept) were considered when planning experiments.
Reagents and antibodies
Cisplatin was purchased from Sigma-Aldrich (USA), dissolved in dimethyl sulfoxide (DMSO, Solarbio, Beijing, China) to a stock concentration of 10 mM, and stored in single-use aliquots at − 80 °C. An anti-MLH1 antibody was purchased from Abcam (ab92312, United Kingdom). Anti-p-c-Abl, anti-cleaved caspase-3, anti-cleaved caspase-9, and anti-cleaved PARP antibodies were purchased from Cell Signaling Technology Inc. (China). Anti-BCL-2 antibody was purchased from Proteintech Group Inc. (USA). An anti-β-actin antibody was purchased from Zhongshan Jinqiao biotechnology Co., Ltd. (Beijing, China).
Real-time polymerase chain reaction (PCR) for measurement of MLH1 transcript levels
Total RNA was isolated using TRIzol (Invitrogen, USA) according to the manufacturer’s instructions. First-strand cDNA synthesis was performed using the Moloney murine leukemia virus (M-MLV) reverse transcriptase enzyme (Invitrogen, USA) according to the manufacturer’s protocol. Transcript levels were quantified using SYBR Premix Ex Taq (Takara Bio Inc., Japan) in an Applied Biosystems StepOne Plus Real-time PCR System. Detailed methods are available for download at www.takara-bio.com. U6 served as an endogenous control. The fold-changes in mRNA expression levels were determined using the 2−△△CT method [11, 12]. Primers were designed by Genepharma Company (Shanghai, China). The primer sequences are presented in Additional file 4: Table S1.
Western blot analysis
Proteins were extracted after treatment using a standard method, and protein concentrations were determined using the Bradford method according to Kruger . Proteins were extracted with RIPA buffer (50 mM Tris pH 8.0, 150 mM NaCl, 1% NP-40, 1 mM ethylenediaminetetraacetic acid (EDTA), and 0.1% sodium dodecyl sulfate (SDS) containing protease inhibitors). Total protein (30 μg) was separated in a 12% SDS gel and transferred to Immobilon-P transfer membranes (Merck Millipore, Ltd. Tullagreen, Carrigtwohill, Co. Cork IRL Rev. Size: 0.2 μm) after electrophoresis. After blocking with 5% nonfat dry milk, the membranes were probed with primary antibodies recognizing human MLH1 (1:1000), p-c-Abl (1:1000), cleaved caspase-3 (1:800), cleaved caspase-9 (1:500), cleaved PARP (1:500), and Bcl-2 (1:1000), followed by a horseradish peroxidase-linked secondary antibody (goat anti-rabbit IgG, 1:1000). β-actin (1:1000) served as an endogenous control. Immunoreactive proteins were visualized using the Immobilon Western Chemiluminescent substrate (ECL; Millipore Corporation, Billerica, MA, USA). The density of all proteins relative to β-actin was quantified with ImageJ (National Institutes of Health, USA). The experiment was repeated in triplicate.
MLH1 overexpression and silencing
The MLH1-pAD-kan adenovirus vector (ADV-MLH1) was utilized to upregulate MLH1 (Additional file 1: Figure S1, Additional file 2: Figure S2 and Additional file 3: Figure S3). ADV-NC served as a negative control (Additional file 4: Table S1). Cells were plated in 6-well plates and infected at 30 to 40% confluence using ADV-MLH1 (1 × 1012 vp/ml, Multiplicity of infection (MOI) = 200) mixed with serum-free medium. The medium was changed after 12 h. After treatment, cells were harvested at different time points and prepared for future use. We selected the optimal MLH1 siRNA fragment to silence MLH1. Cells were transfected with the indicated MLH1-siRNA (MLH1-homo-528/1832/2431) (synthesized by Genepharma, Shanghai) using the Endofectin-MAX Transfection Reagent (Genecopoeia, Rockville, USA) according to the manufacturer’s instructions. The sequences of MLH1-homo-528, MLH1-homo-1832 and MLH1-homo-2431 are presented in Additional file 4: Table S1. Briefly, the cells were plated in 6-well plates. At the time of transfection, cell density was maintained at 50 to 60%, and the medium was changed to complete medium at 6-h intervals. The cells were harvested at different time points for the detection of expression levels.
Cell proliferation assays
An enhanced cell counting kit-8 (CCK-8, Beyotime, Beijing) assay was utilized to determine cell proliferation as previously described . Cells were plated in 96-well plates at a density of 8000 to 10,000 cells/well. Viable cell absorbance values were determined using a NanoQuant Infinite M200 PRP (TECAN, Austria) at 490 nm, and the data were analyzed with SoftMax software. Cell viability was expressed by normalizing untreated cells to 100%. The experiment was performed in triplicate for each cisplatin concentration.
Flow cytometry analysis of the cell cycle and apoptosis
The cell cycle was analyzed using a Cell Cycle Detection Kit (BB-4104, BestBio, Shanghai) according to the manufacturer’s instructions. The cells were harvested by centrifugation and then fixed in 75% cold ethanol overnight after washing three times with PBS. Each sample was washed and resuspended in 500 μl of propidium iodide (PI) working solution for 30 to 60 min in the dark before detection using a FACSCalibur flow cytometer (Becton, Dickinson and Company, USA). The percentage of cells was further analyzed with ModFit LT software. Cell apoptosis was measured using an Annexin V-FITC/PI staining kit (BB-4101, BestBio, Shanghai) according to the manufacturer’s guidelines. The cells were counted and adjusted to 1 × 106 cell/ml. Then, a 2 ml aliquot of the cells was inoculated into each well of a 6-well plate, and the cells were allowed to grow for 24 h prior to drug treatment. Flow cytometry was performed using a FACSCalibur flow cytometer (Becton, Dickinson and Company, USA) and processed using CellQuest Pro analysis software. The experimental methods used for flow-cytometric analysis in our study can be found in previous reports [15, 16].
Animal experiments and immunohistochemistry staining
Six-week-old female BALB/c nude mice weighing 16 to 18 g were purchased from Vital River (Beijing, China). All animal experiments complied with the ARRIVE guidelines and were performed in accordance with the National Institutes of Health Guide for the Care and Use of Laboratory Animals (NIH Publications No. 8023, revised 1978). This experiment was designed for 6 animals per group. Briefly, 1 × 107 Ishikawa cells were suspended in 200 μl of PBS and injected subcutaneously into the left flank of each mouse. When the tumor diameter reached 4 to 6 mm, the mice in each group received the first intratumoral injection of ADV-MLH1 and ADV-NC (each at 2 × 109 VP/tumor). We repeated the intratumoral injection of the same adenoviral vector (2 × 109 VP/tumor) at days 5, 9, 13 and 17. Cisplatin treatment was performed via intraperitoneal injection of 5 mg/kg cisplatin in 0.9% physiological saline at days 4, 11, 18 and 25. Tumor volume was calculated as follows: V (tumor) = D×(d2). (D = length, d = width, d < D).
MLH1 protein expression in mouse tumor tissue from each group (ADV-MLH1 + CDDP-treated mice, ADV-NC + CDDP-treated mice) was assessed by immunohistochemistry staining. Standard three-step, indirect immunohistochemical analysis was conducted in 4-μm tissue sections that had been transferred to glass slides and included the following steps: citrate antigen retrieval, endogenous peroxidase blockage and avidin-binding activity and di-aminobenzidine development . Primary antibody against MLH1 (dilution, 1:200) (Abcam, ab92312, United Kingdom) was used, and the corresponding secondary antibodies were goat anti-rabbit IgG antibodies (cat. no. SP-9000; Zhongshan Jinqiao biotechnology Co., Ltd., Beijing, China). Staining patterns were analyzed by at least two experienced pathologists affiliated with the Department of Pathology of Qilu Hospital of Shandong University.
All statistical analyses were performed in GraphPad Prism version 6.01. Differences between two or three groups were analyzed using Student’s t-test. P-values < 0.05 were considered statistically significant.