1. Patients and breast cancer specimens
This study was approved by the institutional review board in the Affiliated Hospital of Qingdao University. The experiments were undertaken with the understanding and written informed consent of each subject before enrollment in this study. The study conformed with the Code of Ethics of the World Medical Association (Declaration of Helsinki).
All of patients and healthy controls were enrolled in the study from the same geographic area (Qingdao, Shandong) from March 2015 to February 2017. All patients with breast cancer or fibrocystic dysplasia (FCD) were diagnosed according to their clinical and pathologic manifestation, as defined by WHO classification criteria [16]. The patient was considered hormone receptor positive according to the assay results of primary tumor tissue acquired by biopsy. Main exclusion criteria were inflammatory breast cancer; other concurrent or previous malignant disease; life-threatening disease, such as uncontrolled cardiac diseases; or a pregnant or lactating status; radio or chemotherapy administration before surgery. The healthy controls were from people who had physical examination in our hospital, with age and gender matched.
2. Histopathological examination
Surgical specimens or tumor tissues acquired by biopsy were microscopically examined by two or more experienced pathologists, following histopathologic factors were assessed: cell type of the main lesion, primary tumor size, location, multiplicity, bilaterality, margin involvement, lymph-vascular invasion and lymph node metastasis (LNM).
3. RNA extraction and RT-qPCR
Total RNA from cell lines, breast tissues and peripheral blood was extracted using RNeasy Mini Kit (Qiagen). The concentration and quantity of total RNA were determined based on the absorbance at 260 nm using a NANO DROP spectro- photometer (ThermoScientific, USA).
PCR was conducted in 25 µl reaction volume, containing 12.5 µl Maxima SYBR Green qPCR Master Mix (2X) and 300 nM of each primers. The primers sequences were as follows: DTX1, forward primer: 5’- GGGCTGATGCCTGTGA ATG-3’, reverse primer: 5’-CCTGGCGAAACTGGTGC-3’. A pre-incubation at 95℃for 10 min was to activate the Hot Start DNA polymerase and denature DNA, and was followed by 45 amplification cycles of 95℃ denaturation for 10 sec, 60℃ annealing for 20 sec. qRT-PCR was performed in ABI PRISM 7900 Realtime PCR system (Applied Biosystems).
4. Cell culture and Transfection
The human breast cancer cell lines, HCC1937 and BT474, were obtained from American type culture collection (ATCC, Manassas, VA, USA). Each cell line was authenticated using a Short Tandem Repeat (STR) Identifiler kit (Applied Biosystems). HCC1937 and BT474 cells were grown on Dulbecco’s modified Eagle’s medium (DMEM; Thermo Fisher Scientific) with 10% FBS (HyClone, Logan, UT, USA) at 37 °C in 5% CO2.
For knockdown assays, the expression of DTX1 gene was downregulated by ON-TARGET plus siRNA SMART pool RNA (Thermo Scientific, Waltham, MA) using RNAi MAX transfection reagent (Life Technologies, Carlsbad, CA). Non-targeting SMART pool RNA (Life Technologies) was used as a control. The transfection efficiency was confirmed by qRT-PCR.
The ectopic overexpression of DTX1 was achieved with pCMV6-Entry-DTX1 plasmid (RC208338, Origene, Rockville, MD) using FuGENE Extreme 9 transfection reagent (Roche, Nutley, NJ). Empty pCMV6-Entry (PS100001, Origene) was used as a control. Transfection efficiency was confirmed by qRT-PCR.
Drug solutions were prepared fresh before each dose using a dounce homogenizer. For GSI (GSI-IX, MedchemExpress) treatment in vitro experiments, stocks were prepared at 10 mM in DMSO and dilutions were made directly before use. Cells were cultured for 3 days with GSI (compound E, 10uM) to establish a Notch-off state.
5. Cell proliferation analysis
Cell proliferation was analyzed using the ‘Amersham Cell Proliferation Biotrak ELISA, version 2’ system (GE Helathcare, UK) according to manufacturer’s instructions. In short, 5’000 cells were seeded in the well of a 96-well plate and grown for two days, labeled with BrdU for 3–4 h, fixed and labeled with a peroxidaselabeled anti-BrdU antibody. After coloring reaction the optical density was measured with a ‘SpectraMAX 250’ plate reader and analyzed with accompanying ‘Soft Max Pro’ software (Molecular Devices, MDS Analytical Technologies, Toronto, Canada). For cell counting, equal amounts of cells were seeded in triplicates and grown under standard conditions for three days. Cells were then harvested and each biological replicate was counted 3 times using a ‘Neubauer’-chamber (hemacytometer).
6. Transwell migration and scratch test assay
Transwell migration assays were performed using modified Boyden chamber units with polycarbonate filters of 8 mm porosity (Costar, Vitaris, Switzerland). The lower side of the filter was coated with 25 mg/ml collagen 1 (Sigma, St. Louis, USA) for 2 h at 37 °C. The bottom chamber was filled with DMEM containing 10% FCS. Cells (2╳104 per well in serum-free DMEM) were plated in the upper chamber in 100 ml medium and incubated for 24 h in standard conditions (see above). After removal of the remaining cells from the upper surface of the filter insert, migrated cells at the bottom of the filter were fixed with 3.7% formaldehyde in PBS and stained with 0.1% crystal violet. For every individual filter, the cells in 9 fields of view were counted. Every experiment was conducted in triplicates.
For scratch test analysis, cells were grown to above 90% confluency under standard conditions. A wound was inflicted by scratching a 200 ml pipette tip (Starlab, Milton Keynes, UK) over the surface of the culture flask. The wounds were documented immediately after scratching, after 12, 24 and 48 hours. Quantification of wound closing was performed with ImageJ software according to manufacturers’ instructions.
7. Western blot analysis and antibodies
Protein lysates from tissues or cells were resolved on denaturing 8–12% SDS-poly-acrylamide gels and transferred to nitrocellulose membranes (iBlot Gel transfer stacks, Invitrogen). The following primary antibodies were used: anti-Actin (Sigma-Aldrich, St. Louis, USA), anti-phospho-Akt/PKB and anti-total-Akt/PKB (Ser-473) (Millipore), anti-DTX1 (AB Biotech). Decorated proteins were revealed using horseradish peroxidase-conjugated anti-mouse, anti-rabbit, anti-rat (New England Biolabs) secondary antibodies and visualized by the chemoluminescence detection system Super-Signal West Pico (Thermo Scientific). Densitometry of western blots was performed using Image J software according to manufacturers’ instructions.
8. Statistical analysis
SPSS 19.0 was used for statistical analysis. Data were presented as the mean ± standard deviation (SD). Student’s paired or unpaired t test was used to analyze significance between paired or unpaired groups. One-way analysis of variance (ANOVA) test was used to analyze significance between groups of various differentiation. Only significant results P < 0.05 after Bonferroni correction were the focus of this report.