All experiment protocols were approved by the ethics committee of Shengjing Hospital of China Medical University. The experiments involved human beings were guided by the Declaration of Helsinki, with the written informed consents obtained from the donors or their relatives by. The experiments involved animals were employed in accordance with the principles of the National Institutes of Health Guide for the Care and Use of Laboratory.
Cell treatment and transfection
The human normal ovarian epithelial cell line IOSE80 was purchased from Shanghai Huiying Biotechnology Co. Ltd (Shanghai, China) and human OC cell lines A2780, SKOV3, and OVCAR3 were purchased from American Type Culture Collection (ATCC, Manassas, VA, USA). These cell lines were all cultured in Roswell Park Memorial Institute (RPMI-1640) or Dulbecco's Modified Eagle’s Medium (DMEM) (Gibco BRL, Grand Island, NY, USA) that containing 10% fetal bovine serum (FBS; Gibco) at 37℃ with 5% CO2. When the OC cells were in logarithmic phase, the cells were trypsinized and inoculated into 6-well plates with 1 × 105 cells/well. After 24 h of conventional culture, the OC cells were transfected with plasmids according to the instructions of Lipofectamine 2000 (Invitrogen, Carlsbad, CA, USA) when cells reached about 75% confluence. The used plasmids included shRNA against ASH1L-AS1 (sh-ASH1L-AS1), CNRIP1 overexpression plasmid (oe-CNRIP1), or the corresponding negative control (NC) plasmids (sh-NC and oe-NC). Then, the cells were further treated with 10 μmol/mL cisplatin.
Clinical sample collection
From October 2019 to December 2020, 57 cases of tumor tissue samples were collected from OC patients underwent surgery in Shengjing Hospital of China Medical University, while 10 cases of normal ovarian tissue samples were obtained from patients with benign uterine diseases (uterine leiomyoma) underwent hysterectomy and bilateral salpingo-oophorectomy. All enrolled 67 individuals were female, with a mean age of 58 ± 7 years old (46 – 69 years old), who received no anti-tumor therapies before surgery. The tissue samples were confirmed by pathological examination after operation.
RNA isolation and quantification by reverse transcription-quantitative polymerase chain reaction (RT-qPCR)
Trizol (15596026, Invitrogen) was used for total RNA extraction from tissues and cells. RNA was then reversely transcribed into complementary DNA (cDNA) using the PrimeScript RT reagent Kit (RR047A, Takara, Tokyo, Japan). Next, the cDNA was subjected to real-time qPCR analysis using a Fast SYBR Green PCR kit (Applied biosystems, Foster City, CA, USA) on the ABI PRISM 7500 RT-PCR system (Applied biosystems). The used sequences are listed in Table 1. The relative expression levels of mRNAs were normalized to the expression of glyceraldehyde-3-phosphate dehydrogenase (GAPDH) or Actin, followed by 2-ΔΔCt calculation. The experiment was repeated three times.
Protein isolation and quantification by Western blot assay
The total protein was extracted from tissues and cells by radio-immunoprecipitation assay (RIPA) lysate buffer (BOSTER Biological Technology Co., Ltd., Wuhan, Hubei, China) containing protease inhibitors. A bicinchoninic acid (BCA) assay kit (BOSTER Biological Technology Co., Ltd.) was then applied for detection of protein concentration. Next, the protein samples were subjected to 10% sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) separation and transferred onto a polyvinylidene fluoride (PVDF) membrane by electrophoretic transfer method. Following this, the protein samples were sealed with 5% bovine serum albumin (BSA) at room temperature for 2 h in order to block the non-specific binding. The PVDF membrane was subsequently incubated at 4℃ overnight with diluted primary antibodies against Ki67 (4928, 1: 1000) and PCNA (4621, 1: 1000), with β-actin (ab5441, 1: 1000, Abcam; Cambridge, UK) as internal control. Following this, the membrane was further incubated with horseradish peroxidase-labeled goat anti-rabbit secondary antibody (ab205719, 1: 2000, Abcam) at room temperature for 1 h. The enhanced chemiluminescence detection solution (Millipore, Bedford, MA, USA) was used to visualize the samples in the X-ray film, followed by Image J software quantification. The relative protein expression was expressed by the ratio between the gray value of target protein band to that of β-actin protein band. The experiment was repeated three times.
RNA immunoprecipitation (RIP) assay
The binding between ASH1L-AS1 and EZH2 proteins was identified by using a Magna RIP RNA-Binding Protein Immunoprecipitation Kit (Millipore). The cells washed by pre-cooled phosphate buffered saline (PBS) solution were lysed using RIPA (P0013B, Beyotime Biotechnology, Shanghai, China) on ice for 5 min, followed by 10-min centrifugation at 14000 rpm at 4℃. The supernatant was randomly separated into two parts, a part of which was used as an input and the other part was co-precipitated with antibodies. RNA was isolated from the input and samples after digestion by Protease K, which was analyzed by RT-qPCR assay for ASH1L-AS1 expression. The used antibodies were rabbit anti-EZH2 (ab16046, 1: 100, Abcam) at room temperature for 30 min or immunoglobulin G (IgG) (ab109489, 1: 100, Abcam) as NC. The experiment was repeated three times.
Soft agarose colony formation assay
The experiment was carried out in a 6-well plate with a layer of agarose gel (about 2000 - 4000 cells per well). The cells were incubated in a humidified incubator with 5% CO2 at 37℃ for 12 - 14 h, followed by renewal of medium every two days. When the colonies grown enough to count, the culturing can be stopped. After that, the colonies were fixed with 4% paraformaldehyde for 10 min, and then stained with 1% crystal violet dye. The number and size of the stained colonies were calculated by Fiji software.
Cell proliferation determination
A cell counting kit-8 (CCK-8) Kit (Dojindo Laboratories, Kumamoto, Japan) was applied to evaluate the cell proliferative ability. After 48 h of transfection, the cells were reacted with CCK-8 solution for 1 - 4 h, with the optical density (OD) measured at 450 nm wavelength by an enzyme labeling instrument. The experiment was repeated three times
Cell apoptosis determination
Terminal deoxynucleotidyl transferase-mediated dUTP-biotin nick end labeling (TUNEL) staining assay was employed for cell apoptosis evaluation. After 48 h of transfection, the cells were subjected to three PBS washes and 30-min fixation using 4% paraformaldehyde, which were then treated with 0.5% TritonX-100 for 10 min. Following this, 50 μL of TUNEL probes (C10618, Invitrogen) was added for 4 h-co-culturing with cells at room temperature. Then, the cells were stained by 4’6-diamidino-2-phenylindole (DAPI) for 3 min, followed by microscopical observation to count the TUNEL-positive cells (green fluorescent stained cells).
Chromatin immunoprecipitation (ChIP) analysis
According to the instruction of a ChIP kit (P2078, Beyotime), the cells were fixed with 1% formaldehyde, which were then subjected to ultrasonic treatment on ice to produce 500 - 1000 bp fragments. Next, the fragments were incubated overnight with H3K27me3 antibody (ab6002, 1: 200, Abcam) and normal IgG (ab172730, 1: 100, Abcam) as isotype NC, respectively. Then, the magnetic bead was used for complex precipitation, which was analyzed by RT-qPCR.
Cell migration detection
After 48 h of cell transfection, the serum of cells was starved overnight, and then separated from the culture plate by trypsinization. Then, 1 × 105 cells were seeded in a 200 mL of serum-free RPMI1640 medium in an 8 mm Transwell upper chamber. Meanwhile, 650 mL of RPMI1640 medium containing 10% FBS was put into the lower chamber. After 12 h of cell culturing, the cells migrated through the membrane were counted.
CNRIP1 promoter methylation detection
Genomic DNA was extracted and quantified by a Biospin Genomic DNA Extraction Kit (BSC05S1). Then, we used an EpiTect Bisulfite Kit (59104; QIAGEN, GmbH, Hilden, Germany) to modify DNC with bisulfite. The used PCR primers (CNRIP1-F: 5’-GAGTAGTAGTGTTTATTTTA-3’; CNRIP1-R: 5’-ACAATTTAAACTCCTCCTCTAAAAC-3’) were synthesized by Sangon Biological Engineering Technology & Services Co., Ltd. (Shanghai, China). The PCR products were ligated into the clone T vector, and the successfully cloned PCR products were selected for sequencing by Sangon. Finally, according to the sequencing data, we calculated the sequence similarity to target sequence (%), C-T conversion efficiency (%), methylation status of each CG pair, as well as methylation rate of each CG pair (%).
Glycolysis Stress test
For Glycolysis Stress test, the cells were plated in a 96-well XF analyzer and XF Cell Glycolysis Kit (Seahorse; Agilent Technologies, Palo Alto, CA, USA). The XF sensor was incubated with the provided calibrator at 37℃ overnight without CO2 12 h before the experimental analysis. Next, the cells were re-placed into the 96-well XF analyzer (about 3 × 104 cells/well). The plate was subsequently washed using medium and incubated at 37℃ without CO2 again 30 min before measurement. Following this, 2 mM of oligomycin, 500 nM of FCCP, and 1 mM of mixture of antimycin A and rotenone were added to the appropriate port of the kit and calibrated. After the calibration, the cell culture plate was loaded into the instrument and measured according to the standard template.
Lactate content determination
The lactate production was determined with the use of a Lactate Assay Kit (#MAK064; Sigma). In brief, 5 × 105 cells were inoculated into a 35 mm culture plate for 48 h, which were then lysed in lactate analysis buffer. After that, 5 - 10 μL lysate was taken from each sample, and added with a reaction mixture consisting of assay buffer solution, substrate, and developing agent. Finally, the OD value was measured using a microplate reader at 570 nm wavelength to calculate the lactate content.
In vivo animal experiments
Forty-eight 6-8-week-old healthy nude mice (Institute of Materia Medica, Chinese Academy of Medical Sciences, Beijing, China) were housed individually under specific pathogen-free (SPF) conditions, with free access to food and water. The experimental environment was maintained as: 60% - 65% humidity, 22 - 25℃ temperature, and 12 h of light and dark cycle. Before the experiment started, the mice were subjected to a week of adaptive feeding, and the health status was observed. The nude mice were assigned randomly into 8 groups with 6 mice per group, including sh-NC, sh-ASH1L-AS1, sh-ASH1L-AS1 + oe-CNRIP1, sh-NC + cisplatin, sh-ASH1L-AS1 + cisplatin, sh-ASH1L-AS1 + oe-CNRIP1 + cisplatin, oe-ASH1L-AS1 + oe-CNRIP1, and oe-ASH1L-AS1 + oe-CNRIP1 + cisplatin. In brief, the A2780 cells stably transfected with above mentioned plasmids (1 × 109 cells/100 μL) were subcutaneously injected into the back of the nude mice, twice a week for 6 weeks. After 14 days of tumor formation, cisplatin (2 μg/mg body weight) was given into the mice intraperitoneally for one week. Finally, the mice were euthanized to measure the tumor size and weight, and the expressions of Ki67 and PCNA and drug resistance were analyzed.
Data analyses were processed by SPSS 21.0 statistical software (SPSS, IBM, Chicago, IL, USA). The measurement data were expressed as mean ± standard deviation. Comparisons between the two groups were conducted using unpaired student’s t-test, while differences among multiple groups were compared by one-way analysis of variance (ANOVA) with Tukey’s post hoc test. Kaplan-Meier method was used to calculate the survival rate of enrolled patients. Log-rank test was applied for univariate analysis. Pearson correlation analysis was employed to analyze the correlation of the observed indexes. p < 0.05 indicated the difference was statistically significant.