Tissues samples
A total of 57 pairs of breast cancer and normal breast tissues were obtained from the tissue bank at West China Hospital (Chengdu, China). All patients were female, who underwent surgery between February 2010 and May 2015. The diagnosis of breast cancer was confirmed by two patologists at West China Hospital, following biopsy. The expression of human epithelial growth factor receptor-2 (Her-2) was defined as positive by immunohistochemistry (3+) or fluoresence in situ hybridisation examination (+). Ki-67 levels were considered high if the percentage was ≥14% detected by immunohistochemistry.
No radiotherapy or chemotherapy had been received prior to operation. The age of patients ranged from 25–81 years, with a median age of 52 years. There were 54 cases of invasive ductal carcinoma, 2 cases of invasive lobular carcinoma and only 1 case of mucinous carcinoma.
Reagents
The polyclonal rabbit anti-human PARP9 antibody (cat. no. 17535-1-AP) was purchased from Proteintech, Inc. (Wuhan, China). The monoclonal mouse GAPDH (cat. no. 200306) and actin (cat. no. 250132) antibodies, and horseradish peroxidase-conjugated goat anti-mouse IgG (cat. no. 511103) and goat anti-rabbit IgG (cat. no. 511203) secondary antibodies were purchased from Zen Bioscience Co., Ltd. (Chengdu, China).
Western blot analysis
Tissues were lysed with ice-cold lysis buffer containing 1% Triton X-100, 40 mM Hepes pH 7.5, 120 mM NaCl, 1 mM EDTA, 10 mM pyrophosphate, 10 mM glycerophosphate, 50 mM NaF, 0.5 mM orthovanadate, PMSF and aprotinin (Roche Diagnostics, Basel, Switzerland). Tissue lysates were ground for 30 sec/time (4 times) until the tissues were totally triturated, then the mixture were incubated on ice for 30 min and centrifuged for 15 min at 14,000 × g to remove cell debris. The supernatants were harvested and protein concentration was using a BCA assay (Thermo Fisher Scientific, Inc., Waltham, MA, USA). The proteins were heated at 100°C in 4× loading buffer [containing 200 nM Tri-HCl, 40% glycerol, 10% SDS (w/v), 400 nM DTT, 0.04% Bromphenil blue (w/v)] for 10 min, samples containing 20 µg protein were resolved by 10% SDS-PAGE, then transferred to a polyvinylidene difluoride membrane (Millipore; Merck KGaA, Darmstadt, Germany). Each membrane was blocked with 5% non-fat dry milk in Tris buffered saline with Tween-20 (TBST) for 1 h at room temperature. The membranes were then incubated with PARP9 (1:1,500), actin (1:1,000) and GAPDH (1:1,000) primary antibodies overnight at 4°C and the appropriate secondary antibodies (1:1,500) for 1 h at room temperature. Detection was performed using a chemiluminescence kit (BeyoECL plus; cat. no. P0018; Beyotime Biotechnology, Shanghai, China). Images were collected by Alpha Innotech's FluorChem imaging system (Alpha Innotech, San Leandro, CA, USA). GAPDH was used as an internal control. Western blots were subjected to densitometric analysis using Quantity One software (version 4.6.2; Bio-RAD, Hercules, CA, USA). The PARP9/GAPDH ratio in all breast cancer samples was calculated. The level of PARP9 expression was considered high if the PARP9/GAPDH ratio exceeded the median value in these samples.
Cell culture
The breast cancer cell line, HCC1806, was a gift from Professor Ceshi Chen (Kunming Institute of Zoology, Chinese Academy of Sciences, Shanghai, China). The cells were maintained in Dulbecco's minimal essential medium (Life Technologies, Grand Island, NY, USA) containing 10% fetal bovine serum (Thermo Fisher Scientific, Inc.). Cells were incubated at 37°C in a humidified atmosphere with 5% CO2.
RNA interference
All small interfering RNAs (siRNAs) were synthesized by GenePharma, Co, Ltd. (Shanghai, China). The target sequence used for knockdown of PARP9 was 5′-GGACAGAGTTAGAGATTGAAAC-3′, and the sequence of si-Control was 5′-ACGUGACACGUUCGGAGAATT-3′. siRNA (10 µM) were transfected into HCC1806 cells (~7,000 cells per well) by Lipofectamine RNAimax reagent (Thermo Fisher Scientific, Inc.), according to the manufacturer's protocol. Cells were cultured for 48 h prior to subsequent western blotting.
Wound-healing assay
Cells were seeded in 6-well plates at 60,000 cells/well. The cells were cultured in DMEM medium containing 5% fetal bovine serum for 24 h. To inhibit cell proliferation, the cells were treated with 2 µg/ml mitomycin C at 37°C for 96 h. 24 h after the transfection of si-PARP9 or si-Control, a 1-mm scratch was made in the confluent cultures with a pipette tip, and washed twice with PBS to remove debris. The area of the scratch was measured by taking images under a phase-contrast microscope. To ensure the consistency of observation, specific observing points along the wound were labeled, and the same fields were observed at multiple time points.
Statistical analysis
Statistical analysis was conducted using SPSS software (version 17.0; SPSS, Inc., Chicago, IL, USA) and Graphpad Prism software (version 5.0; GraphPad Software, Inc., La Jolla, CA, USA) was used to produce graphs. Fisher's exact test was applied to determine statistical significance on the relationship between PARP9 and clinicopathological parameters. Student's t-test was used to determine the statistical difference in migration rate between siControl- and siPARP9-transfected cells. P<0.05 was considered to indicate a statistically significant difference.