Ethics statement
All patients included in this experiment signed informed consent, which was approved by the ethics committee of the First Affiliated Hospital of Soochow University. Animals were treated in consistence with the ethical requirements of animal experiments.
Experimental subjects
Epithelial OC tissue specimens were collected from 52 OC patients who received surgery in the First Affiliated Hospital of Soochow University from May 2016 to June 2018 (aging 33-65 years with a median age of 53.4 years). Normal ovarian epithelial tissues (n = 27) were resected by appendectomy from patients with uterine fibroids or adenomyosis (aging 32-61 years, with a median age of 52.6 years). All tissue specimens were stored in liquid nitrogen in small pieces at -80℃.
Immunohistochemistry
Fixed with 4% paraformaldehyde, the specimens were paraffin-embedded and sectioned into 4 µm, followed by dewaxing and dehydration. Then, the sections were retrieved by high temperature and high pressure and sealed in 3% H2O2 to eliminate endogenous peroxides. Following blockade in 10% goat serum, the sections were probed with SEPT6 antibody (1:400, Proteintech, Chicago, USA), DNMT1 antibody (1:500, Abcam, Cambridge, MA, USA) and with biotinylated II antibody (ZSGB-Bio, Beijing, China). The sections were successively treated with diaminobenzidine solution, hematoxylin solution and gradient alcohol, followed by permeabilization with xylene and mounting with neutral gum for observation under an optical microscope.
Cell culture
Human OC cells SKOV3 were cultured with 10% fetal bovine serum (FBS; Gibco, Grand Island, USA)-McCoy's 5A medium (Invitrogen, CA, USA), H08910 cells in 10% FBS-Roswell Park Memorial Institute (RPMI)-1640 medium (Gibco), A2780 cells in 10% FBS-Dulbecco’s modified Eagle medium (DMEM; Gibco) and immortalized human fallopian tube epithelial cells FTE187 in Medium 199 and MCDB105 (both from Sigma, MO, USA) containing 10% FBS. All cells were provided by the Cell Bank of Type Culture Collection of Chinese, Academy of Sciences (Shanghai, China).
Cells were cultured in a medium supplemented with 100 U/mL penicillin and 100 U/mL streptomycin (Beyotime Institute of Biotechnology, Shanghai, China) to attain 80-90% confluence. Then, cells were detached by 0.25% trypsin and passaged for experiments.
Cell transfection
SKOV3 cells were grew to 70% confluence in 6-well plates at 5 × 105 cells/well. The transfection mixture was configured in compatibility with the procedures of LipofectamineTM 2000 kit (Invitrogen). pcDNA3.1, pcDNA3.1-ADAMTS9-AS2, sh-negative control (NC), sh-DNMT1, sh-ADAMTS9-AS2 + sh-NC, sh-ADAMTS9-AS2 + sh-DNMT1, overexpression (Oe)-NC, Oe-SEPT6, sh-ADAMTS9-AS2 + Oe-NC or sh-ADAMTS9-AS2 + Oe-SEPT6 were transiently transfected into SKOV3 cells. At 24 h post transfection, cells were cultured in the renewed 10% FBS-McCoy's 5A medium for another 48 h. All the sequences were from GenePharma Ltd. Company (Suzhou, China).
Reverse transcription quantitative polymerase chain reaction (RT-qPCR)
Total RNA extraction from tissue specimens and cells was implemented through Trizol extraction kit (Life technology, CA, USA) and afterwards RNA concentration and purity were measured. ADAMTS9-AS2, SEPT6 and DNMT1 primers were synthesized by Sangon Biotech Co., Ltd. (Shanghai, China) (Table 1). Reverse transcription of RNA was conducted with the PrimeScript RT kit (Takara, Dalian, China) and fluorescence quantitative PCR was practiced on the ABI PRISM® 7300 system (Applied Biosystems, Massachusetts, USA). Taking glyceraldehyde-3-phosphate dehydrogenase (GAPDH) as the loading control, gene expression was calculated by 2−△△Ct method [16].
Table 1
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Genes
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Primer sequence (5’-3’)
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ADAMTS9-AS2
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Forward: 5’-AGGCTGAAGTTACAGGTC-3’
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Reverse: 5’-TTGGCTCCCAGTGTCTTA-3’
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SEPT6
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Forward: 5’-CAGATAGTGCGAAGAGCTGT-3’
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Reverse: 5’-CCGTGCATGTATGTGGTGGTAGT-3’
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DNMT1
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Forward: 5’-GGAGGGCTACCTGGCTAAAG- 3’
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Reverse: 5’-CTCTCCATCGGACTTGCTCC-3’
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GAPDH
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Forward: 5’-TCTCCCTCACAATTTCCATCCC- 3’
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Reverse: 5’-TTTTTGTGGGTGCAGCGAAC-3’
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| Note: ADAMTS9-AS2; long non-coding RNA ADAM metallopeptidase with thrombospondin type 1 motif, 9 antisense RNA 2; SEPT6, septin 6; DNMT1, DNA methyhransferase l; GAPDH, glyceraldehyde-3-phosphate dehydrogenase |
Western blot assay
After total protein extraction, protein concentration was determined and adjusted. Protein samples were denatured with the loading buffer, separated with electrophoresis, transferred onto a nitrocellulose membrane and blocked in 5% skim milk powder. Next, the protein membrane was reacted with the primary antibodies SEPT6 (1:1000, Cell Signaling Technology, Beverly, MA, USA), DNMT1 (1:1000, Abcam) and GAPDH (1:1000, Santa Cruz Biotechnology, Santa Cruz, CA, USA), and with a horseradish peroxidase labeled secondary antibody, immunoglobulin G (IgG) (1:1000, Cell Signaling Technology). Afterwards, the membrane was immersed in enhanced chemiluminescence solution (Pierce Company, IL, USA) and exposed. GAPDH was implicated as a loading control. Protein markers were supplied by Piercenet (#84785), and protein images were analyzed with ImageJ2x software (National Institutes of Health, Maryland, USA).
3-(4, 5-dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide (MTT) assay
Cells (6 × 104 cells/mL, 100 µL) were incubated in each well of 96-well plates for 48 h. Thereafter, MTT (Sigma) solution (20 µL, 5 mg/mL) was added into each well for incubation of 4 h. With the removal of the supernatant, the cells were reacted with 100 µL dimethyl sulfoxide (Sigma) and tested for optical density (OD 490nm) values on a microplate reader (BioRad, CA, USA).
5-Ethynyl-2’-Deoxyuridine (EdU) assay
Seeded in 24-well plates, cells were supplemented with 5 µL EdU solution for 2-h culture and fixed with ice methanol. Followed by that, cells were dyed with the EdU kit (Guangzhou RiboBio Co., Ltd., Guangdong, China) and observed under a laser confocal microscope (Wolfbn, Suzhou, China).
Scratch test
Cells seeded in 6-well plates at 5 × 105 cells/well were grew to cover the bottom. A 200-µL pipette tip was adopted to scratch lines vertically along a sterilized ruler and 3 scratches in parallel were drawn of each well. The cells were cultured with serum-free medium and photographed under a microscope after 24 h. Image J was utilized to count cell migration rate.
Transwell assay
Matrigel (Nobleryder, Beijing, China) that was thawed overnight was diluted 1:5 with serum-free medium. The diluted Matrigel (100 µL) was spread over the polycarbonate membrane in the Transwell chamber with a pore size of 8 µm. SKOV3 cells were resuspended in serum-free medium to 2.5 × 104 cells/mL, and 100 µL cell suspension was added into each well in 24-well plate. The low chamber was fulfilled with 500 µL medium containing 20% FBS. After incubation of 48 h, the cells in the upper chamber were wiped off while those in the lower chamber were fixed with 4% ice paraformaldehyde, stained with crystal violet staining solution, photographed under a microscope and counted.
Flow cytometry
Propidium iodide (PI) single staining tested cell cycle distribution: Cells were centrifuged at 1000 rpm, resuspended with 300 µL phosphate-buffered saline (PBS), fixed with 70% ice-ethanol and centrifuged at 1000 rpm once again. Resuspended in 400 µL PBS, cells were added with 50 µL Rnase A (2.5 mg/mL) to attain 250 µg/mL and incubated for half an hour. Then, the cell suspension was added with 50 µL PI (1 mg/mL) to reach 100 µg/mL and reacted for 30 min without light exposure. Since that, the cellular DNA distribution was tested within 1 h by flow cytometry and the data were evaluated by Modfit software (Verity Software House, Maine, USA).
AnnexinV-fluorescein isothiocyanate (FITC)/PI double staining tested cell apoptosis: Cells were successively treated with ethylene diamine tetraacetic acid-free trypsin, centrifuged at 2000 rpm twice and suspended in 500 µL Binding Buffer. Subsequently, cells were stained by 5 µL Annexin Ⅴ-FITC (BD Company, New Jersey, USA) and 5 µL PI, and detected on a flow cytometer (Beckman Coulter, CA, USA) within 1 h.
Experimental animals
Nude mice BALB/c (n = 50), 4 weeks old, were supplied by Beijing Vital River Laboratory Animal Technology Co., Ltd. (Beijing, China). Those mice were reared in a laminar flow rack of specific pathogen-free grade at 24-26℃, with 45-55% humidity. Foodstuff and drinking water were sterilized with high temperature, and cages and padding were replaced regularly.
Tumor xenografts in nude mice
Athymic nude mice were reared in an isolated and sterile environment. Raised for 4 weeks, mice (5 in each group) were subcutaneously injected with the treated OC cell suspension (50 µL serum-free medium containing about 2 × 107 cells) into the left armpit. The collected tumors after 6 weeks were measured and weighed.
Fluorescence in situ hybridization (FISH) assay
Bioinformatics software http://lncatlas.crg.eu/ was applied to predict the subcellular localization of ADAMTS9-AS2.
A slide (10 mm × 10 mm) was placed in a 24-well culture plate and seeded with cells at 6 × 103 cells/well. At 24 h post culture, cells were fixed with 4°C paraformaldehyde, permeabilized with 0.1% Triton X-100 (Guangzhou RiboBio) and pre-hybridized with the pre-hybridization solution. Then, cells were hybridized with 18S, U6, ADAMTS9-AS2 anti-sense and ADAMTS9-AS2 sense probes (Guangzhou RiboBio) for 16-20 h and rinsed with 2 × sodium citrate buffer. Finally, cells were stained with 4',6-diamidino-2-phenylindole fluorescent staining solution in the hybridized area and observed under a fluorescent microscope (Olympus, Japan, Tokyo).
RNA imunoprecipitation (RIP) assay
RNA binding protein immunoprecipitation kit (Millipore, MA, USA) was applied for RIP assay. SKOV3 cells were lysed with RNA lysis buffer, after which the whole cell lysate was incubated with RIP buffer containing magnetic beads and anti-human anti-Ago2 antibody (NC group was incubated with normal mouse IgG). The protein sample was then treated with proteinase K and the RNA concentration was measured with a spectrophotometer. The purified RNA was tested by RT-qPCR.
RNA-pull down assay
ADAMTS9-AS2 transcription in vitro was facilitated by MEGAscript® Kit (Ambion, Austin, Texas, USA), which was followed by biotin labeling. Then, RNA purification was conducted by MEGAclearTM Kit (Ambion). Two equal volume of Streptavidin Beads were prepared and washed with 100 × binding buffer. The protein lysate was mixed with one of the above-mentioned Beads which were not blocked with bovine serum albumin and tRNA. The Beads were incubated for 15 min and centrifuged at 3000 rpm to collect the supernatant. Biotin-RNA (5 µL) was supplemented with binding buffer to attain 100 µL, followed by incubation at 56°C for 5 min and at 37°C for 10 min. Afterwards, the RNA and the cell lysate were incubated for 15 min. The Beads were eluted with 1 × loading buffer (30 µL). Finally, sodium dodecyl sulphate polyacrylamide gel electrophoresis was performed on the obtained sample.
Statistical analysis
All data were processed by SPSS 22.0 statistical software (IBM, IL, USA). The measurement data were expressed in the form of mean ± standard deviation. Discrepancy between two groups was conducted by t test while that among multiple groups by one-way analysis of variance (ANOVA). Tukey method was utilized for pairwise comparison. P < 0.05 was hinted with statistical significance.