Cell culture and transfection
The ovarian cancer paclitaxel resistant A2780/Taxol cells and the parental A2780 cells were cultured in RPMI 1640 medium (Hyclone, UT, USA) supplemented with 10% fetal bovine serum (Gibco Life Technologies, NY, USA) and 1% penicillin/streptomycin (Hyclone, UT, USA). Besides, A2780/Taxol cells maintained with the above median containing 800ng/mL paclitaxel (Sigma–Aldrich, MO, USA) which was withdrawn one week prior to the experiments. SiRNAs of ABCC5 and siRNA normal control were synthesized by GeneCreate Biotech (Wuhan, China) named as ABCC5-siRNA-1, ABCC5-siRNA-2 and ABCC5-siRNA-3 which were transfected into A2780/Taxol cells using Lipofectamine 3000 (Invitrogen, CA, USA). All cells were cultured at 37°C with the presence of 5% CO2 and saturated humidity. The transfected cells cultured for 48h before the following experiments.
Cell proliferation assay
The A2780/Taxol cells were transfected with ABCC5-siRNA-3 and siRNA NC for 48h, and the treated cells were seeded into 96 well plates and cultured in incubator at 37℃ with the presence of 5% CO2 for 0 h, 24 h, 48 h and 72 h. The cell proliferation was assessed using the CCK-8 assay (Dojindo, Kumamoto, Japan) according to the manufacture, and the absorbance of each well measured at the wavelength of 450nm was read on a spectrophotometer (XFLUOR4 Version: V 4.51).
Cell apoptosis
A2780/Taxol cells were plated in a 6 well plates at a density of 2×105 cells/2mL for 12h. After transfection of ABCC5-siRNA-3 and siRNA NC for 48h, A2780/Taxol cells were cultured with medium containing 200 nmol / L paclitaxel. After 24 h, the cells were collected and washed with PBS twice for apoptosis. Apoptosis was stained with Annexin V-FITC cell apoptosis detection kit (BD, New Jersey, USA) and detected by flow cytometry (BD FACSAriaTM Fusion).
Wounding healing assay
After transfection, A2780/Taxol cells in logarithmic growth phase were collected and seeded to 6-well plates at 5×105 cells per well. When cell proliferation to the full confluence, a uniform scratch was made on the cells with a 200ul sterile pipette tip, gently rinsed 3 times with PBS to eliminate detached cells, a mark along the edge of the scratch was made on the bottom of the plate for visualization. Medium was refreshed and cells were incubated at 37℃ in a 5% CO2 incubator. Images were taken under a microscope (Olympus, Tokyo, Japan) at time points of 0h, 24h, and 48h.
Clone formation ability test
The cells after transfection were harvested, digested with 0.05% trypsin and blown into single cells, and the cells were suspended in 10% FBS medium and seeded in 6-well plates at a density of 1000 cells and placed in a 5% CO2 incubator at 37 °C for 2 weeks. The culture was terminated when macroscopically visible clones appeared in the dish. The supernatant was discarded and cells were carefully washed twice with PBS. Fixation with 4% PFA was added for 15min, and the fixative solution was discarded. An appropriate amount of crystal violet staining solution was added for 30 mins, then the staining solution was washed away using running water. Then the cells were air dried and colony formation were calculated and the difference of which was compared.
Western Blot
Protein of the cells was harvested using RIPA buffer (ThermoFisher Scientifific) and the concentrations were determined by a BCA Protein Arry kit (Biosharp, Guangzhou, China).Then the samples( 150 μg) of each group were boiled with 6 × SDS loading buffer for 10 min before electrophoresis on 12% sodium dodecylsulfate-polyacrylamide gel electrophoresis (SDS-PAGE) gels, and resolved proteins were transferred onto polyvinylidene difluoride (PVDF) membranes (Millipore, Billerica, MA, USA) using electro-transfer. After blocking with 5% skimmed milk, the membranes were incubated with anti-ABCC5 (Abcam, Cambridge, UK, 1:50), and anti-GAPDH antibodies (Abcam, Cambridge, UK, 1:500), followed by incubation with HRP-conjugated anti-rabbit (Abcam, Cambridge, UK,1:2000). Bands were visualized with ECL system and were quantifified using Chemi-Doc XRS imaging software (Bio-Rad).
Gene expression of ABCC5
Total RNAs of all the cell group were isolated using the Trizol reagent (Invitrogen, CA, USA) according to the protocol. The quality and quantity of the purified RNA was determined by measuring the absorbance at 260 nm and 280 nm (A260 and A280) using a SmartSpec Plus Spectrophotometer (Bio-Rad Laboratories, Inc., Hercules, CA, USA). All RNA samples were stored at -80 °C for future use. The cDNA was generated using the PrimeScriptVR RT reagent Kit (Takara, Dalian, China). The expression level of ABCC5 gene was detected by qRT-PCR using the SYBR Select Mas ter Mix (Applied Biosystems Life Technologies) on the ABI PRISM 7300 Sequence Detection System (Applied Biosystems Life Tech nologies). The Actin gene of human (species) was used as a control. The relative gene expression was measured using 2−ΔΔCt method (Livak and Schmittgen 2001). All reactions were performed in triplicate for each sample.
Bio-informatics analysis
Oncomine is currently the world's largest oncogene chip database and integrated data mining platform(https://www.oncomine.org/resource/login.html). The oncomine database was used to analyze the differential expression of ABCC5 between ovarian cancer tissues and normal tissues.
Cancer Genome Atlas (TCGA) is a cancer genomics project using genome analysis technology based on large-scale sequencing (https://portal.gdc.cancer.gov/). Datasets of RNAseq of ovarian cancer patients in TCGA database were selected and analyzed.
Gene Expression Omnibus (GEO) is an international gene expression database created and maintained by the National Biotechnology Information Center which was founded in year of 2000 (https://www.ncbi.nlm.nih.gov/geo/). TCGA and GEO data base were selected in the analysis of correlation between ABCC5 gene expression and pathological stage of ovarian cancer.
Tissue microarray and immunohistochemistry
Human ovarian cancer tissue microarray was purchased from Shanghai Outdo Biotech (sample No. hovac154su01, Shanghai, China) which included 2 cases of benign ovarian tumors and 152 cases of ovarian cancer. All the cancer patients were followed up for 5-9 years. According to the NCCN guidelines, 60 cases of chemo-sensitive and 31 cases of chemo-resistant of serous epithelial ovarian cancer tissues were obtained for the study. This study was approved by the ethics committee of the First Affiliated Hospital of Xi'an Jiaotong University (approval No.: 2020-G143) and the ethics committee of Shanghai oudu biotechnology company (approval No.: YB M-0502). Staining of ABCC5 was performed using ABCC5 rabbit anti-human polyclonal antibody (Abcam, Cambridge, UK, 1:50). The color intensity of cytoplasmic expression of ABCC5 was divided into 4 levels. To be specific, no staining scored as 0, light yellow as 1, brown yellow as 2 and dark brown as 3. At the same time, the positive percentage of cells was calculated as following: the positive cell rate < 5% was 0, 5 ~ 25% was 1, 26 ~ 50% was 2, 51 ~ 75% was 3, and more than 75% was 4. The result was the staining intensity score multiplied by the positive cell rate score: 0 ~ 2 was negative (-), 3 ~ 4 was weakly positive (+), 5 ~ 8 was slightly positive (+ +), 9 ~ 12 was strongly positive (+ + +). Two independent observers read and made a score decision to control the reading error.
Statistical Analysis
Statistical analysis was completed by applying SPSS package (version 19.0, IBM SPSS, IL, USA), the data were presented as mean ± standard deviation. Chi-square test was used to analyze the relationship between ABCC5 and clinicopathological feature of EOC patients including age, tumor stage (FIGO), histological grade, nodal status, tumor diameter, metastasis and chemo-resistance. Further, the multivariate logistic regression analysis model was used to analyze the above factors for predicting the chemo-resistance of EOC. Then the multivariate Cox proportional hazard model was applied for analyzing the prognostic factors of overall survival (OS) and disease-free survival (DFS) for the EOC patients. Finally, Kaplan–Meier analysis was used for estimating the OS and DFS for the patients.