A total number of 25 paired OC and the adjacent tissues were collected from The People’s Hospital of Linan City between May 2016 and Nov 2016. The samples were stored in liquid nitrogen immediately after surgery. This study has been proved by the ethical committee of The People’s Hospital of Linan City and written informed consent was obtained from each patient.
The total RNAs were extracted from 3 OC tissue and 3 normal tissue by TRIzol (Invitrogen). The tissue samples were then sent to Vazyme Biotech (Nanjing, China) for the sequencing analysis.
Ovarian cancer (OC) lines (SKOV-3, Caov-3, OVCAR-3 and HO-8910) as well as normal ovarian epithelial cell line (IOSE80) were obtained from the ATCC (Manassas, VA, USA). OC cells were cultured in RPMI Medium 1640(Gibco, CA, USA) and NHBE was cultured in Dulbecco's Modifed Eagle's Medium (DMEM) (Gibco, CA, USA) all containing 10% FBS (fetal bovine serum, Gibco, CA, USA) supplemented with 100 U•mL−1 penicillin, and 100 mg•mL−1 streptomycin (Invitrogen, CA, USA) in humid conditions with 5% CO2 at 37°C.
MiR-203 inhibitor and inhibitor control were synthesized by GenePharma (Shanghai, China). Overexpressing vector for CircTADA2A and pre-miR-203, control vector, short hairpin RNA (shRNA) targeting circTADA2A and its negative control were purchased from Ribobio (Guangzhou, China). Cells were seeded on 6-well plates at the density of 8×105, followed by getting cultured in RPMI-1640 without FBS at a temperature of 37°C for 12 h prior to the transfection. Cell transfection and co-transfection were carried out with the use of Lipofectamine 2000 (Invitrogen, CA, USA) in accordance with the manufacturer's instructions. 6 h following the transfection, the cell culture medium was replaced with RPMI-1640 medium, which was supplemented with 10% FBS.
24 h following the transfection, cells were collected and resuspended in culture medium, followed by seeding on 96-well plates at a density of 1x104 cells/well. Moreover, 10 µl CCK-8 solution was added to each well and incubated for another 2 hours at a temperature of 37°C. Absorbance at 480 nm was detected using a microplate Reader (Biorad, CA, USA). Each sample was performed for a minimum of 3 times.
Subsequent to the transfection, 1×105 of cells suspended in 200μL RPMI-1640 medium were seeded onto the upper transwell chamber (Corning, NY, USA). Following 24 hours of incubation at 37°C, the cells on the surfaces of lower chambers were fixed using 20% methanol followed by staining with 1% crystal violet (Beyotime, Shanghai, China). Eventually, the cell colonies were both photographed and counted.
Wound healing assay
Subsequent to the transfection, the cells were seeded onto 6 well plates followed by culturing in serum free medium for a period of 24 hours. Thereafter, cell monolayers were wounded using a 10μl pipette tip. After that, cells were replaced with fresh medium and cultured at 37°C. The pictures were taken 48 after the wounding for the purpose of determining the wound-closing procedure.
Following the transfection, the cells were seeded in 12 well plates at the density of 100/well. Subsequent to the incubation for 2 weeks, cells were fixed in 10% formaldehyde, together with staining with 1% crystal violet (Beyotime, Shanghai, China). Images were photographed under a microscope (Leica, Germany), besides counting the colonies that contained more than 50 cells.
SKOV-3 cells were harvested and resuspended in RPMI-1640 medium. The nude mice were injected with a total number of 3 × 106 /100 μL cells subcutaneously at the posterior flank. In addition, tumour size was monitored through the measurement of the length (L) and width (W) using callipers every 3 days. Following a period of 28 days, the tumours were excised out from the sacrificed mice and weighed.
Total RNA was extracted with the use of TRIzol reagent (Invitrogen, CA, USA) in accordance with the manufacturer's instructions. Afterwards, RNA was reverse transcribed to cDNA with the help of a PrimeScript RT Reagent kit (Takara, Dalian, China). The quantitative real-time PCR (qRT-PCR) analysis was carried out with the help of a SYBR-Green PCR Master Mix (Thermo Fisher, MA, USA) in a 7900HT PCR System (ABI, CA, USA). The use of GAPDH and U6 was made as internal controls for the mRNA and miRNA analysis, correspondingly. The relative expression levels were assessed with the use of the 2−ΔΔCt methodology. All of the reactions were carried out in triplicate.
The total protein was extracted from cells with the help of the RIPA lysis buffer (Beyotime, Shanghai, China). The protein concentration was detected in a nanodrop system (Thermo Scientific, MA, USA). 40ug protein was separated by 10% SDS-PAGE, followed by transferring to polyvinylidene difluoride membranes (Millipore, CA, USA). Subsequently, the blots were blocked using 5% non-fat milk for a period of 2 hours at the room temperature followed by the incubation at 4°C overnight with primary antibodies. Thereafter, the incubation of membranes was performed with a corresponding horseradish peroxidase-conjugated secondary antibody for 4 hours at the room temperature. The bands were detected with the use of an enhanced chemiluminescence solution (Pierce, MA, USA), together with imaging using the FluorChem imaging system (Alpha Innotech, San Leandro, CA, USA). The use of GAPDH was made as the internal control. All of the reactions were carried out in triplicate.
Fluorescence in situ hybridization (FISH)
Alexa Fluor 555-labeled circTADA2A probes were designed and synthesized by RiboBio (Guangzhou, China). FISH experiment was carried out using a fluorescent in Situ Hybridization Kit (RiboBio, Guangzhou, China). 1×105 cells were seeded onto the autoclaved glass slides and cultured for a period of 24 hours. Subsequent to fixing with 4% paraformaldehyde for 20 minutes, followed by the permeabilization with 0.5% Triton X-100 for 10 minutes, the cells were cultured at 37℃ overnight. Eventually, the incubation of slides was carried out with DAPI for the purpose of staining the cell nuclear and observed under a fluorescence microscope (Leica, Wetzlar, Germany).
RNA pull down
The biotin labelled circTADA2A as well as miR-203 probes along with theri control probe were synthesized by Sangon Biotech (Shanghai, China). In addition, the probe-coated beads were generated through the co-incubation of the probe with the streptavidin-coated beads (Invitrogen, CA, USA) at 25℃ for 2 hours. Cells were gathered and incubated with specific probes overnight at 4℃. Following that, the beads were eluted and the complex was purified with TRIzol (Takara, Dalian, China). Afterwards, the abundance of both circTADA2A and miR-203 was analysed by the qRT-PCR.
The tumour tissues were fixed in 4% paraformaldehyde for 24 hours, followed by dehydrating in a graded alcohol series and embedding in paraffin, followed by cutting into 5μm sections. The sections were deparaffinised, rehydrated with a graded alcohol series and then incubated in 96℃ with 0.01 mol/l sodium citrate buffer for the antigen retrieval. Following the incubation in 5% H2O2 for a period of 2 hours, the sections were incubated using primary antibodies including ki67 and SMAD1 (Abcam, England) overnight at 4℃. Immunostaining was carried out with the use of streptavidin-peroxidase and diaminobenzidinef (DAB) following the manufacturer's instructions (Beyotime, Shanghai, China). Eventually, the sections were not only observed under a fluorescence microscope (Leica, Wetzlar, Germany) but also imaged.
Luciferase reporter assay
Cells were transfected with miR-203 mimics or mimic control and co-transfected with pGL3 reporter vectors (Promega, CA, USA) that contained the wild-type (Wt) or mutated (mut) potential binding sequence of circTADA2A as well as SMAD1. 48 hours following the transfection, cells were gathered and analysed with the help of a Dual-Luciferase Reporter Assay kit (Promega, CA, USA). Luciferase activity was detected through the use of a GloMax fluorescence reader (Promega, CA, USA). Renilla luciferase activities were put to use as an internal control. Each of the assays was carried out in a minimum of triplicate.
All data are presented as the mean s± standard deviation (SD). The statistical analyses were performed using SPSS 20 software (Abbott Laboratories, Chicago, IL, USA). Data were analysed with one-way ANOVA and Student's test. p<0.05 was considered to be statistically significant.