Cell lines and culture
Human breast cancer MCF-7, T47D, SKBR3, MDA-MB-453, MDA-MB-435S, and BT549 cell lines, a human breast non-tumorigenic MCF-10A cell line, and a human mammary epithelial (HMEC) cell line were obtained from the American Type Culture Collection (ATCC, Manassas, VA, USA) and cultured under the ATCC-recommended conditions. All cells were maintained in a humidified incubator at 37°C and 5% CO2.
Breast tissue specimens
In this study, we collected two different cohorts of human breast tissue specimens, i.e., for in situ hybridization and immunohistochemistry, we recruited 127 patients with breast cancer and 50 patients with breast benign disease who underwent surgical treatment at The First Affiliated Hospital, Anhui Medical University (Hefei, China) between 2003 and 2006; for qRT-PCR, fresh tissue specimens from 30 breast cancer and 25 breast benign disease patients were prospectively collected between 2010 and 2011 from the same hospital. The fresh tissue specimens were immediately placed in a cryovial after surgery, snap-frozen, and stored in liquid nitrogen until use. Breast cancer patients who had undergone chemotherapy or radiation therapy before surgery were excluded. All breast cancer patients were female and received radical mastectomy or modified radical mastectomy. These 127 breast cancer patients were followed-up for a median 60 months. A protocol to use patient samples was approved by the Biomedical Ethics Committee of Anhui Medical University and a written informed consent was obtained from each patient.
In situ hybridization and immunohistochemistry
Formalin-fixed and paraffin-embedded tissue specimens from 127 breast cancer patients and 50 breast benign disease patients were used to construct tissue microarrays and cut into 4-μm-thick sections. For in situ hybridization, digoxin-labeled antisense oligonucleotide probes for BCL6 cDNA were obtained from Boshide Biotech Co. (Wuhan, China). The probe sequences were 5′-GACAGCTGTATCCAGTTCACCCGCCATGCCAGTGA-3′, 5′-TTCTATAGCATCTTTACAGACCAGTTGAAATGCAA-3′, and 5′-ATCCTGCAGATGGAGCATGTTGTGGACAGTTGCCG -3′.
For immunohistochemical analysis of BCL6 expression in tissue samples, a rabbit anti-BCL6 polyclonal antibody was obtained for Santa Cruz Biotechnologies (Santa Cruz, CA, USA) and used at a dilution of 1:100 according to our previous studies [15, 16].
Expression of BCL6 mRNA and protein in breast tissue specimens were reviewed and scored by two pathologists (QW and ZSW) using a light microscope (Olympus) using the staining intensity and percentage of tissue staining, i.e., 10% percent or more tumor cells stained were considered as positive, whereas <10% tumor cells stained with any intensity was considered as negative.
Plasmid constructions and generation of stable BCL6-expressing cell lines
The coding sequence of human BCL6 transcript variant 1 (GenBank accession #NM−001706) was cloned into a mammalian expression vector pReceiver (GeneCopoeia, Guangzhou, China) according to the manufacture’s protocol. After DNA sequence confirmation, this vector was named as pReceiver-BCL6 (BCL6). MCF-7 cells were then stably transfected with pReceiver-BCL6 or the empty pReceiver plasmids (VEC) to establish stable cells, MCF-7-BCL6, with forced expression of BCL6 and their control cells, MCF-7-VEC, respectively.
Transfection of siRNA and miRNA
To knockdown BCL6 expression or manipulate miRNA expression, we choose T47D and MCF-7 cells as a pair of model cell lines for gene transfection. Briefly, cells (1.0 ×105 /well) were seeded in 6-well plates and transiently transfected with BCL6 small interfering RNA (siRNA) or control scrambled siRNA duplex (GenePharma, Shanghai, China) or with 2’-O methylated single-stranded miR-339-5p antisense oligonucleotides (ASO) vs. its negative control or miR-339-5p mimics (all from GenePharma) vs. its negative control using Lipofectamine 2000 (Invitrogen, Carlsbad, CA, USA) according to the manufacturer’s instructions. The sequences of BCL6 siRNA and scrambled control siRNA duplex were listed in Additional file 1: Table S1.
RNA isolation and quantitative polymerase chain reaction
Total cellular RNA was isolated using a Trizol reagent (Invitrogen) according to manufacturer’s instructions. qRT-PCR was then performed to detect expression of BCL6, GAPDH, miR-339-5p, U6, and common tumor-related genes as described previously [15, 17, 18]. The sequence of the primers used for qRT-PCR was summarized in Additional file 1: Table S2.
Protein extraction and Western blot
Total cellular protein and western blot analysis were performed according to previous studies [15, 19]. The antibodies used were as follows: a rabbit anti-BCL6 polyclonal antibody (Santa Cruz Biotechnologies), a mouse anti-cyclinD1 monoclonal antibody (Santa Cruz Biotechnologies), a rabbit anti-CXCR4 (Bioss, Beijing, China), and a mouse anti-GAPDH monoclonal antibody (Santa Cruz Biotechnologies).
Assays for cell phenotypic changes
Cell phenotypic changes after gene manipulations included proliferation, soft agar colony formation, cell migration and invasion in MCF-7 and T47D/MDA-MB-453 cells and the corresponding assays were performed as described previously [15, 17–19]. In addition, we performed the cell wound healing assay to analyze tumor cell migration capacity. Briefly, T47D cells were seeded into 6-well plates and transfected with a BCL6 siRNA or NC vector. Upon cells reached totally confluence, scratching was done using a plastic tip. The wounded monolayers were incubated at 37°C in 1640 containing 10% FBS with or without mitomycin C (10 μg/ml, Sigma, St Louis, MO, USA) to block mitosis. Photos were taken at different periods of time under a microscope and the wound healing after scratched was measured daily.
Flow cytometry assay
Cell apoptosis was assayed using the Annexin V-Apoptosis Detection kit (BestBio, Shanghai, China) according to the manufacturer’s instructions. All the experiments were performed using a FACScalibur cytometer (BD Biosciences, San Jose, CA). Cell cycle distribution was analyzed using the PI method. Each experiment was performed in triplicate and repeated at least once.
Nude mouse breast cancer cell xenograft assay
All animal work was performed according to the animal care and use regulations of Anhui Medical University with the approved protocol by Biomedical Ethics Committee of Anhui Medical University. Briefly, 5 × 106 MCF-7-VEC and MCF-7-BCL6 cells were suspended in 120 μl Matrigel/PBS at a radio of 1:1 (v/v) and then injected into the mammary fat pad of female BALB/c-nu (Slaccas, Hunan, China). The day before injection, one estrogen pellet (17β-estradiol, 0.72 mg/pellet, Innovative Research of America, Sarasota, FL) was implanted into each mouse. Tumor growth was detected by measuring the tumor mass twice a week using a formula = (length x width2)/2. The mice were ultimately sacrificed on Day 27 after implantation. Primary tumors and tumors metastasized to other organs, such as the lung and liver, were collected for further analysis.
Luciferase reporter assay
The 3’UTR region of BCL6 was cloned to the psiCHECK-2 vector, including luciferase reporter gene. BCL6 3’UTR was amplified with primers of 5’-CCAGCCACAAGACCGTCCAT-3′ and 5′-CTCCGCAGGTTTCGCATTT-3′ and then inserted into the XhoI and NotI sites of the psiCHECK-2 vector. A psiCHECK-2 construct containing 3’UTR of BCL6 with a mutated sequence of miR-339-5p was also generated. All constructs were verified by DNA sequencing. After that, psiCHECK-2-BCL6 3’UTR and psiCHECK-2-mut-BCL6 3’UTR were co-transfected with 20 pmol miRNA-339-5p mimics or its negative control into breast cancer cells using Lipofectamine 2000 as described previously [15, 17]. Firefly luciferase activity was normalized to Renilla luciferase activity. All experiments were performed in triplicate and repeated twice.
All statistical analyses were performed using SPSS software for Windows (version 13.0; SPSS, Chicago, IL, USA). Differences between groups were compared using Pearson’s chi-square test for qualitative variables and Student’s t-test for continuous variables. Kaplan-Meier curves were constructed to determine patient relapse-free survival (RFS) and overall survival (OS). The statistical differences in survival among subgroups were compared using the log-rank test. P value <0.05 was considered statistically significant.