Human samples
Tissues were collected from patients who underwent surgery at the Department of Obstetrics and Gynecology of Harbin Medical University Cancer Hospital between 2012 and 2013, including 40 epithelial EOC tissues and 20 normal epithelial ovarian tissue sections. None of the patients were treated with chemotherapy or radiotherapy before they were subjected to operation. The histopathological diagnoses were performed according to the World Health Organization criteria. All fresh specimens were stored at −80 °C for further use. This study was approved by the Medical Ethics Committee of Harbin Medical University Cancer Hospital and all patients were provided informed consent.
Immunohistochemistry (IHC)
Tumor samples were fixed in 4 % formaldehyde, embedded in paraffin wax, and then cut into 5 µM sections. Samples were de-paraffinized in xylene and rehydrated. After blocking endogenous peroxidase and performing antigen retrieval, tissue slides were blocked in goat serum for 30 min and incubated with antibodies against KIAA0101 (1:100 dilution, Santa Cruz, Santa Cruz, CA, USA) overnight at 4 °C, followed by biotinylated secondary antibody (Santa Cruz) for 30 min. Staining was performed in parallel using a Vectastain ABC kit (Vector Laboratories, Burlingame, CA, USA).
Cancer cell lines and culture
The human EOC cell lines SKOV3 and COV644 were supplied by China Center for Type Culture Collection (CCTCC). EOC cells were cultured in Dulbecco’s modified Eagle’s medium (DMEM; Gibco-BRL, Gaithersburg, MD, USA), supplemented with 10 % fetal bovine serum and antibiotics (Gibco). All cells were incubated at 37 °C under a humidified atmosphere containing 5 % CO2.
Quantitative real-time PCR (qRT-PCR)
qRT-PCR was performed as previously described [22]. Briefly, total RNA was extracted using Trizol reagent (Invitrogen, Carlsbad, CA, USA). qRT-PCR analyses for mRNA of KIAA0101 were performed by using QIAGEN OneStep RT-PCR kits (Qiagen, Valencia, CA, USA). The mRNA level of β-actin was measured as an internal control. To measure miR-429 expression, total RNA was polyadenylated and reverse transcribed using TaqMan MicroRNA Reverse Transcription Kit and TaqMan miRNA assay (Applied Biosystems, Foster City, CA, USA) according to the manufacturer’s instructions. The expression of U6 small nuclear RNA was used as the internal control. RT-PCR was performed in triplicates. Relative expression of the tested genes was calculated and normalized using the 2−ΔΔCt method. Primers were as follows: KIAA0101 forward, 5′TCCTGAAGAGGCAGGAAGCAG T 3′, reverse, 5′ TTGTGTGATCAGGTTGCAAAGGA 3′; β-actin forward, 5′ TGACGGGGTCACCCACACTGTGCCCATCTA3′, reverse, 5′ CTAGAAGCATTTGCGGTGGACGATGGAGGG 3′.
Transfection and luciferase assay
All oligonucleotides were transfected into EOC cells at a final concentration of 50 nM using HiPerFect transfection reagent according to the product manual (Qiagen). The wild type full-length 3′UTR of KIAA0101 gene containing the putative miR-429 biding sites was amplified by PCR and was inserted into the psiCHECK2 vector (Promega, Madison, WI, USA). The mutant KIAA0101 3′-UTR was generated from the KIAA0101 3′-UTR using the QuikChange® Multi Site-Directed Mutagenesis kit (Stratagene, La Jolla, CA, USA). The coding sequences of KIAA0101 were generated by PCR and cloned into pCDNA3.1 (+) vector (Invitrogen) to generate pCDNA3.1-KIAA0101. The luciferase reporter vector (KIAA0101 3′UTR and TOP flash), pCDNA3.1-KIAA0101 and Renilla plasmid were all transfected using Lipofectamine LTX according to the manufacturer’s instructions.
Cells were seeded in triplicate in 24-well plates 1 day before transfection for the luciferase assay. 48 h after transfection, the cells were harvested and lysed, and the luciferase activity assayed using the dual-luciferase assay kit (Promega). Normalized luciferase activity was reported as luciferase activity/Renilla luciferase activity. Three independent experiments were performed.
Cell proliferation assay
The cell growth rate with Cisplatin treatment were determined by MTT assay (Promega). Briefly, at 48 h after transfection, the cells were seeded into 96-well plates at 5000 per well in a final volume of 100 μl with different concentration of Cisplatin (0, 4, 8, 16, 32 and 64 µM). Then at 48 h, 25 µl of MTT stock solution was added to each well and incubated for 4 h. The absorbance was measured at 570 nM. The assays were performed in triplicate.
Colony formation assay
The transfected EOC cells were seeded in 6-well plates (400 cells per well) and cultured for 10 days with different concentrations of Cisplatin (0, 8 and 32 µM). The cells were fixed with 10 % formalin, and stained with 0.5 % crystal violet (Sigma, St. Louis, MO, USA). The assay was performed in five replicates.
Transwell migration and invasion assays
In vitro cell migration and invasion assays were performed using 24-well Transwell chambers (8-µM pores, BD Biosciences, San Jose, CA, USA). The transfected EOC cells (5 × 104 cells per well) were cultured in the top chamber with 100 µl medium containing 1 % FBS. 500 μl complete media with 10 % FBS was added into the lower chamber. After 24 h of cultivation, the medium from the chamber and the Transwell were removed, and the chamber was gently wiped with a cotton swab. The migrated cells were fixed in 4 % paraformaldehyde, and stained with crystal violet solution. Six fields were randomly selected and counted. The procedure for the cell invasion assay was similar to the cell migration assay, except that the Transwell membranes were pre-coated with Matrigel (BD Biosciences). The assays were performed in triplicate.
Western blot
Western blot was performed as described previously [23]. Briefly, Total protein was extracted by RIPA buffer. Nuclear protein was extracted using nuclear extracted kit (Beyotime, Beijing, China). The total extracts were separated using 10 % SDS–polyacrylamide gels and transferred onto polyvinylidene difluoride (PVDF) membranes (Bio-Rad, Hercules, CA, USA). The membranes were probed with a primary antibody against human KIAA0101, β-catenin, Axin2, c-myc, Histone or β-actin (Santa Cruz), respectively. After several washes with TBST, the membranes were incubated with (HRP)-conjugated secondary antibody (Santa Cruz). Bound antibody was detected using the Supersignal West Pico ECL chemiluminescence kit (Thermo scientific, Rockford, IL, USA).
Statistical analysis
Statistical analyses were performed by SPSS Windows version 19. Data was expressed as mean ± SD of the experiments performed in triplicate. One-Way ANOVA was performed to determine the significance of difference among groups. Differences were considered significant at *p < 0.05, **p < 0.01 and ***p < 0.001.