Patients and specimens
Patients with TNBC who were hospitalized at the Department of Medical Oncology or the Department of Breast Oncology of Sun Yat-sen University Cancer Center between January 2007 and December 2016 were retrospectively selected. The inclusion criteria were as follows: (1) patients underwent modified radical mastectomy; (2) the diagnosis of TNBC was confirmed by molecular biology and pathology; and (3) patients had complete follow-up information and pathological specimens available. The appropriate pathological samples for each patient were acquired from the Pathology Department. The final follow-up date was December 2019. Overall survival (OS) was defined as the time between diagnosis and death or the last follow-up visit. Disease-free survival (DFS) was calculated from the time of diagnosis to the date of relapse or metastasis.
Staining and evaluation
Paraffin-embedded tissues were cut at 2 mm thickness. The slides were dewaxed and rehydrated, and endogenous peroxidase activity was blocked. The specimens were boiled in ethylenediaminetetraacetic acid (EDTA) at full power for 5 min and medium heat for 20 min for antigen retrieval. Common goat serum was used to suppress nonspecific binding. Then, polyclonal rabbit anti-ANGPTL4 antibody (diluted 1:100; Abcam, #ab115798, USA) and a secondary antibody were used. Subsequently, we applied horseradish peroxidase. Finally, hematoxylin was used to counterstain the nuclei.
All sections were separately evaluated by two independent pathologists. The percentage was determined as follows: 0–5% was scored as 0, 6–25% was scored as 1, 26–50% was scored as 2, and more than 50% was scored as 3. The intensity was calculated as follows: 0 score for no staining, 1 score for weak staining (light yellow), 2 score for yellowish brown staining, and 3 score for brown staining. The scores of proportion and intensity were added to obtain an overall score, which ranged from 0 to 6 (17). A receiver operating characteristic (ROC) curve was applied to obtain an optimal cut-off score for overexpression of ANGPTL4 using the 0, 1 criterion.
The SPSS 22.0 statistical software package (SPSS, Inc., Chicago, IL, USA) was used to analyze all data. The variances between each groups were calculated by Student’s t-test and p value <0.05 was regarded as statistical significance.
Cell culture
The breast cancer cell lines used in this research were all from the American Type Culture Collection and were identified by DNA (STR) profiling. MDA-MB-231 cells were grown in DMEM (Gibco, #41965-039) with 10% FBS (Gibco, #10270-106). BT549 cells were grown in RPMI-1640 (Gibco, # A1049101) with 10% FBS. These cell lines were cultured at 37°C under a humidified atmosphere containing 5% CO2.
Transfection and establishment of stable cell lines
The lentivirus carrying the pEZ-Lv105 plasmid encoding the full-length ANGPTL4 ORF sequence (NM_139314.3) and empty vector were purchased from GeneCopoeia. Lentivirus was transfected into cells, and puromycin (A1113803, Invitrogen; Thermo Fisher Scientific, Inc.) selection (10 µg/ml) started 24 h after transfection. Stable ANGPTL4-overexpressing MDA-MB-231 and BT-549 cells (ANGPTL4 OE) and empty vector cells (Vec) were established from isolated colonies and grown for the next assay. Untreated MDA-MB-231 and BT-549 cells were referred to as the negative control group (NC). The efficiency of ANGPTL4 gene transfection was verified by qPCR and Western blotting.
Immunoblotting
Total cellular protein was lysed by using RIPA buffer (1:10, Cell Signaling Technology, 9806S). The BCA method (Thermo, USA) was used to calculate the protein concentrations. With a Mini Trans-Blot Electrophoretic Transfer Cell System (Bio-Rad), equal amounts of protein were subjected to gel electrophoresis. Proteins were transferred onto a PVDF membrane (Merck Millipore, Merck KGaA, Darmstadt, Germany). The membrane was blocked with 5% nonfat milk in Tris-buffered saline containing 0.1% Tween-20 for 1 h at room temperature and then incubated overnight at 4°C with specific primary antibodies. HRP-conjugated anti-rabbit or mouse (Santa Cruz) secondary antibodies were used and visualized using a ChemiDoc imaging system.
Wound healing assay
Cells were cultured on plastic in 6-well plates until 100% confluence, and a scratched area was created using a blue pipette tip. The gap was photographed at 0 and 16 h. The migration distance was measured in five fields randomly from every triplicate sample and is expressed as the mean versus that of the control.
Matrigel invasion assay
A Matrigel invasion assay was performed by using Transwell polycarbonate membrane filters with a pore size of 8.0 μm (Costar, Cambridge, MA). Briefly, Matrigel was thawed, diluted and placed into the upper chamber of a 24-well Transwell. After 4 h of incubation for Matrigel gelling, the cells were harvested, resuspended in 1% FBS media at a density of 106 cells/ml and placed onto the Matrigel. The lower chamber was filled with 600 μl of 10% FBS media. After a 6-h incubation at 37°C, the nonmigrated cells on the upper surface of the membranes were eliminated by cotton swabs. The membranes were fixed with pure methanol and stained with a solution of crystal violet. Cells migrating to the substratum of the membrane were counted.
Attachment assay
A 48-well plate was coated with 200 µl/well of 10 µg/ml fibronectin (Sigma, #F0895) in Ca and Mg-free PBS (pH 7.40) at 4℃ overnight and blocked with 200 µl of 1% heat-denatured BSA in PBS for 60 min at 37℃. The blocking solution was discarded, and the wells were rinsed with PBS three times. Then, 100 µl of adhesion buffer was added to each well. The cells were harvested, and 50,000 cells were plated in each well and incubated for 20–30 min at 37°C. The wells, except the standard control wells, were washed very gently 3 times until no cells were visible in the BSA-coated control wells. Then, 100 µl of adhesion buffer and 15 µl of MTT dye were added to the wells, and the plate was incubated for 4 h at 37°C in the dark. Next, the MTT-treated cells were lysed in 100 µl of DMSO, and absorbance was measured at 570 nm on a spectrophotometer.
Expression analysis by RNA sequencing
Total RNA from MDA-MB-231 cells with stable ANGPTL4 overexpression or the vector or NC was extracted using TRIzol (Invitrogen) according to the manufacturer's instructions. RNA-Seq was then performed at Novogene Co., Ltd. (Beijing, P.R. China). After library preparation and cluster generation, transcriptome sequencing was performed on an Illumina HiSeq platform. Differences in the gene expression of the three groups were determined using the DESeq2 R package (1.10.1). mRNAs with an adjusted p value less than 0.05 detected by DESeq2 were considered differentially expressed.
Quantitative real-time PCR
Total RNA from three groups of MDA-MB-231 cells (231 NC, 231 Vec and 231 ANGPTL4) was reverse transcribed into complementary DNA (cDNA). Then, qRT-PCR amplification was performed using FastStar Universal SYBR Green Master Mix (Roche Diagnostics GmbH, Mannheim, Germany) on an ABI 7500 Real-Time PCR Instrument (Applied Biosystems). The raw cycle number (Ct) values for each specimen were determined by the software supplied with the instrument. The relative gene expression level was computed by ΔCt (ΔCt = Ctsample − CtACTN). The discrepancy between the ΔCt of the target gene and reference gene was computed by ΔΔCt (ΔΔCt = ΔCttarget gene – ΔCtreference gene). Expression fold changes were calculated by 2-ΔΔCt counts.