MCF-10A，MCF-7 and MDA-MB-436 cell lines that provided by Cell Bank of the Chinese Scientific Academy were cultivated in DMEM with 10% FBS. Cells were seeded in a six-well plate (105 cells per well). Upon reaching 80% confluence, cells were transfected with overexpression plasmid using Dharmafect 1 (Dharmacon). OPN and its control was cloned into pCMV3-C-his vector (OPN-OE or vector, Sino Biological). After 48h, RT-qPCR was utilized to test transfection efficiency . LY294002 was provided by Selleck.
Total RNA was harvested by Trizol reagent (Gibco, Grand Island, NY, USA) in light of the recommended protocol. Reverse transcriptase reaction was operated with Brilliant II Fast SYBR green QPCR master mix (Agilent Technologies, Santa Clara, CA, USA). RT-qPCR was operated utilizing SYBR Premix Ex Taq TM (Takara Bio, Inc., Otsu, Japan) and measured by 7500 FAST Real-Time PCR System (Bio- Rad Co., USA). The mRNA expressions were valued by 2−ΔΔCt method and GAPDH was used to normalize the data.
The level of cell viability was assayed employing a CCK8-Kit in light of standard protocol. The OD values were assessed at 450 nm.
The inoculation of cells (2×105cells/well) into 6-well plates was carried out. When cells reached 90% confluence, a 200 µl plastic tip was used to make a straight line in the monolayer, and plates rinsed by PBS. The cells were observed under an inverted microscope coupled to a camera (Leica DMI 4000) at 25°C, and this time was designated at 0 h. Afterwards, cells were maintained in an incubator. The migrative cells were evaluated applying Image J (v 1.5.1, National Institutes of Health, USA).
Cells were suspended in serum-free medium and then seeded into the upper chamber of a transwell with an 8-µm pore (Corning, Inc.). The upper chambers were pre-coated with (Transwell invasion assay) or without (Transwell migration assay) Matrigel (1 mg/ml), while lower chambers were decorated with medium carrying 10% FBS. The chambers were adopted for cell cultivation. The cells were subsequently exposed to 4% polyoxymethylene fixation as well as 1% crystal violet staining. Cells were visualized employing a light microscope (Leica DM 4000).
Tumor-Sphere Formation Assay
The cells were inoculated into 6-well plates with 1000 cells per well after corresponding treatment. After 14 d cultivation of cells, tumor spheres were formed. The tumor spheres were observed and documented under an optical microscope (DM4M, Leica, Solms, Germany) at a magnification of 200 ×.
The lysis of cells was carried out employing RIPA buffer (Shanghai Ruji Biotechnology Development Co., Ltd.). Subsequently, lysates were centrifuged, the supernatant was recovered and used for protein quantification by a BCA kit（Beyotime）. Then 30 μg of protein were loaded onto PVDF membranes (Merck Millipore) which were impeded by 5% milk in PBS-Tween 20 and cultivated with corresponding primary antibodies, after which was the exposure to secondary antibodies HRP-linked anti-rabbit IgG (1:5000, Abcam). The relative density of each band was decided employing ImageJ software. Then the relative protein expression was presented as the ratio of the density values of bands between experimental and control samples.
In vitro HUVEC tube formation assay
To solidify the gel, 300 μL of growth factor-reduced Matrigel (BD Biosciences, USA) was added into precooled 48-well plates and incubated for 30-60 min at 37 °C. Subsequently, human umbilical vein endothelial cells (HUVECs) that inoculated into a 48-well plate (300 µL/well) were suspended in breast cell-derived conditioned medium carrying 10% FBS for cultivation. The tube forming ability of cells was captured applying an inverted light microscope.
The overexpression and corresponding controls of OPN (OPN-OE#1, OPN-OE#2 and Vector) were synthesized by GenePharma Co. 69 (Shanghai, China). After that, the transfection of these vectors into cells was conducted adopting Lipofectamine 3000 reagent (Beijing Ya'anda Biotechnology Co., Ltd.) in light of recommended protocol.
Mice xenograft models
Nude mice were intramuscularly injected with MDA-MB-231 cells in the right hind thigh at the density of 2 × 106 per ml. The transplanted nude mice were separated into 3 groups at random: Vector, OPN-OE and OPN-OE+ LY294002, n =5. OPN-OE overexpression plasmid was injected into the tail vein of mice in OPN-OE and OPN-OE+ LY294002 group. Vector group was injected with blank control plasmid via tail vein. Control mice were left untreated. Mice in OPN-OE+ LY294002 group were injected with 10 µ LY294002 once every 4 days. All mice were examined every 3 days and sacrificed 21 days after tumor inoculation.
Tissue sections were prepared and subjected to immunohistochemical analysis. Anti-VWF was used as primary antibody. HRP-conjugated secondary Ab served as secondary antibody. Photographs were captured employing an Olympus-IX71 microscope at 40 × 10 magnification.
Measurement of MDA conten and GSH in tumor tissue
The enzymatic activities of MDA (Cat.no.A003-1-2) and GSH (Cat.no. A005-1-2) were assessed with different commercial assay kits which were supplied by Nanjing Jiancheng Bioengineering Institute.
All data that displayed in the form of mean ± SD got analyzed utilizing SPSS 20.0 software. Student’s t-test and one-way ANOVA were applied for the demonstration of comparisons. P < 0.05 meant that the experimental figures exhibited statistical significance.