Patients and specimens
Blood samples were collected from 57 breast cancer patients diagnosed by histopathological examination at The Central Hospital Of Enshi Tujia And Miao Autonomous Prefecture. All blood samples were centrifuged at 3000 g for 10 min after collecting for 1 h, and the supernatant serum was collected using RNase-free tubes and stored at -80℃ until used. All patients only received trastuzumab-based neo-adjuvant chemotherapy and were classified into trastuzumab-resistant (Non-response, N=30) and tamoxifen-sensitive (Response, N=27) depending on the sensitivity to trastuzumab.
Cell culture
Human breast cancer cell lines SKBR3 and BT474 were purchased from Shanghai Academy of life Science (Shanghai, China) and cultured in RPMI-1640 medium (Gibco, Carlsbad, CA, USA) harboring with 15% fetal bovine serum (FBS) and 0.1 IU/mL insulin at 37℃ with 5% CO2. Trastuzumab resistant breast cancer cells, named SKBR3-TR and BT474-TR were established by continuously exposing parental cells to increasing concentration of trastuzumab (Sigma, St. Louis, MO, USA) for more than 6 months until cells displayed resistance to trastuzumab. Trastuzumab resistant cells were maintained in the same media supplemented with 3 μg/mL trastuzumab.
Cell viability assay
Parental or resistant cells (5000 cells/well) were seeded in 96-well plates overnight. Following transfection or trastuzumab treatment (0, 0.3125, 0.625, 1.25, 2.5, 5, or 10 μg/mL) for additional 48 h, cells in 96-well plates were incubated with 10 μL cell counting kit-8 solution (Sigma) for 4 h at 37℃. The optical density (OD) at 450 nm was determined by a microplate reader and the half maximal inhibitory concentration (IC50) value of trastuzumab was assessed on the basis of the relative survival curve.
Cell transfection
The miR-381-3p mimic (miR-381-3p), small interfering RNA (siRNA) targeting OIP5-AS1 (si-OIP5-AS1#1, si-OIP5-AS1#2, si-OIP5-AS1#3), pcDNA3.1 OIP5-AS1 overexpression vector (oe-OIP5-AS1), pcDNA3.1 HMGB3 overexpression vector (HMGB3) and their corresponding negative control (miR-NC, si-NC, Vector) were synthesized by Genepharma (Shanghai, China). The transfection of cells was performed using LipofectamineTM 2000 transfection reagent (Invitrogen, Carlsbad, CA, USA).
Quantitative real-time polymerase chain reaction (qRT-PCR)
Whole-RNA extracts from parental or resistant cells were prepared using TRIzol reagent (Invitrogen), and exosomal RNAs were isolated with the exoRNeasy Midi Kit (Qiagen, Valencia, CA, USA) according to the standard procedure. Complementary DNA (cDNA) was synthesized using SuperScript III® (Qiagen), and quantitative PCR was performed using SYBR Premix Ex Taq (Qiagen) on the Bio-Rad CFX96 Sequence Detection system (Bio-Rad, Hercules, CA, USA). The expression levels were detected by 2-△△Ct method with glyceraldehyde 3-phosphate dehydrogenase (GADPH) or U6 small nuclear B noncoding RNA (U6) serving as an internal reference. The primer sequences were listed as follows: OIP5-AS1: F, 5’-TGCGAAGATGGCGGAGTAAG-3’ and R, 5’-TAGTTCCTCTCCTCTGGCCG-3’; miR-381-3p: F, 5’-TAATCTGACTATACAAGGGCAAGCT-3’ and R, 5’-TATGGTTGTTCTGCTCTCTGTCTC-3’; GADPH: F 5’-GAGAAACCTGCCAAGTATGATGAC-3’ and R 5’-GGAGTTGCTGTTGAAGTCAC-3’, U6: F, 5’-CTCGCTTCGGCAGCACA-3’ and R, 5’-AACGCTTCACGAATTTGCGT-3’.
Colony formation assay
Transfected parental or resistant cells (5000/well) suspended in RPMI-1640 medium with 0.5 μg/mL trastuzumab were seeded in 6-well plates. After 21 days of cultures at 37℃ with 5% CO2, cell colonies were fixed with methanol and stained with 0.1% crystal violet. Finally, the number of visible colonies (≥50 cells) was counted.
Cells migration assay
The migratory capacity of cells was performed by a 24-well transwell chamber (8 μm; Corning Costar, Cambridge, MA). Transfected cells suspended in serum-free RPMI-1640 medium were filled the top chambers. Then 500 μL RPMI-1640 medium mixed with 10% FBS was added into the lower chambers. 24 h later, cells on the lower face of the membranes were fixed and stained. Finally, migrated cells in five random fields were counted with a microscope.
Flow cytometry
Annexin V-fluorescein isothiocyanate (FITC)/propidium iodide (PI) apoptosis detection kit (BD Biosciences, San Jose, CA, USA) was employed to detect cell apoptosis. In brief, after transfection with the designed vector for 48 h, cells were interacted with 5 μL FITC annexin V and 10 μL PI. Finally, the apoptotic rate was measured by a flow cytometer.
Exosome (exo) isolation
Exosomes were isolated from serum samples or cells using ultracentrifuge method. Cell culture fluid from exosome-depleted medium or serum was centrifuged at 3000 g for 30 min at 4℃ to remove cell fragments. Then the resulting supernatant was further centrifuged at 100,000 g for 70 min at 4℃, and filtered using 0.22 μm filtration. Subsequently, pelleted exosomes was washed with PBS, and centrifuged at 100,000 g for 70 min again. Finally, purified exosomes were resuspended in PBS for the detection of the size and quality of exosomes using nanoparticle tracking analysis (NTA) software or for functional assays. Exosomes pellets were interacted with Trizol reagent to isolate RNA, and were lysed with RIPA lysis buffer used for protein detection. For blocking of exosome release, parental or resistant breast cancer cells were treated with GW4869 (10 μM) or Vehicle (as control) for 48 h. For exosome co-cultures, exosomes (50 μg/ml) were incubated with parental SKBR3 and BT474 cells (5 × 105) in a 6-well plate with 10% FBS exosome-depleted culture medium for 48 h.
Transmission electron microscopy (TEM)
Purified exosomes were dropped on the carbon-coated copper grid and allowed to absorb for 5 min at 37℃, and then stained with 2% phosphotungstic acid solution for 2 min, followed by washing with PBS for 3 times. After air-dried, the grid was visualized using a transmission electron microscope (TEM) (JEOL, Akishima, Japan).
Western blot
Proteins were extracted from cells or exosomes using RIPA lysis buffer (Beyotime, Beijing, China) and quantified determined using a bicinchoninic acid (BAC) Protein Assay Kit (Beyotime). Extractive protein was loaded on sodium dodecyl sulfate polyacrylamide gel electrophoresis for separation, and then shifted onto polyvinylidene fluoride membranes. Afterwards, membranes were interacted with CD81 (ab79559, 1: 1000, Abcam, Cambridge, MA, USA), CD63 (1:2000, ab68418, Abcam), TSG101 (ab125011, 1:5000, Abcam), HMGB3 (1:1000, #6893, Cell Signaling Technology, Beverly, MA, USA) and the secondary HRP-conjugated antibody (1:1000, ab9482, Abcam). The β-actin (1:1000, #4970, Cell Signaling Technology) was used as an internal reference. The protein bands were visualized using the Image J software.
Dual-luciferase reporter assay
The predicted potential binding sequences of miR-381-3p in OIP5-AS1 and HMGB3 3’-untranslated (3’UTR) regions and their mutated sequence were separately cloned into pmirGLO Dual-luciferase vectors (Promega, Madison, WI, USA). Subsequently, these constructed vectors were co-transfected into SKBR3 and BT474 with miR-381-3p mimics or miR-NC using LipofectamineTM 2000 (Invitrogen). The luciferase activities were detected using a dual luciferase assay kit (Promega).
Pull-down assay
Biotin (Bio)-miR-381-3p and Bio-NC synthesized by Genepharma Company were transfected into SKBR3 and BT474 for 48 h. Then, cells were lysed, and the lysates were incubated with M-280 streptavidin magnetic beads (Invitrogen). After elution, the bead-bound RNA complex was purified and subjected for qRT-PCR analysis.
Xenograft experiments in vivo
Female BALB/c mice (Five-week-old) from Jinan Pengyue Animal Center (Jinan, China) were randomly divided into four groups (N = 5 each). Each group was injected with BT474 cells (1 × 106) in the flank region of mice. When tumors grew to 100 mm3, groups 2, 3, and 4 were intratumorally injected with trastuzumab (3mg/kg) every 2 days, and the negative control of group 1 was injected with PBS; besides, isolated exosomes (10 μg) from BT474-TR cells loaded with si-OIP5-AS1 lentiviral vector (si-OIP5-AS1#1) or si-NC lentiviral vector (si-NC) were injected into the center of tumor of groups 4 and 3 every two days, respectively. Tumor volume was calculated every 4 days. At day 32, all mice were sacrificed and tumor masses were weighed and harvested for further molecular analysis.
Statistical analysis
Numerical results from three independent experiments were manifested as the mean ± standard deviation (SD). The statistical difference between each group was analyzed by Student’s t test or one-way analysis of variance (ANOVA) with GraphPad Prism 7 software. Receiver operating characteristic (ROC) curves were plotted to analyze the diagnostic value of exosomal OIP5-AS1. P values less than 0.05 was considered as statistically significant.