Clinical cancer samples and cell lines
All cancer samples were obtained from the Affiliated Tumor Hospital of XiangYa Medical School of Central South University, and stored at -80°C until analyzed. All experiments were conducted in accordance with the Declaration of Helsinki and were approved by the Xiangya Hospital Medical Ethics Committee in Central South University.
Breast cancer cell lines MCF-7,MDA-MB-231,SK-BR-3,T47D, ZR-75-1, MCF-10A and human embryonic kidney cell HEK-293 were used in the study. MCF-7 and MDA-MB-231 were obtained from NeuronBiotech (Shanghai, China). SK-BR-3, T47D, ZR-75-1 and MCF-10A were obtained from Dingguo, Co. (Beijing, China). HEK-293 was obtained from Xiangya experiment center (Changsha, China). All the cells were cultured in complete DMEM high glucose medium (Hyclone, Logan, UT, USA) supplemented with 10% FBS (Hyclone) and 1% penicillin and streptomycin sulfate (Solarbia, Co., Beijing, China). Cells were incubated at 37°C with 5% CO2 and medium was changed every 2 or 3 days.
Virion and cell line constructions
To establish the miRNA overexpression cell lines, partial mir-96, mir-182 and mir-183 pri-microRNA sequences flanked by EcoRI and AgeI restriction sites were inserted into the CMV promoter of lentivirus infectious virions pLKD-CMV-G&PR-U6-shRNA (Hpcoo3) (Additional file 1: Figure S1A). MCF-7 or T47D cells were infected with these viruses and selected under the pressure of 1 μg/ml puromycin (Invitrogen, San Diego, CA, USA). The green fluorescent protein (GFP) signal of the infected cells was detected under microscope (Additional file 1: Figure S1B), and the expression of the miR-183/-96/-182 cluster in each cell line was measured by reverse transcription (RT)-PCR (Additional file 1: Figure S1C).
To disrupt the activity of the miR-183/-96/-182 cluster, we generated miR-183/-96/-182 cluster sponge lentivirus virion. Basically, 10 copies each of complementary sequences to miR-183, miR-96 and miR-182, each with mismatches at positions 9 to 12 for improved stability [20],[21], were introduced into the pLOV-CMV-eGFP-EF1a-PuroR lentivirus infective virion (Additional file 2: Figure S2). A moderate multiplicity of infection (MOI) of 1 was used for transduction. The infection efficiency and cell morphology were monitored under microscope every day. After 3 days of transduction, cells were collected for cell cycle analysis and RNAs were collected for real-time PCR.
To research the function of transcription factors, the coding sequences of HSF2 and ZEB1 flanked by XhoI and KpnI restriction sites were inserted into vector GV219. The plasmids were transfected into MCF-7 cells and the cells were selected with a culture medium containing 600 μg/ml G418-Geneticin (GenView, Galveston, TX, USA) for 2 months.
LNA-based Northern Blotting
Total RNAs were extracted from cancer samples with the mirVanaTM miR isolation kit and 10 μg of total RNA was used for each assay. All procedures followed manufacturer's instructions for the miRCURY LNA™ microRNA detection probes (Exiqon, Woburn, MA, USA). After fractionation by electrophoresis on a denaturing 12% polyacrylamide gel containing 8 M urea, RNAs were transferred to Nytran N membrane (Amersham Biosciences, Piscataway, NJ, USA) and fixed by UV crosslinking. Blots were prehybridized for 1 h at 45°C in PerfectHyb™ Plus Hybridization Buffer (Sigma, St Louis, MO, USA) and hybridized overnight at 45°C in hybridization buffer containing 0.1 nM probe, then washed twice for 30 minutes at 65°C in 0.1SSC/0.1% SDS. As the probes were 5'-DIG labeled, we detected the signal by PhototopeR-Star Kit (New England BioLabs Inc, Ipswich, MA, USA), and the densities were quantified by the Image J program. Because the miR-183, miR-96 and miR-182 sequences are similar, we tested the probe specificities before doing the experiments (Additional file 3: Figure S3). Mimic oligonucleotides were designed based on miRNA sequences registered in the miRBase Sequence Database (see Additional file 4: Table S1).
RT-PCR and real-time PCR
For mRNA RT-PCR and real-time PCR, total RNAs were extracted from cancer samples or cultured cells with Trigol (Dingguo, Co.) reagent. Primer sets were designed within the exon junction areas listed in Additional file 4: Table S2. For miRNA real-time PCR, miRNAs were extracted from cells using a mirVana miRNA isolation kit (Ambion, Austin, TX, USA). All primers, including the YRBIO™ miRNA qPCR Detection primer sets and U6 snRNA PCR primer set were purchased from Yingrun Biotechnology (Changsha, China).
In brief, mRNA and miRNA were reverse-transcribed with an M-MLV First Strand kit (Invitrogen). Then 50 ng cDNA was mixed with All-in-one™ qPCR Mix (Genecopoeia, Rockville, MD, USA) and the target gene primer set (final concentration: 1 μM for each primer) to produce a 20-μl reaction mixture. All real-time PCR experiments were carried out with an ABI Step One Plus Real-time PCR System (Applied Biosystems, Carlsbad, CA, USA). All real-time PCR reactions were done in triplicates, and the average ΔCT (Δ cycle threshold) for the triplicates was used in subsequent analysis.
Plasmid, miR-Down™ antagomir and transfection
Large-scale plasmids were extracted by PureYield™ Plasmid Midiprep System (Promega, Madison, WI, USA), and small-scale plasmids were extracted by Mini DNA purification kit (Dingguo). Chemically modified antisense oligonucleotides (miR-Down™ antagomir, GenePharm Co. Ltd, Shanghai, China) were used to inhibit miR-96, miR-182 and miR-183 expression. A scrambled oligonucleotide was used as control. Plasmid and miR-Down™ antagomir transfections were conducted with Lipofectamine™ 2000 reagent (Invitrogen).
Luciferase reporter assays
For promoter analysis, promoter region sequences or their mutants flanked by XhoI and KpnI restriction sites were inserted into the upstream region of luciferase reporter gene in pGL3-Basic vector (Promega). MCF-7 cells were transfected with 200 ng reporter construct and 1 μg GV219 vector with or without transcription factor sequence. Also, 40 ng of pRL-CMV-Renilla plasmid was transfected as an internal control.
For target analysis, 33 bp of RAB21 3'-UTRs including the seed sequence were flanked by XbaI and FseI restriction sites and inserted between the Luciferase coding sequence and SV40 polyadenylation element in pGL3-Promoter vector (Promega). HEK-293 cells were transfected with 200 ng reporter construct and 1 μg Hpcoo3 vector with or without partial pri-microRNA sequence of miR-183/-96/-182 cluster. Also, 40 ng of pRL-CMV-Renilla plasmid was transfected as an internal control.
The luciferase reporter assays (Promega) were performed 48h after transfection, and luciferase activity was determined with a GloMax 20/20 Luminometer (Promega). Relative luciferase activities were calculated as ratios of firefly to renilla luciferase activities.
Assays: 3-(4, 5-dimethyl-2-thiazolyl)-2, 5-diphenyl-2H-tetrazolium bromide (MTT)
Cells were seeded on 96-well plates (5 × 103 cells per well) and incubated for 24 h in 0.2 ml medium. After reaction with 20 μl 5 mg/ml sterile MTT (Sigma) for 4 h at 37°C, culture media was removed and 150 μl of dimethyl sulphoxide (DMSO) was added. The absorbance was measured with the ELISA reader (BioTek, Vermont, VT, USA) at 490 nm and 540 nm and the reactions were performed in triplicates.
Cell wound-healing assays
Cells were seeded on 6-well plates (5 × 105 cells per well) and incubated for 24 h. Adherent cell monolayers were scratched with a 10-μl pipette tip and cultured in 2 ml DMEM high-glucose medium without FBS or antibiotics. Cell migration was monitored under microscopy later.
Colony formation assays
The culture dish was covered by 2 ml bottom gel (0.5% basic agar in RPMI medium 1640 (Invitrogen) supplemented with 10% FBS and 1% penicillin/streptomycin) and 1.5 ml top gel (0.7% agar in RPMI-1640 medium supplemented with 10% FBS and 1% penicillin/streptomycin) mixed with 10,000 cells. Cells were incubated for 16 days and the colonies were stained with 0.5ml 0.005% crystal violet overnight followed by washing with PBS (Hyclone) three times. The pictures of cell colonies were taken by a digital camera.
Cell cycle analysis
Cells were digested with 0.05% trypsin (Thermo Scientific, Logan, UT, USA) for 2 minutes to dissociate them from the plates. After fixation in 70% pre-chilled (−20°C) ethanol in PBS at 4°C overnight, cells were treated with 10 μg/ml of RNase (Auragene, Co., Shenzhen, China) in PBS at 37°C for 2 h and stained with 50 μg/ml of propidium iodide (PI) (Sigma) for 5 minutes. Flow cytometry was conducted on a BD FACSCalibur flow cytometer (BD Biosciences, Franklin, IN, USA) and data were analyzed by ModFit LT software.
Western blotting
Total proteins were lysed in RIPA buffer (150 mM NaCl, 0.1% SDS, 0.5% sodium deoxycholate, 1% NP-40 and 50 mM Tris-HCl, pH 7.6) with a proteinase inhibitor cocktail (Roche, Mannheim, Germany). After separation by 15% polyacrylamide gels and transfer to 0.45 μM membrane (Millipore, Billerica, MA, USA), proteins were detected by anti-RAB21 (Abcam, HongKong, China) and anti-β-tubulin (Sigma) antibodies.
Phalloidin and 4',6-diamidino-2-phenylindole (DAPI) staining
For imaging of fixed cells, cells were seeded on acid-washed, glass coverslips coated with 5 μg/ml of collagen. Cells were then fixed with 3.7% paraformaldehyde in PBS permeabilized with 0.2% Triton X-100 in PBS for 15 minutes. Then we co-stained the cells with fluorescein isothiocyanate (FITC)-conjugated phalloidin (Beyotime, Shangai, China) to detect the F-actin, and with DAPI (Invitrogen) to detect the nuclear. Coverslips were mounted with Microscopy Aquatex® mounting medium (Merck, Darmstadt, Germany), and then detected under the Leica Tcs-sp5-II confocal microscope (Leica, Wetzlar, Germany).
Statistical analysis
Data were expressed as means ± SD, and the statistical software SPSS 11.5 (IBM, Armonk, NY, USA) was used for analysis of variance (ANOVA) and analysis using Student's t-test. Statistical probability (P) in tables, figures, and figure legends are expressed as follows: *P <0.05, **P <0.01, *** P <0.001.