Cell lines and breast tumor specimens
The cell lines MDA-MB-231 (HTB-26™), MDA-MB-436 (HTB-130™), MDA-MB-453 (HTB-131™), MDA-MB-468 (HTB-132™), BT-549 (HTB-122™), ZR-75-1 (CRL-1500™), ZR-75-30 (CRL-1504™), T47D (HTB-133 ™), MCF-7 (HTB-22™), SK-BR-3 (HTB-30™) and MCF-10A (CRL-10317™) were obtained from American Type Culture Collection (ATCC) and maintained in complete growth medium according to the ATCC-recommended culture conditions. HBL-100 was obtained from the Shanghai Cell Bank Type Culture Collection Committee (CBTCCC, Shanghai, China) and cultured in DMEM (Gibco, Gaithersburg, MD, USA) supplemented with 10% fetal bovine serum (FBS) (Gibco). Mycoplasma testing was routinely conducted by HD Biosciences. All cells were stored in liquid nitrogen and used for no more than 6 months after being thawed.
Patients with locally advanced breast cancer (>5 cm in diameter or with clinically palpable axillary adenopathy) received preoperative chemotherapy, weekly paclitaxel (80 mg/m2) and carboplatin (AUC = 2). All human breast cancer specimens were obtained from patients who were diagnosed with breast cancer and underwent surgery at the Shanghai Cancer Center during 2003–2009 (n = 110). All specimens contained over 90% tumor cells and were stored in liquid nitrogen until analysis. The study was approved by the Ethics Committee of the Cancer Hosipital, Fudan University and each patient gave written informed consent to participation in this research.
siRNA, miRNA, plasmid construction, transfection, and luciferase assays
Specific siRNAs and scrambled siRNA were purchased from Genepharma, and miR-200a mimics, antagomiRNA, and negative controls were obtained from Ribobio. MicroRNA-200a represents miR-200a-5p in the current study. Transfection of siRNA were same as previously described [10]. The negative controls had no detectable effects in human cell lines or tissues (Additional file 1: Table S1).
The 3’-UTR of human TP53INP1 mRNA was cloned into the XhoI/NotI sites of the psiCHECK2 vector (Promega) using the In-Fusion® Advantage PCR Cloning Kit (Clontech). Luciferase assays were conducted as we previously reported [10]. The primers are listed in Additional file 1: Table S1.
Immunoblotting
Immunoblotting was performed using a standard method [10]. Primary antibodies used were: anti-p73, anti-cleaved caspase-3, and anti-Bim (Epitomics); anti-Noxa (AbD Serotec); anti-YAP1, anti-Bax, and anti-GAPDH (Proteintech); and anti-TP53INP1 (Prosci). Secondary antibodies used were horseradish peroxidase-conjugated anti-rabbit and anti-mouse IgG (Proteintech). Quantity One software (BioRad) was used to quantify protein expression.
RNA extraction and quantification of miRNAs
The tissues were homogenized with Polytron PT100. Total RNA was extracted using the Trizol reagent (Invitrogen), according to the manufacturer’s instructions. cDNA was synthesized from total RNA using a specific stem-loop RT primer (50 nM) with the ReverTra Ace®qPCR RT Kit (Toyobo). The primers for miR-200a and U6 detection assays were purchased from Ribobio. Real-time PCR was performed using the SYBR® Green Real-time PCR Master Mix (Toyobo) and conducted on the 7900HT Fast Real-Time PCR System (Applied Biosystems). The relative amount of miRNA or mRNA in each sample was calculated using the comparative CT method as described in our previous study [10].
Establishment of gemcitabine resistant cell line
MDA-MB-231 cells were selected using 1 μM gemcitabine for 24 h, and then they were washed with PBS and recultured in drug-free medium. The treated cells were pulsed with 1 μM gemcitabine during periods of exponential growth. This cycle of treatment was repeated six times. The cell lines were cultured in 7 μM gemcitabine to generate gemcitabine-resistant sublines. Prior to the start of the experiments, the MDA-MB-231 GR cells were cultured in a gemcitabine-free medium.
TUNEL assays
Synthetic miR-200a- and miR-control-transfected cells were plated onto poly-lysine-treated cover clips in six-well plates. Then, cells were treated with 5 μM cis-platin for 24 h. Apoptosis was detected using the in situ cell death detection kit with TMR red (Roche). Phalloidin-TRITC (10 μg/ml) (Sigma) was used to stain for actin, and DAPI was used to stain the nucleus. Confocal microscopy was then used to analyze the results.
Proliferation assays
Cells were plated in 96-well plates. Following a 12-h culture, drugs were added. Each condition was repeated in triplicate, and untreated cells served as controls. After treatment for 72 h, 10 μl per well of WST-8 (Dojindo) was added to analyze cell viability. After culturing for 3 h, the absorbance at 450 nm was recorded using a 96-well plate reader. The growth inhibition ratio was calculated as follows: (%) = (OD. control well – OD. treated well) / OD. control well *100%.
miRNA targets prediction
The following online software programs were used: TargetScan 5.2, and BioGRID 3.1 [11, 12]. The predicted targets of miR-200a, including the p53 family members as well as binding proteins that may regulate the p53 pathway were exported into Cytoscape and analyzed for evidence of intersection [13].
Statistical analysis
The results of at least 3 experiments are expressed as the mean ± SD. An ANOVA and the Student’s t test were used to compare values between the test and control samples. Fisher’s exact χ2 test was used for categorical patient variables. Statistical significance was represented as P values <0.05. The optimal cut-off value for miR-200a expression was calculated by a X-tile program [14]. All statistical calculations were performed using STATA software.