A total of 114 breast cancer patients recruited from the First Affiliated Hospital of Nanjing Medical University from 2013 to 2016 were included in this study. Samples consisted of 114 of breast cancer tissues and 30 of adjacent normal tissues. All tissues were histopathologically confirmed by a pathologist who selected tumor areas with higher tumor cell density that was suitable for RNA isolation and immunohistochemistry (IHC). All the samples were pathologically examined and stored in liquid nitrogen for miRNA analysis. Ethical approval for the study was granted by the Clinical Research Ethics Committee, Nanjing Medical University. Written informed consent was taken from each participant.
SK-BR-3 human breast cancer cell line was purchased from the Cell Bank of Shanghai (Shanghai, China). BT-474 breast cancer cell line was kindly gifted from Dr. Tiansong Xia (First Affiliated Hospital of Nanjing Medical University). Cells were cultured in RPMI 1640 or DMEM medium, supplemented with 10% fetal bovine serum (Hyclone, Logan, UT, USA), at 37 °C in a humidified atmosphere with 5% CO2.
Dual-luciferase activity assay
The 3′-UTR of human ERBB2 containing the putative target site for the miR-1296-5p was chemically synthesized and inserted at the XbaI site, immediately downstream of the luciferase gene in the pGL3-control vector (Promega, Madison, WI) by Integrated Biotech Solutions Co., Ltd (Shanghai, China), respectively. Twenty-four hours before transfection, cells were plated into 24-well plates (1.5 × 105 cells/well). PGL3-ERBB2-3′-UTR (200 ng) and pRL-TK (80 ng, Promega) were transfected in combination with 60 pmol miR-1296-5p mimic or miRNA mimic control using Lipofectamine 2000 (invitrogen). Luciferase activity was measured 24 h after transfection using the dual luciferase reporter assay system (Promega). Firefly luciferase activity was normalized to renilla luciferase activity for each transfected well. Three independent experiments were performed in triplicate.
Western blot analysis
SK-BR-3 or BT-474 cells were plated in 6-well plates (6 × 105 cells/well). Seventy-two hours after the transfection of miR-1296-5p mimic or miRNA mimic control, cells were harvested and homogenized with lysis buffer. Total protein was separated by 10% denaturing SDS–polyacrylamide gel electrophoresis. Western blot analysis was performed as described . The primary antibodies for ERBB2, S6, Phospho-S6, beta-actin, and GAPDH were purchased from cell signaling technology (Danvers, MA). The expression levels of indicated proteins were normalized to GAPDH or beta-actin.
Quantitative real-time PCR analysis for miRNA
Breast cancer tissues and cells were isolated with Trizol reagent (Invitrogen, Carlsbad, CA). MiRNA fraction was purified using a mirVana™ miRNA isolation kit (Ambion, Austin, TX). The concentration and purity of the RNA samples were determined spectroscopically. The SYBR and U6 gene were used for detecting the gene amplification and normalizing the each sample, respectively. The primers of reverse transcription and polymerase chain reaction were purchased from RiboBio Co., Ltd (Guangzhou, China). QRT-PCR was performed according to the protocol of primer sets. PCR product amplification was detected by the level of fluorescence emitted by SYBR Green (SYBR® Premix Ex Taq™ II, TaKaRa) which intercalated into double stranded DNA . The ΔCt method was used for miRNA expression analysis of biopsy specimens. The cycle number at the threshold level of fluorescence (Ct) for each sample was determined, and then calculating the ΔCt value. The ΔCt value was the subtraction between the Ct value of miR-1296-5p and the Ct value of U6: ΔCt = Ct (miR-1296-5p) − Ct (U6). The fold-change for miRNA compared to each controls was calculated using the 2−ΔΔCt method. PCR was performed in triplicate.
SK-BR-3 or BT-474 cells were transfected with miR-1296-5p mimic or miRNA mimic control and plated into 6-well plates at a density of 1000 cells/well, incubated at routine culture condition for 2 weeks, fixed and stained with crystal violet. The number of colonies were counted under a microscope from three independent replicates.
SK-BR-3 cells were plated into 6-well plates (6 × 105 cells/well). Forty-eight hours after the transfection of miR-1296-5p mimic or miRNA mimic control, flow cytometry was used to detect apoptosis of the transfected SK-BR-3 cells by determining the relative amount of annexin V-FITC-positive and propidium iodide (PI)-negative cells. Annexin V-FITC and PI were purchased from BD biosciences (San Jose, CA).
RhoA and Rac1 activation assay
For RhoA and Rac1 activation assays, breast cancer cells were plated into 35 mm dishes and transfected with miR-1296-5p mimic or miRNA mimic control. The experiments were then performed according to the manufacturer’s protocol (Cytoskeleton Inc., Denver, CO, USA).
In vitro drug sensitivity assay
SK-BR-3 or BT-474 human breast cancer cells were plated into 35 mm dishes (6 × 105 cells/dish), 100 nmol/L of the miR-1296-5p mimic or 100 nmol/L miRNA mimic control were transfected in SK-BR-3 cells, using lipofectamine 2000 (Invitrogen, Long Island, NY, USA). The miR-1296-5p mimic, miRNA mimic control were chemically synthesized by Shanghai GenePharma Company (Shanghai, China). Twenty-four hours after transfection cells were seeded into 96-well plates (5 × 103 cells/well). Forty-eight hours after the drug treatment, cell viability was assessed by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-tetrazolium bromide (MTT, Beyotime Biotechnology, Nantong, China) assay. The absorbance at 490 nm of each well was read on a spectrophotometer. The concentration at which drugs produced 50% inhibition of growth (IC50) was estimated by the relative survival curve. Three independent experiments were performed in quadruplicate.
Each experiment was repeated at least 3 times. Numerical data were presented as mean ± SD. The difference between means was analyzed with Student’s t test. All statistical analyses were performed using SPSS 13.0 software (Chicago, IL). Differences were considered significant when p < 0.05.