The antibiotic solution (containing 10,000 U/mL penicillin and 10 mg/mL streptomycin), trypsin-EDTA mixture (containing 0.05% trypsin and EDTA), FBS (fetal bovine serum), donkey anti-mouse-Alexa 488, goat anti-rabbit-Alexa 488, and goat anti-mouse-Alexa 594 were obtained from Invitrogen (Carlsbad, CA, USA). Rabbit polyclonal anti-MDR (H-241, sc-8313) was purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA). Mouse monoclonal anti-actin, clone C4 (MAB1501) was purchased from Millipore (Billerica, MA, USA). The mouse monoclonal anti-CD63 monoclonal (H5C6) antibody was obtained from BD Pharmingen (San Diego, CA, USA). Hoechst 33258 dye was obtained from Dojindo (Kumamoto, Japan). Antigen activation of the tissue microarray was achieved using a protease (#415231, Nichirei, Japan). The duplexes of each siRNA targeting human CD63 mRNA (si CD63-1, GGUGGAAGGAGGAAUGAAAdTdT, UUUCAUUCCUCCUUCCACCdTdT; CD63-2, GGCAGCAGAUGGAGAAUUAdTdT, UAAUUCUCCAUCUGCUGCCdTdT; CD63-3, GUGGCUACGAGGUGAUGUAdTdT, UACAUCACCUCGUAGCCACdTdT) were purchased from BONAC Corporation (Fukuoka, Japan). The siRNA duplexes targeting human RPN2 mRNA (GGCCACUGUUAAACUAGAACA, UUCUAGUUUAACAGUGGCCUG) were purchased from Sigma-Aldrich (St. Louis, MO, USA), and the AllStars Negative Control siRNA was obtained from Qiagen (Valencia, CA, USA).
MDA-MB-231-luc-D3H2LN (MM231-LN) cells were purchased from Xenogen (Alameda, CA), and multidrug-resistant MCF7-ADR cells were provided by Shien-Lab, Medical Oncology, National Cancer Center Hospital of Japan. These cells were maintained in RPMI 1640 medium (Invitrogen, Carlsbad, CA, USA) supplemented with 10% heat-inactivated FBS and antibiotic-antimycotic at 37°C in 5% CO2.
Transient transfection assays
Transfection of siRNA was accomplished using DharmaFECT transfection reagent (Thermo Scientific, Waltham, MA, USA) according to the manufacturer’s protocol. AllStars Negative Control siRNA was used as the negative control (N.C.).
Cell proliferation assay (MTS assay)
Five thousand cells per well were seeded in 96-well plates. The following day, the cells were transfected with siRNAs. After 1, 2 or 3 days of culture, cell viability was measured using a cell counting Kit-8 (Dojindo, Kumamoto, Japan) according to the instructions of the manufacturer. The absorbance at 450 nm was measured using an Envision multilabel plate reader (Wallac, Turku, Finland).
MM231-LN and MCF7-ADR cells (2 × 105 cells in a 6-well plate) were transfected with control, RPN2 or CD63 siRNA as described above. After 2 days in culture, cells were collected, and proteins were extracted with M-PER (Thermo Scientific). Caspase-3/7 activity was assessed using an Apo-ONE Homogeneous Caspase-3/7 Assay system (Promega, Wisconsin, USA) at an excitation wavelength of 480 nm and an emission wavelength of 520 nm using an Envision system (Wallac).
Transwell invasion assay
Breast cancer cell invasion was assayed in 24-well Biocoat Matrigel™ invasion chambers (8 μm; BD Pharmingen, San Diego, CA, USA) according to the manufacturer’s protocol. Briefly, after the transfection of siRNA into the cells, 20,000 cells were plated in the upper chamber containing RPMI 1640 medium without FBS on the following day. The lower chambers were filled with RPMI 1640 medium with 10% FBS as a chemoattractant. Twenty-two hours later, the low-invasive cells were removed with a cotton swab. The cells that migrated through the membrane and adhered to the lower surface of the membrane were fixed with methanol and stained with Diff Quick staining solution (Sysmex, Kobe, Japan). For quantification, the cells in four random fields were counted using a microscope. All assays were performed in triplicate, and the invasive values were normalized to the values from cells transfected with the AllStars Negative Control siRNA.
Isolation of mRNAs and quantitative real-time PCR (qRT-PCR)
Total RNA was extracted from cultured cells using a miRNeasy Mini Kit (Qiagen, Valencia, CA, USA) according to the manufacturer’s protocol. The qRT-PCR method has previously been described . PCR was performed in 96-well plates using a 7300 Real-Time PCR System (Applied Biosystems, Foster City, CA, USA), and all reactions were performed in triplicate. TaqMan® qRT-PCR kits and human-CD63 and human-β-actin TaqMan® Expression Assays were purchased from Applied Biosystems (Foster City, CA, USA). Reverse transcription (Applied Biosystems, Foster City, CA, USA) and TaqMan® quantitative PCR (Applied Biosystems, Foster City, CA, USA) were performed according to the manufacturer’s instructions. SYBR® Green I qRT-PCR was performed, and the β-actin housekeeping gene was used to normalize the variation in the cDNA levels. The primer sequences are as follows (shown 5′ to 3′): human β-actin, GGCACCACCATGTACCCTG (Forward) and CACGGAGTACTTGCGCTCAG (Reverse); and human RPN2, ATCTAACCTTGATCCCAGCAATUGTG (Forward) and CTGCCAGAAGCAGATCTTTGGTC (Reverse).
SDS-PAGE gels were calibrated using Precision Plus protein standards (161–0375) (Bio-Rad, Hercules, CA, USA), and anti-CD63 (1:200) and anti-actin (1:1,000) were used as the primary antibodies. The dilution ratio of each antibody is indicated in parentheses. A peroxidase-labeled anti-mouse secondary antibody was used at a dilution of 1:10,000. The bound antibodies were visualized using chemiluminescence with an ECL Plus Western blotting detection system (GE HealthCare, Piscataway, NJ, USA), and luminescent images were captured using a LuminoImager (LAS-3000; FujiFilm Inc., Tokyo, Japan).
After being washed three times with PBS, the cells were fixed in 4% paraformaldehyde (Wako, Japan) and incubated in 0.1% BSA containing primary antibodies (anti-CD63 (1:500) and anti-MDR (1:500) for 1 hour. The cells were then incubated in 0.1% BSA containing Alexa Fluor fluorescent secondary antibodies. Nuclei were visualized with Hoechst 33258 dye (Dojindo, Kumamoto, Japan). All staining was observed using a confocal microscope (FluoView FV1000; Olympus, Tokyo, Japan).
Cell membrane labeling
MDA-MB-231-luc-D3H2LN and MCF7-ADR cells were transfected with control or RPN2 siRNA. After 2 days in culture, cells were labeled with a PKH26 red fluorescent labeling kit (Sigma Aldrich). Cells were observed using confocal microscopy (FluoView FV1000; Olympus, Tokyo, Japan). Nuclei were visualized via Hoechst 33258 (Dojindo, Kumamoto, Japan) staining.
The tissue arrays of breast cancer samples (BR1503b, BR10010a) were purchased from Biomax US. The company provided certified documents that all human tissue samples were collected with informed consent from the donors or their relatives. Detailed information on all tumor samples can be found at http://www.biomax.us/. The tissue microarrays were incubated in 0.1% BSA containing primary antibodies, including anti-CD63 (1:500) and anti-MDR (1:500), for 1 hour after a 5-min protease treatment (Nichirei, Tokyo, Japan). The cells were then incubated in 0.1% BSA containing Alexa Fluor fluorescent secondary antibodies. Nuclei were visualized using Hoechst 33258 (Dojindo, Kumamoto, Japan) staining for observation using a confocal microscope (BZ-9000; Keyence, Tokyo, Japan).
The data presented in bar graphs are the mean and s.e.m. of at least three independent experiments. Statistical analyses were performed using Student’s t-test. Associations between lymph node metastasis or MDR expression and CD63 expression were assessed by means of a chi-square test. The statistical analysis was two-sided, and P < 0.05 was considered to be significant.