Breast cancer cell lines including BT474 and MCF7 used in the study were primarily purchased from ATCC. The cells were cultured according to the standard protocols. Cells were cultured in DMEM-F12 or 1640 with 10% fetal bovine serum with penicillin (100 U/ml), streptomycin sulfate (100 µg/ml), with or without EGF and insulin. Fibroblasts (NHDF) were purchased from Jingkang (Shanghai, China) and cultured in DMEM-F12 with 10% fetal bovine serum with penicillin (100 U/ml), streptomycin sulfate (100 µg/ml). The cells were incubated in a humidified incubator at 37 °C with 5% CO2.
Breast cancer tissues were splice into small pieces and digested with collagenase I for overnight, the cells were cultured and the conditioned medium were collected. Fibroblasts educated by TGF-β1 or cancer cells and the cell conditioned medium was collected. The conditioned medium was filtered through a 0.22-µm filter (Merck Millipore, Massachusetts, USA) to remove cellular debris, and then exosomes were extracted using the kit from Invitrogen.
Cell Proliferation Assay
Breast cells were treated with exosomes from CAFs with miR-3613-3p down-regulation and the controls. The cells were seeded in 96-well plates and the cell survival ability were assayed by CCK8 at the indicated time. The survival rates were analyzed. The cells were seeded in 6-well plates for two weeks and the colonies with 50 cells were counted and analyzed.
Cultured cells were harvested and lysed with RIPA buffer containing the protease inhibitors on ice for 30 min. Equal protein was separated by SDS-PAGE. The protein was transferred onto PVDF membrane using and probed with primary antibodies and then horseradish peroxidase–labeled secondary antibodies. The protein band signals were visualized using an ECL.
Real Time RT-PCR
Total RNA from cells or tissues was extracted using EZ total RNA isolation kit based on the manual. All the primer sequences were ordered from Ruibo (Shanghai, China). The PCR was run on the 7500 Real-Time PCR system using the following thermocycling parameters: 95 °C for 10 minutes, 40 cycles at 95 °C for 10 seconds, 60 °C for 30 seconds, 72 °C for 10 seconds, followed by a melting curve analysis. The expression of miRNA and mRNA was analyzed by comparing Ct values. GAPDH or U6 snRNA was the internal controls.
Lentivirus carrying miRNA or miRNA inhibitor
The lentiviral particles with miR-3613-3p were ordered from Genechem (Shanghai, China). Fibroblasts were transfected with the lentivirus and the knocking down effect in cells was evaluated by real time RT-PCR.
Wound Healing Assay
BT474 and MCF7 cells were treated with exosomes from CAFs infected with lentivirus with miR-3613-3p overexpression and the controls. The cells were seeded in 12-well plates. The cells were made a wound using 200 ul tips. The wound widths were measured and the migration ability was analyzed.
BT474 and MCF7 cells were treated with exosomes from CAFs infected with lentivirus with miR-3613-3p overexpression and the controls. The invaded cells were fixed in 100% methanol and then dyed with 0.1% crystal violet. The photos of the invaded cells in three fields were taken and cell invasion ability were analyzed.
A total ROS detection kit (Enzo Life Sciences) was used to detect intracellular ROS and of BT474 and MCF7 cells according to the manufacturer’s protocol . At the end of treatment, cells were stained with ROS detection solution at 37 °C for 1 h and then observed under a fluorescence microscope.
Cancer cells with 3 × 104 cells in every well were grown in a 24-well plate. The cells were co-transfected with luciferase reporter (200 ng per well), miR-3613-3p (200 ng per well), and 10 ng Renilla luciferase vector (pRL-CMV; Genomeditech, China) using Lipofectamine™ 3000 (Invitrogen, USA). The cells were cultured in the regular condition for 48 h and then the luciferase and renilla activity of these samples were measured by a Dual-Luciferase Reporter Assay kit (Promega, USA).
All quantitative experiments were repeated at least three independent biological repeats and are presented as the means ± SD (standard deviation). Quantitative data were analyzed by either one-way analysis of variance (ANOVA) (multiple groups or parametric generalized linear model with random effects. p value less than 0.05 was considered statistically significant.