Ethical considerations
This study was approved by the Medical Ethics Committees of the affiliated Research Center and Hospital. All the patients participated in the current study, signed a consent form freely after explanation of aims and methods according to their knowledge. Also, all of the authors adhered to the 1975 Declaration of Helsinki and its next revisions.
Patients and samples
Inclusion criteria for sample selection considered as any patients previously diagnosed with breast cancer through histopathological evaluations performed on tissues obtained from core/fine needle biopsies. Also, a positive history of chemotherapy, autoimmune diseases, radiotherapy, and any chronic diseases were defined as exclusion criteria. For the included patients, all of the histopathological evaluations were performed by two well-experienced pathologists on core/fine needle biopsy samples as well as provided tissues following the surgeries. Also, the control sample for each patient obtained from the histopathological approved tumor-free margin (paired control) and samples obtained from the reduction mammoplasty (unpaired controls). All the samples stored at -80º C in liquid nitrogen for the investigations.
Cell culture
In order to compare TSGA10 expression levels, MCF-7 [13] and MDA-MB231 [14] as breast cancer and MCF10A [15] as a normal cell line were cultured (all purchased from Pasture Institute, Tehran, Iran). The cells were cultured in DMEM/F12 medium supplemented with 10% bovine calf serum (BCS) containing 100 U/ml penicillin and 100 µg/ml streptomycin. Cells were maintained at 37°C in a humidified incubator with 5% CO2.
Induction of hypoxia
In order to induce hypoxic situation, cells were treated with cobalt (II) chloride hexahydrate (CoCl2.6(H2O), MW=237.92 g/mol. MCF-7, MDA-MB23, and MCF10A cells were counted in equal numbers using hemocytometer and planted on a 6 well plate in two replications. They have cultured in the DMEM/F12 medium supplemented with 10% BCS containing 100 U/ml penicillin and 100 µg/ml streptomycin and synchronal treated with 100 µM cobalt (II) chloride hexahydrate for 24 hours [16]. After the mentioned time period RNA extraction was performed.
Inhibition of histone deacetylase
In this study, sodium valproate was used as a histone deacetylase inhibitor (HDACI) in order to evaluate the role of epigenetic changes in the expression of TSGA10 [17]. The malignant cells were treated with different concentrations (0.1, 0.2 and 0.4 mM) of this HDACI in the same situation as previous sections. After 24 hours of treatment with sodium valproate, the BCS free medium was added and RNA extraction performed 18 hours later.
Induction of oxidative stress
The cells were counted in equal numbers using hemocytometer and planted on a 6 well plate in two replications in DMEM/F12 medium supplemented with 10% BCS containing 100 U/ml penicillin and 100 µg/ml streptomycin for 24 hours. Then, the same BCS-free medium containing different concentrations of H2O2 (10, 100, and 200 μM) was added for induction oxidative-stress. The RNA extraction was performed separately in 30 minutes and 24 hours after the treatment.
Lactate assay
In order to investigate cellular proliferation lactate levels were evaluated in each of the three cell lines (MCF-7, MDA-MB231, and MCF-10A). Cells were cultured as same as the previous section but under both hypoxic and normoxic situations. In each of the mentioned situations, all the cultured cells were treated with low and normal amount of glucose. After 24 hours the supernatant was removed and the same media but without BCS was added. Following another 24-hour interval, lactate levels were evaluated according to the manufacture’s instruction (Greiner lactate colorimetric LOD-PAP-Test, Monoreagent, Germany).
Real-time PCR
All of the obtained tissues from surgeries (tumor tissue and controls), as well as cultured cells (with and without oxidative-stress, hypoxic induction, and low-glucose condition), were washed with sterile phosphate-buffered saline (PBS) and homogenized using liquid nitrogen. Total RNA extraction was performed using TRIzol® reagent (Invitrogen, Carlsbad, CA, USA) according to the manufacture’s instruction.
Sense and antisense primers for TSGA10 and HIF-1α were designed by robust primer design software as shown in Table 1. The real-time polymerase chain reaction (PCR) was performed using Takara Kit by Rotor Gene 6000 system (Corbett Research, Australia) on the collected samples and the mentioned 3 cell lines. This method was performed as we have previously discussed in a same research [6]. After data gathering, the collected real-time PCR results from both normal and cancerous cells were compared and evaluated as normalized ratio.
Immunoblotting
For the evaluation of protein levels, western blotting protein analysis was used. For this aim, 40 µg of whole-cell total protein of each sample was separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) under reducing condition in 8–10% gradient gel. Then, the proteins were transferred to a polyvinylidene fluoride membrane and incubated for 2 hours at 24ºC with blocking buffer (5% non-fat milk dissolved in PBS). For the detection of TSGA10, goat anti-TSGA10 antibody (Santa Cruz, USA) was used and the membranes were incubated for 24 hours at 4°C. After two times of membranes washing with PBS (5 minutes), membranes have been incubated with the secondary antibody (Santa Cruz, USA) for 2 hours at room temperature. Then after, they were re-washed and using horseradish peroxidase-conjugated reaction the TSGA10 bond visualized. The quantitation of TSGA10 band intensity was performed by TotalLab Life Science Image Analysis Software, Version 2.0 (TotalLab Ltd, Newcastle upon Tyne, UK) through normalization with β-actin protein.
Statistical analysis
Data analyses were performed by GraphPad Prism software 5.0 (GraphPad Software, San Diego, CA). Analyzed data reported as mean±standard deviation (SD) and evaluated by the t-test, one way or two-way analysis of variance (ANOVA) followed by the Tukey test. Any P-value less than 0.05 was considered to be statistically significant. In the figures showing the significance levels were followed by *P < 0.05, ** P < 0.01, ***P < 0.001.
The specificity and sensitivity index was obtained using the Receiver operating characteristic (ROC) curve by X software version Y (Company, Country). ROC curve data were interpreted by calculation of area under the curve (AUC) through the Youden index with following criteria: [1-0.9): excellent, (0.9-0.8]: good, (0.8-0.7]: fair, (0.7-0.6]: poor and 6< were considered as fail.