Ethics statement
Written informed consents were acquired from all patients before this study. The protocol of this study was confirmed by the Ethic Committee of Sichuan Academy of Medical Sciences ༆ Sichuan Provincial People's Hospital and based on the ethical principles for medical research involving human subjects of the Helsinki Declaration.
Study subjects
Paired OC tissues and adjacent normal tissues were harvested from 160 patients that accepted surgery in Sichuan Academy of Medical Sciences ༆ Sichuan Provincial People's Hospital between January 2017 and January 2019 and immediately preserved in liquid nitrogen for subsequent use. Age of the patients ranged from 18 to 80 years and the primary OC was confirmed pathologically by veteran pathologists. Additionally, patients with other malignant diseases or previous neoadjuvant chemotherapy or radiotherapy were excluded [19, 20].
Cell culture
Four OC cell lines (OVCAR3, SKOV3, A2780, and HO-8910) and human ovarian immortalized nontumorigenic ovarian surface epithelial cells (IOSE) were acquired from American Type Culture Collection (VA, USA) and cultured in Dulbecco’s modified Eagle medium (DMEM) (TransGen Biotech Co., Ltd., Beijing, China) containing 10% fetal bovine serum (Gibco Company, NY, USA) and 1% penicillin/streptomycin (Sigma-Aldrich Chemical Company., MO, USA). All the cells were incubated in humidified chambers [20].
Cell transfection
SKOV3 and A2780 cells were severally transfected with OIP5-AS1 silenced vector, miR-92a mimic/inhibitor, ITGA6 silenced vector or their respective negative control (NC) (Shanghai GenePharma Co., Ltd., Shanghai, China). The cells were cultured in 6-well plates until the cell confluence reached 70%-80%, and then transfected with plasmids by Lipofectamine 2000 (Invitrogen, CA, USA) according to the protocols.
Cell counting kit-8 (CCK-8) assay
The cell viability was determined in strict line with CCK-8 method (Dojindo Molecular Technologies Inc. Kumamoto, Japan). OC cells in each group were incubated on 96-well plates at 1000–3000 cells/well and the optical density was measured using a FLx800 Fluorescent Microplate Reader (BioTeke Corporation, Beijing, china).
Colony formation assay
The colony formation ability of cells was assessed in accordance with CytoSelect™ 96-well cell transformation assay (standard Soft Agar kits, Cell Biolabs Inc. CA, USA). Cells in each group were seeded onto 96-well plates at 5000 cells/well and incubated for 2 w. The clonies were detected by CyQuant and analyzed by a FLx800 fluorescent microplate reader (Biotek).
Transwell assay
Cells were suspended with 200 µL serum-free DMEM and seeded onto cell culture insert precoated with 1 µg/µL Matrigel. The basolateral chambers were appended with complete medium. Incubated for 48 h, the cells that did not penetrate through the membrane were removed, while the transmembrane cells were stained by 0.1% crystal violet dye solution. The numbers of migrated and invasive cells in five randomly fields were measured under a light microscope (Olympus Optical Co., Ltd, Tokyo, Japan).
Scratch test
OC cells were seeded and on the second day, cells covered the bottom of the plates. A 200 µL pipette tip was used to vertically scratched along the edge of a disinfected ruler (3 parallel scratches on each well). The supernatant was discarded and the wells were supplemented with serum-free medium to eliminate the effect induced by proliferation. Cells were photographed under a microscope after 24 h.
Flow cytometry
Transfected cells were collected after 48 -h transfection. Fluoresceine isothiocyanate Annexin V Apoptosis Detection Kits (BD Biosciences, Franklin Lakes, NJ, USA) were utilized to assess the apoptosis under a FACScan Flow Cytometer. The apoptosis rate was calculated by Cell Quest software (BD Biosciences).
Reverse transcription quantitative polymerase chain reaction (RT-qPCR)
Total RNA in tissues and cells was extracted by TRIzol kits (Invitrogen). A NanoDrop ND-3000 spectrophotometer (Thermo Fisher Scientific Inc., MA, USA) was employed for RNA quantification and the RNA was reversely transcribed into cDNA by PrimeScript RT Master Mix (TaKaRa Biotechnology Co., Ltd., Liaoning, China). The expression levels of target genes were affirmed by SYBR Premix Ex Taq II kits (TaKaRa) and the StepOnePlus system (Applied Biosystems Inc., CA, USA). Glyceraldehyde phosphate dehydrogenase (GAPDH) was used as the standardized control of OIP5-AS1 and ITGA6, and U6 was used as the standardized control of miR-92a. Data were analyzed using 2−ΔΔCt method and the primer sequences were shown in Table 1.
Table 1
Gene | Primer sequence (5’-3’) |
OIP5-AS1 | F: 5’-TGCGAAGATGGCGGAGTAAG-3’ |
R: 5’-TAGTTCCTCTCCTCTGGGGCCG-3’ |
miR-92a | F: 5’-ACACTCCAGCTGGGAGGTTGGGATTTGTCGC-3’ |
R: 5’-CTCAACTGGTGTCGTGGAGTCGGCAATTCAGTTGAGA-3’ |
ITGA6 | F: 5’-CACATCTCCTCCCTGAGCAC-3’ |
R: 5’-TATCTTGCCACCCATCCTTG-3’ |
U6 | F: 5’-GCTTCGGCAGCACATATACTAAAAT-3’ |
R: 5’-CGCTTCACGAATTTGCGTGTCAT-3’ |
GAPDH | F: 5’-AAGGTGAAGGTCGGAGTCAA- 3’ |
R: 5’-AATGAAGGGGTCATTGATGG-3’ |
Note: F, forward; R, reverse; OIP5-AS1, opa-interacting protein 5 antisense transcript 1; miR-92a, microRNA-92a; ITGA6, integrin alpha 6; GAPDH, glyceraldehyde phosphate dehydrogenase. |
Western blot analysis
The proteins were extracted using radio-immunoprecipitation assay lysis buffer (Beyotime Biotechnology Co., Ltd., Shanghai, China). Equivalent proteins (20 µg) were separated on sodium dodecyl sulfate-polyacrylamide gel electrophoresis gel and transferred onto membranes for incubation with primary antibodies: ITGA6 (1: 10,000, Sigma), E-cadherin, vimentin (both 1: 1,000, Cell Signaling Technology, MA, USA) and GAPDH (1: 1,000, Santa Cruz Biotechnology Inc., CA, USA). Subsequently, the cells were incubated with horseradish peroxidase-conjugated secondary antibody and the bands were developed by Pierce™ enhanced chemiluminescent Western Blotting Substrate (32,109, Thermo Fisher). The signal intensities of proteins were evaluated using Image J software (Promega, WI, USA).
Dual luciferase reporter gene assay
Binding sites of OIP5-AS1 and miR-92a, miR-92a and ITGA6 were predicated by https://cm.jefferson.edu/ra22/Precomputed/ and http://www.targetscan.org/vert_72/. OIP5-AS1 and binding sequence of miR-92a containing its mutant sites were amplified by PCR and the PCR products were connected to pMirReporter plasmid (Sigma) to obtain OIP5-AS1 wild type (WT) vector (wt-OIP5-AS1) and OIP5-AS1 mutant type (MUT) vector (mut-OIP5-AS1). Similarly, ITGA6 WT (wt-ITGA6) and MUT (mut-ITGA6) report vectors were synthetized. Afterwards, Lipofectamine 3000 reagent (Thermo Fisher) was utilized to transfect WT/MUT vectors and miR-92a mimic or mimic-NC into SKOV3 and A2780 cells on 96-well plates for 24 h. The luciferase activity was determined.
RNA pull-down assay
The DNA fragment with OIP5-AS1 or its NC sequence was performed with PCR amplification through the T7-containing primer and bound to GV394 (Invitrogen). Plasmid DNA was digested with restriction enzyme XhoI and the biotin-labeled RNAs were reversely transcripted. Expression of target RNAs was determined by RT-qPCR based on the method in a former publication [21].
Statistical analysis
All data analyses were conducted using SPSS 21.0 software (IBM Corp. Armonk, NY, USA). The measurement data conforming to the normal distribution were expressed as mean ± standard deviation. The unpaired t-test was performed for comparisons between two groups, one-way analysis of variance (ANOVA) was used for comparisons among multiple groups and Tukey’s post hoc test was used for pairwise comparisons after one-way ANOVA. P value < 0.05 was indicative of statistically significant difference.