Place radiosensitive HepG2 cells (Cell Resource Center, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences, Shanghai, China) in 10% fetal bovine serum (Hangzhou Siji Qing Biological Engineering Materials Co., Ltd., Hangzhou, China) and double antibodies (penicillin 100 U/mL, streptomycin 100 µg/mL) in RPMI 1640 medium (Sigma-Aldrich, USA) in 5% CO2, 37 ℃ incubator. Establishment of radiation-resistant cell line (RR-HepG2): Induced by fractional radiotherapy radiation incremental method, using 60Co at a dose of 5 Gy and a dose rate of 0.5 Gy/min to irradiate HepG2 cells to a cumulative exposure of 60 Gy.
When the confluence of HepG2 cells reaches about 70%, refer to the instructions and use TM with Lipofectamine 2000 transfection reagent (Invitrogen, USA). Transfect miR-520d-5p mimics, si-CP, CP-OV into HepG2 cells respectively, continue to culture and discard the transfection medium, and add conventional medium to culture overnight to observe the transfection efficiency under an inverted fluorescence microscope.
RNA isolation and quantitative reverse transcription-PCR (qRT-PCR)
Total RNA from the cells and tissues were extracted by using TRIzol (Invitrogen, USA). Complementary DNA was synthesized from SuperScript first strand synthesis system (Invitrogen). Quantitative PCR analysis was performed on the Applied Biosystems 7300 using IQTMSYBR Green SuperMix (Bio-Rad, Hercules, CA, USA).
Clone formation experiment to detect radio sensitivity
Dilute the gradient multiple dilution method to dilute HepG2 and RR-HepG2 cells to 1 × 104 cells/mL. According to the increase of irradiation dose, different numbers of cells were inoculated overnight, and 24 hours after transfection, irradiation (0, 2, 4, 6, and 8 Gy) was given. The cells were changed every 3 days, cultured for 10 to 14 days, the medium was discarded, and washed twice with PBS; 400 µL of fast Giemsa stain reagent was added to each well for 2 minutes; then, 800 µL Giemsa stain reagent was added to each well to stain for 8 min, rinse the staining solution under running water, and dry naturally. Observe the colonies of ≥ 50 cells under the light microscope, (planting efficiency, PE) = number of clones/number of inoculated cells × 100%, survival fraction (SF2) = number of colonies in the irradiation dose group/( The number of cells inoculated in this group × PE in the unirradiated group). A single-click multi-target model was used to fit the cell survival curve, SF = 1-(1-e-D/D0) N, and Dq = D0 × lnN. Where D is the irradiation dose (Gy), D0 is the average lethal dose, Dq is the quasi-threshold dose (representing the breadth of survival), and N is the extrapolated value. Radiation sensitization enhancement ratio (SER) = D0 in the simple irradiation group/D0 in the combined irradiation group. The mean analysis was carried out with the survival scores of three exposures.
The cells were grown at 2–4 × 104 cells/well in 96-well microplates. CCK-8 assay (Sigma Chemical Co., St. Louis, MO) was subsequently added to medium to (0.5 mg/mL) a final concentration and incubated for 4 h at 37 °C. The absorbance was measured at 450 nm.
Dual-luciferase reporter assay
CP 3’UTR with or without mutation were subcloned into pGL3 promoter plasmid. 3 × 104 cells/mL were seed into 24-well culture plates in phenol red-free medium. After transfection with miRNA precursor control pre-miRNA (pre-miR-co) or pre-miR-520d-5p (Biomics Biotech) for 48 h, the luciferase activity was determined by the Dual Luciferase Assay Kit (Promega, Madison, WI, USA) according to the manufacturer’s instructions.
Flow cytometry to detect apoptosis
Collect cells (1 × 106 cells/mL) 48 h after irradiation, centrifuge at 1000 r/min for 5 min, then discard the medium, wash once with PBS, discard PBS; add 250 µL PBS to resuspend the cells, and finally add 10 µL annexin V-EGFP and 5 µL PI, mix well, incubate at room temperature in the dark for 15 min, and detect by flow cytometry.
Western blotting assay
Total proteins from cells or tissues were western-blotted using the monoclonal antibody against CP, Bcl-2, Bax and caspase-9 (all 1:500, Santa Cruz, USA). β-actin (1:5000, Sigma, USA) served as a loading control. Horseradish peroxidase (HRP)-labeled secondary antibody (1:1000, Sigma, USA) was used and incubated for 1 h at 25 °C. The band densities were quantified by the LICOR Odyssey infrared imaging system (LICOR Bio- science, Nebraska, USA).
To measure differences of groups, one-way ANOVA with a Bonferroni post hoc test, Student’s t-test, or Wilcoxon’s signed rank test was applied. All the experiences were performed at least three times and the results were expressed as means ± standard deviation (SD). P < 0.05 was considered statistically significant. GraphPad Prime 8.0 was used to draw the survival curve and the single-machine multi-target mode and L-Q linear model curve fitting to find the D0, Dq, N, SF2, k, SER.