Fig 1: In radiation-treated HCC cells, miR-216a-3p knockdown reduces oxidative stress and partially decreases the apoptosis induced by Que. (A) The effect of miR-216a-3p knockdown on the mitochondrial membrane potential of Que-treated cells was determined by measuring the cells’ red/green fluorescence ratios; (B) The impact of miR-216a-3p knockdown on HCC cell apoptosis was investigated using flow cytometry and Annexin V-FITC/PI double labeling; (C–E) The effect of miR-216a-3p expression on the levels of Cleaved PARP/PARP, Bax, and Bcl-2 was measured by Western blotting; (F) Immunofluorescence analysis was used to examine the levels of γ-H2AX; (G) The effect of miR-216a-3p levels on ROS levels in HepG2 and Huh-7 cells was measured by flow cytometry; (H–J) ELISA revealed the impact of miR-216a-3p levels on the content of oxidative stress indicators MDA, SOD, and GSH-Px in HepG2 and Huh-7 cells. HCC: Hepatocellular carcinoma; Que: Quercetin; IR: Irradiation; SOD: Superoxide dismutase; MDA: Malondialdehyde; GSH-Px: Glutathione peroxidase. * indicates P<0.05, ** indicates P<0.01, *** indicates P<0.001.
Fig 2: Que influences HCC growth and radiosensitivity by modulating miR-216a-3p. (A) HepG2 cells were implanted into mice to create a subcutaneous transplantation tumor model. Mice were given 50 mg/kg Que and 4 Gy 6MV-X-rays according to the experimental design, and the size of the subcutaneous tumors was evaluated using vernier calipers on the 21st and 28th days of rearing; (B) On day 28, the mice were euthanized, and the tumors were extracted and photographed; (C) The tumors were weighed on day 28; (D) Immunohistochemistry was used to observe the expression levels of Ki67 and Cleaved-caspase 3 proteins in the tumor tissues of mice from each group; (E) TUNEL labeling was used to detect apoptosis in each group’s tumor tissues; (F) Western blotting was used to detect Ki67, Cleaved PARP/PARP, and Cleaved caspase 3/caspase 3 protein levels in tumor tissues; (G) ROS, MDA, SOD, and GSH-Px levels were detected using an ELISA kit in tumor tissues of each group; (H) miR-216a-3p expression was detected using qRT-PCR in the tumor tissues of each group. HCC: Hepatocellular carcinoma; Que: Quercetin; IR: Irradiation; SOD: Superoxide dismutase; MDA: Malondialdehyde; GSH-Px: Glutathione peroxidase. *** indicates P<0.001.
Fig 3: Que accelerates oxidative stress and apoptosis in radiation-treated HCC cells. (A) The ratio of red fluorescence (JC-1 aggregates) to green fluorescence (JC-1 monomers) in HepG2 and Huh-7 cells treated under different conditions was examined using JC-1 fluorescence labeling, and the mitochondrial membrane potential of the cells was determined; (B) Annexin V-FITC/PI double labeling was used in flow cytometry to determine apoptosis in HCC cells under various treatment conditions; (C–E) Western blot analysis was used to assess differences in the expression of Cleaved PARP/PARP, Cleaved caspase 3/caspase 3, Bax, and Bcl-2 between groups; (F) Immunofluorescence was used to examine the levels of γ-H2AX; (G) ROS levels in HepG2 and Huh-7 cells were detected using flow cytometry under different treatment conditions; (H–J) ELISA measured changes in the levels of oxidative stress indicators MDA, SOD, and GSH-Px in HepG2 and Huh-7 cells under various treatment conditions. HCC: Hepatocellular carcinoma; Que: Quercetin; IR: Irradiation; SOD: Superoxide dismutase; MDA: Malondialdehyde; GSH-Px: Glutathione peroxidase. * indicates P<0.05, ** indicates P<0.01, *** indicates P<0.001.
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