OC patients and tissue collections
This study was approved by the Ehics Committee of Hainan People's hospital. Study patients of this study were 60 OC patients (age: 37 to 67 years; mean±S.D. age: 54.1 ± 6.6 years) who were enrolled at aforementioned hospital betweem January 2012 and December 2014. All patients were excluded from other clinical disorders and no therapy was performed on these patients before this study. Patients with a previous history or familly history of malignancies were also excluded. Ovarian biopsy was perfromed on all 60 patients before therapy to collect both adjacent (within 5cm around tumor) non-tumor avarian tissues and OC tissues. Histopathological analysis was performed to confirm correct tissue samples were obtained. All patients signed informed consent.
Treatment and follow-up
According to AJCC system, the 60 patients included 10, 13, 21 and 16 cases at clinical stage I, II, III and IV, respectively. Therapeutic approaches, such as surgical resections, chemotherapy, radiotherapy, and immunotherpay, were performed on these patients according to patients’ clinical stage and health conditions. All patients were followed up for 5 years from the day of admission. Patients’ survival conditions were recorded. All patients completed the 5 year follow-up.
Cell culture and transfection
Human OC cell line UWB1.289 from ATCC (USA) was used. Cell culture medium was composed of 10% FBS and 90% 1:1 mixture of RPMI-1640 medium/ MEGM medium. Cells were cultivated in a 5% CO2 incubator at 37°C with 95% humidity. Subsequent experiments were performed 48h later.
Cell transfections
The construction of expression vectors of CTBP1-AS2 and PTEN was performed using pcDNA3.1 vector (Sigma-Aldrich) as backbone. Mimic of miR-216a and negative control (NC) miRNA were from Invitrogen. UWB1.289 cells were transfected with 10 nM expression vector and/or 50 nM miRNA using Lipofectamine 2000 (Invitrogen, USA). All steps were completed according to manufacturer’s instructions. For controls, cells transfected with empty vector or NC miRNA were NC cells, and untransfected cells were control (C) cells. Subsequent experiments were performed 48h later.
Dual luciferase activity assay
pGL3 vector (Promega Corporation) was used as backbone to establish the luciferase vector of CTBP1-AS2. To analyze the interaction between CTBP1-AS2 and miR-216a, UWB1.289 cells were co-transfected with NC miRNA+ CTBP1-AS2 luciferase vector (NC group) or miR-216 mimic + CTBP1-AS2 luciferase vector (miR-216a group) through aforementioned methods. At 48h post-transfection, luciferase activities of both groups were measured and compared.
RNA isolations
Isolation of total RNAs from OC tissues, non-tumor tissues, and UWB1.289 cells was performed using Ribozol (Sigma-Aldrich). RNA precipitation was performed using 85% ethanol to harvest miRNAs. Genomic DNA was removed by DNase I digestion at 37°C for 2h.
RT-qPCR
The synthesis of cDNA samples was performed using SSRT IV kit (Thermo Fisher Scientific) with total RNA as template. SYBR Green Master Mix (Bio-Rad) was used to perform all qPCR reactions with GAPDH internal control to measure the levels of CTBP1-AS2 and PTEN mRNA expression. To measure the levels of miR-216a expression, poly (A) addition, reverse transcriptions and qPCR reactions were performed using All-in-OneTM miRNA qRT-PCR Detection Kit (GeneCopoeia) with U6 as the internal control of miR-216a. Three replicate reactions were involved in each experiment and gene expression levels were normalized using 2-ΔΔCq method.
Western-blot assay
The isolation of total protein from UWB1.289 cells was performed using RIPA buffer (Invitrogen). Protein concentrations were measured by performing BCA assay (Invitrogen). Protein samples were denatured in boiling water for 10 min, followed by separation of proteins using SDS-PAGE gel (8%). Proteins were transferred to PVDF membranes, followed by blocking in PBS containing 5% non-fat milk for 2h at 25°C. The blocked membranes were first incubated with rabbit primary antibodies of PTEN (ab31392,Abcam) and GAPDH (ab9485, Abcam) at 4°C for 20h, followed by incubation with secondary antibody of lgG-HRP (ab6721, Abcam) for 2h at 25°C. After that, signals were developped using ECL ™ Select Western Blotting Detection Reagent (Sigma-Aldrich). Image J v1.48 software was used to normalize signals.
CCK-8 assay
A Cell Counting Kit-8 (CCK-8) kit from Dojindo (Japan) was used to analyze the proliferation of UWB1.289 cells after transfections. UWB1.289 cells were cultivated in a 96-well plate with 4000 cells in 0.1ml medium per well. Cells were cultivated at 37°C and CCK-8 solution was added to reach 10% final concentration before the measurement of OD values. OD values were measured at 450 nM every 24h for a total of 96h.
Statistical analysis
All experiments were carried out in 3 independent biological replicates. Data were expressed as mean±S.D. values. Paired OC and non-tumor tissues were compared by paired t test. Unpaired t test was used to compare two independent groups. ANOVA Tukey’s test was used to compare multiple groups. With the median expression level of CTBP1-AS2 in OC tissues as cutoff value, the 60 patients were divided into high and low CTBP1-AS2 level groups (n=30). Survival curves were plotted for both groups and compared by log-rank test. p<0.05 was statistically significant.