4.1 Patients and tissue specimens
The specimens of pathologically confirmed BC patients were collected from The First Medical Center of Chinese PLA General Hospital. All the involved patients have signed the consent informs, and this study was approved by Institutional Review Committees of Chinese PLA General Hospital. Generally, 103 patients who had received radiotherapy were involved into the IR group, and 109 patients who had not received radiotherapy were involved into the control group. All the collected BC tissues were made into paraffine sections for CEBPA immunohistochemical (IHC) staining. The CEBPA score was calculated by multiplying the percentage of stained cells (0–100%) by the intensity of the staining (low, 1+; medium, 2+; strong, 3+), giving a total score between 0-3. The cutoff value of the CEBPA IHC scores were determined based on receiver operating characteristic (ROC) curve analysis, resulting in cutoff value to be 0.75. The samples with CEBPA IHC score > 0.75 were named as high CEBPA (CEBPA-high) samples, and samples with CEBPA IHC score ≤ 0.75 were named as low CEBPA (CEBPA-low) samples. In addition, the IR group was divided according to the response of tumor to radiotherapy: complete response (CR) and partial response (PR) patients were defined as IR sensitive (IR-S) patients, while stable disease (SD) and progressive disease (PD) patients were defined as IR resistant (IR-R) patients.
4.2 Cell lines
BC cell lines ZR75-1 and MDA-MB-231 were obtained from American Type Culture Collection (ATCC), and the ZR75-1 CEBPA KO cells were constructed by Genloci Biotechnologies Inc.. All the cells were maintained in DMEM medium supplemented with 10% fetal bovine serum (FBS) and 1% penicillin/streptomycin, and the cell dishes were placed in a humidified incubator at 37 °C and 5% CO2.
The IR resistant (IR-R) clone of MDA-MB-231 was produced by treating the parental cells with a total of 60 Gy of γ rays over 5 weeks, and the surviving cells were designated as MDA-MB-231 IR-R cells. The corresponding radiosensitive (IR-S) clone of MDA-MB-231 was parallelly cultured from the same parental cells.
The lentiviral vector expressing CEBPA was constructed by cloning CEBPA into pCDH Puro (System Biosciences). The recombinant CEBPA lentivirus vector and pPACK packaging plasmid mix (System Biosciences) were co-transfected into HEK293T cells by employing lipofectamine 3000 (Invitrogen) to obtain the lentiviruses. This lentivirus was then utilized to infect ZR75-1 CEBPA-KO and MDA-MB-231 IR-R cells, and stable cell lines overexpressing CEBPA were screened out using puromycin.
4.3 Western blot
Cell samples were lysed on ice with RIPA lysis buffer supplemented with protease inhibitor. Cell lysate samples were mixed with the same amount of 2 × SDS loading buffer, boiled at 100 ℃ for 15 min, and centrifuged at 12 000 rpm for 2 min. The supernatant samples were loaded onto SDS-PAGE gel for separation, and subsequently were transferred to nitrocellulose filter membranes. Tris Buffered saline Tween-20 (TBST) containing 5% skimmed milk was used to block the membranes for 1 h at room temperature. The primary antibodies included human anti-CEBPA antibody (Abcam, ab15048) and anti-β-tubulin mAb (Abmart, M20005), and the secondary antibodies were goat anti-rabbit mouse IgG-HRP (Abmart, M21003). After thrice washing, the membranes were incubated overnight at 4 °C with primary antibody solutions. Subsequently, after twice washing, the membranes were incubated in secondary antibody solutions for 1 h at room temperature. Finally, the target protein bands were visualized through ECL Luminous Liquid (Millipore) and Imaging LabTM software (Bio-Rad).
4.4 Quantitative real time PCR (qRT-PCR) analysis
Total RNA of each sample was extracted using TRizol reagent (Invitrogen). Reverse transcription was performed to obtain cDNA. The gene expression abundance was measured by Quantitative PCR instrument CFX96 touch (Bio-Rad). A total of 20 μL qRT-PCR system was prepared, including 9.2 μL of cDNA template, 10 μL of 2 × SYBR Green qRT-PCR Mix, 0.4 μL of 10 μm Primer Forward, and 0.4 μL of reverse primer. β-actin (Bio-Rad) was used as the internal reference gene to normalize the transcript levels of examined genes. The results were recorded and analyzed using the formula 2-△△Ct. The primers were listed in Supporting Table S1.
4.5 Clonogenic survival analysis
Cell survival was determined by a standard colony-forming assay. The cells were seeded into 6 cm cultural dishes with 200 cell/dish. 24 h after seeding, the cells were exposed to IR with 0, 2, 4, 6 or 8 Gy using a 60Co source, respectively. The 0 Gy groups were considered as control groups. After IR, the cells were further cultured for 14 days to grow into colonies. Subsequently, cells were washed with PBS, fixed with 4% paraformaldehyde, and then dyed with 0.4% crystal violet. The colonies exceeded 50 cells were counted, and the plating efficiency (PE) was determined as
4.6 Cell apoptosis analysis
Cells treated with or without a single dose of 4 Gy IR (a 60Co source) were harvested for testing. Generally, the cells were digested to produce single cell suspensions and labeled with propidium iodide and Annexin V (Beyotime, C1062M) according to the manufacturer’s instructions. Finally, the stained cells were analyzed by a FACSCalibur flow cytometer (Biosciences BD Biosciences Pharmingen).
4.7 Measurement of reactive oxygen species (ROS) generation
The tested cells were digested to produce single cell suspensions. The cells were stained by ROS probe 2’,7’-Dichlorofluorescin diacetate (DCFH-DA) (Solarbio Life Sciences, C1062M) according to the instruction. Briefly, the suspended cell samples were incubated in DCFH-DA solution for 20 min in dark. After incubation, the cells were washed three times and the fluorescence intensity was analyzed by a FACSCalibur flow cytometer.
4.8 Luciferase reporter assay
ARE was inserted into pGL4.0 luciferase vector to produce luciferase reporter construct driven by ARE (ARE-luc). The tested cells were seeded in 24-well plates, and transfected with expression vector of β-galactosidase reporter and expression vector of Nrf2, in combination with empty PGL4.0 vector or ARE-luc vector, respectively. 24 h after transfection, the IR group cells were exposed to 2 Gy radiation, whereas the control group cells were not. 4 h after radiation, the cells were harvested and luciferase activities were determined with a Luciferase Reporter Assay kit according to manufacturer’s instructions. The β-galactosidase activity was used to normalize the luciferase activity.
4.9 Cell Viability Assay
Cells were seeded into 96-well plates. After incubation overnight, a series of erastin solution with concentrations to be 0, 2, 4, 6, and 8 μM were applied to corresponding wells. After 36 h treatment, cell viability was measured by a CCK-8 kit (Psaitong, PS0534-10000T), and finally, the absorbance of each well was measured at 450 nm.
4.10 Measurement of glutathione/glutathione disulfide (GSH/GSSG) ratio, and malondialdehyde (MDA) level
The intracellular MDA concentration was assessed using Lipid Peroxidation MDA assay kit (Beyotime, S0131S). GSH and GSSG were measured using the GSH and GSSG assay kit (Beyotime, S0053). Briefly, cells cultured in 10 cm2 plates were treated with DMSO or 4 μM erastin for 36 h. After treatments, cells were washed with PBS and collected to measure intracellular MDA or GSH and GSSG according to the corresponding kit instruction.
4.11 In vivo radiotherapy study
All the animal experiments were approved and guided by the Ethics Committee of Beijing Institute of Biotechnology. ZR75-1 CEBPA-WT cells, ZR75-1 CEBPA-KO cells, and CEBPA overexpressed ZR75-1 CEBPA-KO cells (CEBPA-KO+CEBPA) were used. Female BALB/C nude mice (4-6 weeks) were subcutaneously injected with 2×106 cells in the back. Radiotherapy was applied when the tumors exceeded 200 mm3. The control group did not get any treatment, while the radiotherapy groups were treated with 2 Gy IR every 2 days by using a 60Co source. The tumor volumes were measured every 3 days using a digital caliper
At the end of the experiment, the mice were sacrificed, and the tumors were isolated for further analysis.
4.12 Tunel staining
Tumor tissues from the in vivo radiotherapy study were fixed, dehydrated, and embedded in paraffin. Paraffin sections were applied to tunel staining. DAPI was used to stain the cell nucleus. Tunel Cell Apoptosis Detection Kit (Servicebio, G1501) was used for the detection of tissue apoptosis according to the manufacturer’s instructions. The stained tumor tissue sections were observed under a fluorescence microscope.
4.13 Bioinformatics analysis
The transcriptome and clinical data of BC were downloaded from TCGA program (https://portal.gdc.cancer.gov), containing 1099 BC patients. Among them, 528 have underwent radiotherapy (with radiotherapy group) and 571 have not (without radiotherapy group). In order to investigate the correlation between the expression of CEBPA and the survival rate, Kaplan-Meier (KM) survival curves were plotted according to the median value of CEBPA expression level. In order to eliminate the effect of drug treatment and cancer subtype, for the BC patients with radiotherapy, the invasive ductal carcinoma (IDC) that only took chemotherapeutic agents were picked out for KM survival curve plotting. For comparison, the BC patients without radiotherapy were also analyzed for KM survival curve.
The WGCNA package in R software was utilized to build a co-expression network about CEBPA by including all the genes with expression level larger than 0.5. Based on this analysis, the protein-protein interaction (PPI) networks were constructed for CEBPA, and then were displayed by Cytoscape version 3.9.1 software. The “ClusterProfiler” package in R software was used for functional enrichment analysis, and GO (Gene Ontology) biological processes and KEGG (Kyoto Encyclopedia of Genes and Genomes) pathways analysis about CEBPA was carried out. The overlapped results between BC patients with or without radiotherapy were plotted.
The gene functions were obtained from the GSEA website MSIGDB database (http://software.broadinstitute.org/gsea/msigdb) and then analyzed by GSEA version 4.3.2 software. The “ESTIMATE” package of the R software was utilized to calculate the “immune score”, “stromal score”, and “ESTIMATE score”. The “CIBERSORT” package of the R software was employed to analyze the infiltration level of immune cells.
4.14 Statistical analyses
All the experiment data were analyzed by SPSS 23.0 and Prism 8.0 (GraphPad). All the quantitative data were presented as mean ± SD and compared by using Students’t-test. All in vitro experiments were repeated three times. P < 0.05 is considered statistically significant.