Cell lines and serum samples
Human breast cancer cell lines (MCF-10A, MCF-7 and T47D) were obtained from the Chinese Type Culture Collection, Chinese Academy of Science. The cells were maintained in DMEM supplemented with 10% fetal bovine serum and grown at 37°C in a humidified atmosphere containing 5% CO2. Tamoxifen-resistant breast cancer cell lines MCF-7/TAM and T47D/TAM were produced from parental MCF-7 and T47D cells, respectively, by persistent exposure to a tamoxifen gradient for approximately 12 months; the gradient was created by using increasing concentrations of TAM from 0.05 µM to 1 µM until the cells acquired resistance to 1 µM tamoxifen (Sigma-Aldrich, St. Louis, MO, USA).
All clinical tissue samples (70 tamoxifen-sensitive patients and 70 tamoxifen-resistant patients) were obtained from the Affiliated Hospital of Weifang Medical University (Weifang, China) between September 2015 and October 2018. Tamoxifen sensitivity was defined as patients without recurrence or with recurrence after 48 months, and tamoxifen resistance was defined as patients with disease progression or recurrence 6 months or less after tamoxifen therapy. The study was approved by the Ethics Committee of the Affiliated Hospital of Weifang Medical University. The samples were retrieved after diagnosis and before surgery and immediately frozen at -80°C. In addition, RNA sequencing and miRNA sequencing data from The Cancer Genome Atlas (TCGA) database (http://tcgadata.nci.nih.gov/) were analyzed to explore the clinical significance of miR10a in ER-positive breast cancer.
Transient transfection
MCF-7, T47D and MCF-7/TAM, T47D/TAM breast cancer cells were respectively transfected with miR10a mimic, miR10a inhibitor, RFPL-3 siRNA or nonspecific siRNA (Gene Pharma, Shanghai, China) using Lipofectamine 2000 (Invitrogen, USA) according to the manufacturer's protocol. The sequences of the siRNA oligonucleotides as follow: siRNA-RFPL-3 F:5’-GUGGGAACAAGCACAGAAUTT-3’, R:5’-AUAUCCACUUGGAACUUCCTT-3’; si-Control F: 5’-UUCUCCGAACGUGUCACGUTT-3’, R: 5’-ACGUGACACGUUCGGAGAATT-3’;
At 48 hours after transfection, RNA and protein were isolated, and the cell viability and cell proliferation were tested.
Cell viability assay
The transfected breast cancer cells (3×103 per well) were plated in 96-well plates, 10 µl of cell counting kit-8 (CCK-8) solution was added per well after 24–96 hours, and the cells were further incubated at 37°C with 5% CO2 for 3 h. Then, the absorbance at 450 nm was recorded using a microplate reader.
Cell proliferation assay
After the transfected cells being digested and centrifuged, the PicoGreen fluorescent reagent was added to the supernatant according to the manufacturer's protocols of Quant-iT PicoGreen dsDNA Reagent and Kit (Invitrogen, USA), and samples were transferred to 96-well plates. Fluorescence was measured with a microplate reader (NanoDrop 3300, Thermo Scientific, USA), and values were calculated using known DNA standards.
Dual-luciferase reporter assay
To validate RFPL-3 as a direct target of miR10a, MCF-7 cells were co-transfected with wild-type or mutant RFPL-3 3′UTR dual-luciferase reporter with miR10a agomirs or NC vector using Lipofectamine 2000 reagent in 96-well plates. After 24 h, the luciferase activity was quantified using a Dual Luciferase Assay Kit (Promega Corp., WI).
RNA extraction and quantitative real-Time polymerase chain reaction (qPCR)
Total RNA was extracted using Trizol reagent (Invitrogen, USA) and total RNA (1 µg) was reverse-transcripted into cDNA under the standard conditions. qPCR was performed by using the SYBR Green kit (Thermo Fisher, USA) according to the manufacturer’s protocol. The primers for qPCR detection are listed as follows:
miR10a F: 5’-ACCCTGTAGATCCGAATTTGTGTAA-3’, R:5’-AGAGCGGAGTGTTTATGTCAACT-3’;
RFPL-3 F:5’-GTCTGCCTCAAGTGCATCAA-3’, R:5’-AGCCTCTCTAGCTGCCGATT-3’; hTERT F:5’-ACGACGTGCTGGTTCACCT-3’, R:5’-CTCCCTGACGCTATGGTTCC-3’;
GAPDH F:5’-AGGTCGGTGTGAACGGATTTG-3’, R:5’-GGGGTCGTTGATGGCAACA-3’
U6: F:5’-TGCGGGTGCTCGCTTCGGCAGC-3’, R:5’-CCAGTGCAGGGTCCGAGGT-3’. Relative expression levels were determined using the comparative threshold cycle method (2−△△Ct).
Western blotting
Proteins from cell were isolated using the RIPA lysis buffer with protease inhibitor and 10% SDS gel electrophoresis, the samples were incubated overnight at 4°C with primary antibodies against RFPL-3 (1:2000), hTERT (1:1000), β-actin (1:3000). All the antibodies were purchased from Abcam. Secondary antibodies were then incubated for 2 h at room temperature. Protein-antibody complexes were visualized with ECL detection reagents (Thermo Fisher Scientific) and an analysis system (Bio-Rad, USA).
Animal model
The MCF-7, MCF-7/TAM and MCF-7/TAM cells with miR10a mimic were resuspended in 200 µl of PBS/Matrigel and injected into the mammary fat pads
of 4-week-old female nude mice (BALB/c) under general anaesthesia (1.2% Avertin,
0.1 ml/10 g). In each group (n = 5), the tumour volume was calculated according to the
following formula: tumour volume = 0.5×(Dmax×Dmin2). The mice were sacrificed
after three weeks, and the primary tumours were removed for further evaluation.
Statistical analysis
All experiments were performed in triplicate. All data were statistically analysed using SPSS version 13.0, and graphs were made using GraphPad Prism version 5.0 software. One-way analysis of variance and Newman-Keuls post hoc tests were used to compare variances between groups. A value ≤ 0.05 was considered to be a significant difference. Differences were considered highly significant when ≤ 0.01.