Study population. This study adhered to the tenets of the Declaration of Helsinki and the research protocol was approved by the National Cheng Kung University Hospital Institutional Review Board (No. B-ER-107-396) and the Institutional Review Board of Chi Mei Medical Center (10808-L03). Informed consent was obtained from patients. The patients with EOC who underwent primary cytoreductive surgery in the National Cheng Kung University Hospital and Chi Mei Medical Center, Liouying, and who had received front-line postoperative platinum-based chemotherapy between January 1, 2010, and December 31, 2016, were considered eligible to participate in this study. Non-cancerous patients who underwent partial oophorectomy for benign ovarian tumors were also included.
The cancerous and benign ovarian tissue samples were obtained intraoperatively and freshly stored in the vapor phase of liquid nitrogen (-196℃) or postoperatively collected from the NCKUH Biobank (Tainan, Taiwan). The medical records of patients were reviewed, and their clinical characteristics and information regarding the cancer stage, front-line chemotherapy, response to chemotherapy, and treatment outcomes were collected. Their follow-up records through October 31, 2021, were reviewed. The overall survival (OS) was calculated according to the date of diagnosis, and the progression-free survival (PFS) and PFI were determined based on the date of the last contact or progression following front-line chemotherapy.
miRNA isolation. Ovarian cancer and non-cancerous (control) specimens were collected and de-identified. Total RNA was extracted from the specimens (100 mg with a tumor cellularity of 70% or greater), using a miRNeasy kit (Qiagen, Germany). The extracted RNA was quantified using a NanoDrop 1000 spectrophotometer (Thermo Scientific). In addition, the integrity of the extracted RNA was determined using an Agilent 2100 Bioanalyzer.
Quantification of miR-509-3p. To obtain the miRNA distribution profile and quantify miR-509-3p in the specimens, we performed a quantitative real-time polymerase chain reaction (qPCR) according to the manufacturer’s instructions (Qiagen). In brief, 100 ng of total extracted RNA was collected and pooled from the samples, and cDNA was synthesized using a miScript Reverse Transcription kit (Qiagen). The cDNA sample was mixed with the qPCR master reagent (Human miScript Assay 384 set v10.1 [Qiagen]) using a Matrix Hydra eDrop (Thermo Scientific). Only wells with single melting-temperature values were included in further analysis. miRNAs were normalized with reference to the global miRNA mean and expression was calculated using the comparative Ct. method. Statistical analysis was performed using the Student’s t-test. P-values < 0.05 were considered statistically significant.
Quantitative reverse transcriptase PCR (RT-PCR). The RNA obtained (5 µg) was used as the template for the cDNA synthesis reactions, together with random primers and superscript III reverse transcriptase (Applied Biosystems). The resultant cDNA solution (at a 1:20 dilution) was used to detect the level of the target gene mRNA using quantitative PCR (qPCR). An accurate quantitation was achieved based on standard curves, which were drawn by serially diluting a known amount of RNA obtained through an in vitro transcription reaction and by performing TaqMan qPCR using these dilutions, in addition to using the patient samples. Quantitative analysis of the mRNA expression was performed using the Light Cycler® 2.0 System (Roche Diagnostics GmbH). The primers and TaqMan probes used for the analyses were designed using the manufacturer’s software, Primer Express. The following primers were used: COL11A1 (HS01097664) and GAPDH (HS99999905). No-reverse-transcription (no-RT) control reactions were performed using 100 ng of the total RNA derived from each individual sample as a template to ensure that the amplification did not occur due to DNA contamination. No signal corresponding to the no-RT controls was detected. The target gene mRNA expression was assessed using real-time RT-PCR. GAPDH was used as an internal control for RNA quality. All quantitative analyses were performed in duplicate to assess the consistency of the results. The relative expression levels of the target gene, which were normalized to those of GAPDH, were calculated as follows: ΔCt = Ct(target) – Ct(GAPDH). The ratio of the number of copies of the target gene mRNA to the number of copies of GAPDH was then calculated: 2− Ct × K (K = 106, a constant). Relative fold changes in gene expression were calculated using the comparative 2−ΔΔCT method.
Cells and media. The OVCAR-3 and OVCAR-8 cell lines were purchased from the National Cancer Institute DTP tumor repository program. The A2780 and A2780CP70 cell lines were provided by Dr. Hsu Keng-Fu (Department of Obstetrics and Gynecology, National Cheng Kung University Hospital, College of Medicine, National Cheng Kung University, Tainan, Taiwan). All cells were cultured in Roswell Park Memorial Institute-1640 medium supplemented with 10% fetal bovine serum and stored according to the manufacturer instructions. Cells used were passaged 5–20 times. Routine cell authentication was performed approximately every 6 months by cell morphology monitoring, growth curve analysis, species verification via isoenzymology, karyotyping identity verification via short tandem repeat-profiling analysis, and contamination checks. The most recent authentication was done in March 2022.
DNA isolation and bisulfite sequencing analysis. Genomic DNA was isolated using a QIAamp DNA Mini Kit (Qiagen). Sodium bisulfite modification of the DNA was performed using an EZ DNA Methylation-Gold Kit (Zymo) according to the protocol of the manufacturer. The two CpG sites of the miR-509-3p promoter region were amplified via PCR using the bisulfite-modified DNA template. The methylated allele (M1) was amplified using the primers miR-509-3p-F (5′-GGTATAGAATATTTAGTATGTGG-3′) and miR-509-3p-R (5′-TTTCTATTTTATTTCTCTTTT-3′) and the methylated allele (M2) was amplified using the primers miR-509-3p-F (5′-AGGAAGAAAGAATAAGTTATTTA-3′) and miR-509-3p-R (5′-AAAACAATTA TTTCTTATATT-3′). The PCR product was analyzed using a BigDye Terminator cycle sequencing kit (Applied Biosystems, Foster City, CA) and an ABI 3730 automated capillary sequencer.
Plasmid constructs and transfection. Small interfering RNAs (siRNAs) directed against human COL11A1 (sc-72956-SH), and a non-targeting negative control target (sc-108060) were purchased from Santa Cruz Biotechnology (Dallas, TX, USA). COL11A1 cDNA plasmid (BC117697, GE Healthcare) was cloned into a pCMV6-AC-GFP vector (PS100010, OriGene), followed by verification via sequencing. SUMO-3 (HG12782-UT) cDNA plasmid was purchased from Sino Biological (Beijing, China). miR-509-3p mimics (MC12984), mimics negative control (4464058), miR-509-3p inhibitor (MH12984), and inhibitor negative control (4464076) were purchased from Ambion (Foster City, CA, USA). A2780CP70 or OVCAR-8 cells were transfected with miR-509-3p mimics and SUMO-3 in combination using Lipofectamine 3000 (Thermo Fisher Scientific).
Western blot analysis, antibodies, and reagents. Following protein extraction, equal amounts of protein were separated using 8–15% sodium dodecyl sulphate-polyacrylamide gel electrophoresis [20]. Antibodies against COL11A1 (GTX55142), DNMT1 (GTX116011), DNMT3A (GTX129125), and DNMT3B (GTX129127) were obtained from GeneTex (Irvine, CA, USA). An anti-β-actin antibody (sc-47778) was purchased from Santa Cruz Biotechnology (Dallas, TX, USA), whereas antibodies against Akt (9272), phospho-Akt (Ser473, 9271), ubiquitin (58395), mouse IgG (7076), and rabbit IgG (7074) were obtained from Cell Signaling Technology (Danvers, MA, USA). An antibody against phospho-DNMT1 (Ser84) was purchased from Affinity Biosciences (Melbourne, Australia). An antibody against phospho-DNMT1 (Ser154) was purchased from Bioss Antibodies (Woburn, MASS, USA). An antibody against SUMO-3 was purchased from Abcam (Cambridge, UK). An antibody against p16 (AF5484) was purchased from Affinity Biosciences (Bath, UK). 5-aza-2′-deoxycytidine (5-aza) and MG132 were obtained from Sigma-Aldrich. Cisplatin (Fresenius Kabi Oncology, Ltd.) was provided by the Cancer Center of National Cheng Kung University Hospital.
Cell proliferation and 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) cytotoxicity assay. Cells (104/well) were seeded on 96-well flat-bottomed microtiter plates, and then transfected with miR-509-3p mimics or miR-509-3p inhibitor and cultured for 24, 48, 72, and 96 h. For co-transfection, before miR-509-3p mimic treatment, the cells in the exponential growth phase were pretreated with SUMO-3 overexpression plasmids for 24 h and cultured for 24, 48, 72, and 96 h. For cisplatin treatment, after transfection with miR-509-3p or miR-509-3p inhibitor for 24 h, cells were treated with different cisplatin doses. After 48 h of incubation, the in vitro cytotoxic effects of these treatments were determined using an MTT assay (at 570 nm) and the cell viability was expressed as the percentage of control (untreated) cells (% of control). The MTT analysis was conducted as previously reported [19].
Transwell invasion assay. The Transwell cell invasion assay was performed using polycarbonate membranes with 8 µm pores (Costar, Cambridge, MA, USA). Cells (5 × 104) were seeded on the membrane of the upper chamber of the Transwell pre-coated with rat collagen I (60 µg/Transwell). Fibronectin in medium (0.6 mL) was added to the lower chamber as the chemoattractant in a 24 h assay at 37°C under 5% CO2. The remaining cells in the upper chamber that did not migrate were removed using a cotton swab. The filters were fixed in 95% ethanol and stained with 0.005% crystal violet for 1 h. Migrated cells were counted using a phase-contrast microscope (Olympus, Lake Success, NY, USA). The mean of 10 contiguous fields represented the cell number. Each experimental condition was assayed in triplicate. used. The invasive capacity of cells was normalized to that of each corresponding control. One-sample unpaired Student's t-test was conducted to analyze the differences between the normalized invasive capacities obtained from the three independent experiments and the hypothetical value (which was set to 1).
Luciferase reporter analysis. The SUMO-3 3′-UTR fragments with wild-type miR-509-3p binding sites (Wt) or mutated binding sites (Mut) were inserted into a pGL4 vector (Promega). The SUMO-3 3′-UTR PCR product was cloned into the SacI/EcoRV site of the pGL4 vector. The following primers were used to target the SUMO-3 3′-UTR: forward, 5′-TTCACCACGATGATTTTCCT-3′ and reverse, 5′-GCACACAAAAGTACCCACAATATC-3′. The resultant construct was confirmed using DNA sequencing. Site-directed mutagenesis was performed to generate SUMO-3 3′-UTR constructs containing miR-509-3p mutant-binding sites by using the following complementary oligonucleotides: forward, 5′-CTGTAACTTAAATTGGGTTAATCAG-3′ and reverse, 5′-CTGATTAACCCAAT TTAAGTTACAG-3′. A2780CP70 or OVCAR-8 cells were transfected with the vector and the miR-509-3p mimics in combination. We performed luciferase assays 48 h post-transfection using a dual-luciferase reporter assay system (Promega). The normalized luciferase activity was reported as the ratio of luciferase activity to β-galactosidase activity. The activities of firefly luciferase and Renilla luciferase were measured as described previously [19].
Chromatin immunoprecipitation (ChIP) assays. Native protein–DNA complexes were cross-linked via treatment with 1% formaldehyde for 15 min, and ChIP assays were performed as previously reported [20]. Briefly, equal amounts of isolated chromatin were subjected to immunoprecipitation using anti-DNMT1, anti-DNMT3A, anti-DNMT3B, and IgG monoclonal antibodies. Primers with the following sequences were used for the ChIP assays: miR-509-3p forward, 5′-GGTACAGAACATTCAGCATGTGG-3′ and reverse, 5′-AGAAAACTAGAAAAC TGTACAAA-3′.
Statistical analysis. Data were analyzed using SPSS statistical software (version 21.0, IBM Corp., Armonk, NY, USA). Categorical variables are presented as frequencies and percentages and were analyzed using Chi-square test or Fisher’s exact test. Continuous variables are expressed as the mean ± standard deviation or as the median ± interquartile range. Interval variables were analyzed using Student’s t-test or Mann-Whitney U test. The cut-off values obtained based on the receiver operating characteristic curve for miR-509-3p, COL11A1 and miR-335 were optimized for their diagnostic sensitivity and specificity in predicting cancer progression or death. Survival was estimated using the Kaplan–Meier method and was compared using the log-rank test. Two-sided P-values < 0.05 were considered statistically significant. Cox proportional hazards models were implemented to estimate the hazard ratios (HRs) and 95% confidence intervals (CIs).