Coexpression of EphA10 and Gli3 promotes breast cancer cell proliferation, invasion and migration

Patients and tissue samples

One hundred and twenty-four BC tissues, 50 pericancerous tissues, 50 lobular hyperplastic tissues and 30 normal breast tissues from benign lesions were obtained at the Second and Third Xiangya Hospitals, Central South University, from January 2000 to December 2002 and were histologically identified by two pathologists. The tumors were restaged according to the seventh TNM Classification of Malignant Tumors. Classification and histological grade of the cancerous tissues were determined with reference to the WHO classification of tumors.

Clinicopathological data of the intraductal carcinomas and invasive carcinomas are summarized in table 1. Survival data of the 124 patients with invasive BC were collected. The follow-up time for patients was 13 years. Twenty-six patients who survived longer than 13 years were included in the analysis as censored cases.

Pericancerous tissues were collected from the above 50 patients with invasive BC. The age of these 50 patients ranged from 32 to 70 (46.5±9.4) years. The pathological examination showed 18 normal breast tissues, 14 mild dysplasia, 10 moderate dysplasia and 8 severe dysplasia cases. Lobular hyperplastic tissues were collected from 50 patients aged from 28 to 60 (36.7±8.4) years. The pathological examination showed 16 normal lobular tissues, 19 mild dysplasia, 10 moderate dysplasia and 5 severe dysplasia cases. Normal breast tissues were collected from normal tissues beside 30 breast fibroadenoma and pathologically identified.

All tissues were made into paraffin sections for immunohistochemistry.

Immunohistochemistry

EnVision Detection Kit (Dako, Carpinteria, California, USA) was used in the experiment. Briefly, the paraffin-embedded tissues were cut into 4 μM thick sections. Deparaffinized tissue sections were treated with 3% H₂O₂ in dark for 15 min, followed by heat-induced epitope retrieval using sodium citrate buffer at 96°C for 30 min. Primary rabbit anti-human EphA10 and Gli3 antibodies (1:100, Dako) were incubated with the tissues for 2 hours. The tissues were treated with Solution A for 30 min before 3,3′-diaminobenzidine staining and hematoxylin counterstaining. Ten random fields of each section (500 cells per field) were examined by two observers independently. The final data took an average of the two observations. Positive cases were represented by 25% or more positive cells.

Cell culture

Invasive BC cell lines (MDA-MB-231, BT20 and Hs578T) and normal human mammary epithelial cells (MCF10A) were provided by Shanghai Institute of Cell Research, Chinese Academy of Sciences. Dulbecco's Modified Eagle Medium (DMEM, Gibco, Grand Island, New York, USA) used for cultivation of the cells were supplemented with 10% fetal bovine serum (FBS), 1% penicillin and 1% streptomycin.

Cell transfection

MDA-MB-231 and BT20 cells were transfected with sh-EphA10 (20 µL), sh-Gli3 (20 µL) or the negative control (sh-NC, 20 µL) (GenePharma, Shanghai, China) using LipoFiter reagent (Hanbio, Shanghai, China). The following experiments were conducted 24 hours after triplicate transfection.

qRT-PCR

TRIzol reagent (Invitrogen, Carlsbad, California, USA) was used for extraction of total RNA from cells. Concentration and purity of the RNA samples were measured. The concentration of qualified RNA samples was adjusted, and the RNA was reverse-transcribed using a reverse transcription kit (Takara, Tokyo, Japan) and random primers according to the instructions. Gene expression was detected by LightCycler 480 (Roche Diagnostics, Indianapolis, Indiana, USA), and the reaction conditions were set according to the instruction of the fluorescent quantitative PCR kit (SYBR Green Mix, Roche Diagnostics). Each sample had three duplicates. The internal reference gene of mRNA was GAPDH. The 2^−ΔΔCt method was used for data analysis. Sequences of the primers are listed in table 2.

Western blotting

Cells lysed in radioimmunoprecipitation assay lysis buffer (Beyotime, Shanghai, China) were centrifuged for protein extraction. The protein concentration of each sample was measured using a BCA kit (Beyotime) to ensure the same loading amount of each protein sample. Corresponding volume of proteins was mixed with the loading buffer (Beyotime) and heated in a boiling water bath for 3 min. A 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) was performed for protein separation using a SDS-PAGE gel preparation kit (Beyotime). The electrophoresis (1–2 hours) was initiated at 80 V and switched to 120 V after bromophenol blue entered the separation gel. The proteins were transferred to a membrane in an ice bath under a 300 mA current. The transfer of protein was sustained for 60 min. After being rinsed for 1–2 min, the membrane was placed in blocking buffer at room temperature for 60 min or at 4°C overnight. Primary rabbit anti-human GAPDH antibody (internal reference, 5174S, 1:1000, Cell Signaling Technology, Boston, Massachusetts, USA), anti-EphA10 antibody (ab106437, 1–2 µg/mL, Abcam, Cambridge, Massachusetts, USA) and anti-Gli3 antibody (sc-74478, 1:1000, Santa Cruz, Texas, USA) were incubated with the membrane on a shaker at room temperature for 1 hour. The membrane was washed with washing buffer for 3×10 min. The secondary antibody (horseradish peroxidase-labeled goat anti-rabbit IgG, 1:5000, Beijing ComWin Biotech, Beijing, China) was incubated with the membrane at room temperature for 1 hour, followed by another round of membrane washing. The membrane was added with color developing solution, and the protein expression was detected by the chemiluminescence imaging system (Bio-Rad, Hercules, California, USA).

CCK-8 assay

Suspension of transfected MDA-MB-231 or BT20 cells (100 µL) was transferred to a 96-well plate. Each group had three replicate wells. The cells were incubated for 0, 24, 48 and 72 hours before treatment with CCK-8 reagent (10 µL per well, Dojindo, Tokyo, Japan) for 1–4 hours. The absorbance value was measured at 450 nm.

Transwell assay

Transwell inserts (−20℃, Corning, New York, USA) were taken out to thaw the Matrigel at room temperature. The Transwell inserts and 24-well plate were added with 0.5 mL of serum-free medium. The culture fluid was removed after 2 hours. MDA-MB-231 and BT20 cells grown at the logarithmic phase were suspended and cultured in 6-well plates. The cells were transfected as mentioned above after reaching 70%–90% confluence. Each group had three replicate wells. After 24 hours of cultivation, the cells were trypsinized and washed twice with phosphate buffer saline. The cells were resuspended in serum-free DMEM with an adjusted concentration. The basolateral chamber was added with 600 µL of medium containing 10% FBS, and the apical chamber was added with 100 µL of the cell suspension. After 24 hours, the Transwell inserts were taken out and the supernatant was discarded. Cells remaining in the apical chamber were cleaned using a cotton swab. Cells that invaded through the membrane were fixed with 4% paraformaldehyde for 20 min before Wright–Giemsa staining. Number of the cells in five random fields of view was counted under a microscope at high magnification and the cells were photographed.

Scratch assay

MDA-MB-231 and BT20 cells grown at the logarithmic phase were suspended and evenly inoculated in six-well plates. The cells were transfected as stated after 24 hours. A line was vertically scratched in the cell layer using a sterile pipette (100 µL). The scratch width of each group was basically the same. The scratch area at start was recorded as the control. The cells were photographed before and after 24-hour cultivation.

Statistical analysis

SPSS V.17.0 and GraphPad V.7.0 were used. Data are shown in the form of mean±SD. χ² test was used for analysis of the relationship between EphA10/Gli3 and clinical factors of BC. Kaplan-Meier and log-rank tests were applied for analysis of the patient survival. A Cox proportional hazards model was established for multivariate analysis. t-test and one-way analysis of variance were used for comparisons between two groups and among multiple groups. Tukey’s multiple comparisons test was performed for post hoc multiple comparisons. Significant statistics were represented by p<0.05.