Linear accelerator

Cells were seeded at a density of 5×10³ cells/well in 96-well cell culture plates and incubated overnight in 100 μL of RPMI 1640 medium. In the following days, DMSO, IH, FZ or IH+FZ at different concentrations were added 4 hours before irradiation (0 Gy, 2 Gy, 4 Gy and 6 Gy) with 6 MV X-rays at a dose rate of 2 Gy/min generated by a linear accelerator (Siemens, Erlangen, Germany). To measure the cell viability at the indicated time point, 10 μL of CCK-8 reagent (Dojindo Laboratories, Kumamoto, Japan) was added to each well, and the cells were incubated at 37°C for 2.5 hours. Optical density (OD) values at a wavelength of 450 nm were measured by a microplate reader (Thermo Fisher Scientific).

Human breast cancer cell lines MCF-7 (ATCC HTB-22) and MDA-MB-231 (ATCC HTB-26) were provided by the Breast Cancer Institute, Cancer Hospital, Fudan University. They were originally purchased from the American Type Culture Collection (ATCC, Manassas, USA). The MCF-7 cells were ER⁺ and HER2⁻, and the MDA-MB-231 cells were ER⁻ and HER2⁻. The MDA-MB-231 cells were cultured in RPMI 1640 medium, and the MCF-7 cells were cultured in Dulbecco’s modified Eagle’s medium (DMEM). All culture media were supplemented with 10% fetal bovine serum, 100 U/mL penicillin, and 100 mg/mL streptomycin. Both cell lines were grown as attached cultures at 37°C in a humidified atmosphere containing 5% CO₂. During irradiation, the culture flasks were placed flat on a treatment bed on the top of a plexiglass plate with a thickness of 1.3 cm. Irradiation was performed using a linear accelerator (Siemens, Munich, Germany) with 6 MV-X ray irradiation and a source skin distance of 100 cm. The angle of the accelerator during irradiation was 180°, and the rays entered through the bottom of the flask. The actual dose absorption rate was 3–5 Gy/min measured using a dosimeter before irradiation.

Linear accelerator (Siemens, Munich, Germany) was used for irradiation. Cells were treated with 200 cGy/min dose rate in room temperature to reach a required total dose using. IR group contain fifteen mice, consist of five randomly selected nude mice in each different miR-30a-5p expression group (10/group). Tumor-bearing mice in the IR group were treated with 2.0 Gy irradiation for 5 consecutive days from day 21 to 25, and achieved a total dose of 10.0 Gy irradiation.