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The Molecular Mechanism of Circ_DONSON Targeting MiR-671-5p on the Radiosensitivity of Lung Cancer Cells |
QIN Bo, LU Lu, ZHAO Tao |
The People's Hospital of Lichuan, Hubei Lichuan 445400, China |
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Abstract Objective: To investigate the effect of circ_DONSON on the radiosensitivity of lung cancer cells and its possible mechanism. Methods: A total of 43 cases of lung cancer tissues and adjacent tissues were collected, and real-time quantitative polymerase chain reaction (RT-qPCR) was used to detect the expression of circ_DONSON and microRNA-671-5p (miR-671-5p) in the tissues. Lung cancer cells A549 were cultured in vitro and irradiated with different doses (2 Gy, 4 Gy) of X-ray, and then RT-qPCR was used to detect the expression of circ_DONSON and miR-671-5p in the cells. After A549 cells transfected with si-NC, si-circ_DONSON, miR-NC, miR-671-5p mimic, or si-circ_DONSON and miR-671-5p inhibitor were irradiated with 4 Gy X-ray, cell counting kit-8(CCK-8) method and flow cytometry were used to detect cell proliferation activity and apoptosis, respectively; the colony formation experiment was used to detect the number of colonies formed, and the western blot method was used to detect the expression of Cleaved-caspase3 protein in cells. The dual luciferase reporter gene experiment verified the regulatory relationship between circ_DONSON and miR-671-5p. Results: The expression of circ_DONSON in lung cancer tissues was 4.27±0.38, which was higher than that of adjacent tissues (0.80±0.13, P<0.05), but the expression of miR-671-5p was 0.37±0.07, which was lower than that of adjacent tissues (0.37±0.07, P<0.05). Compared with the A549 cells of control group, the expression of circ_DONSON in A549 cells irradiated with different doses (2 Gy, 4 Gy) of X-ray were increased (3.07±0.142 Gy/3.69±0.124 Gy vs 1.00±0.00, P<0.05), but the expression of miR-671-5p were decreased (0.23±0.022 Gy/0.45±0.034 Gy vs 1.00±0.00, P<0.05). Compared with 4 Gy X-ray irradiated A549 cells of control group or A549 cells transfected with si-NC, the cell proliferation activity and the number of colony formation of 4 Gy X-ray irradiated A549 cells transfected with si-circ_DONSON were decreased (0.48±0.04 vs 1.04±0.11,1.01±0.09; 41.33±1.70 vs 85.67±2.05,86.33±1.25; P<0.05), but the apoptotic rate and the expression of Cleaved-caspase3 protein were increased [(31.18±1.39)% vs (14.24±0.80)%, (14.30±0.84)%;0.90±0.06 vs 0.41±0.04, 0.40±0.05; P<0.05]. Compared with 4 Gy X-ray irradiated A549 cells of control group or A549 cells transfected with miR-NC, the cell proliferation activity and the number of colony formation of 4 Gy X-ray irradiated A549 cells transfected with miR-671-5p mimic were decreased (0.63±0.05 vs 1.01±0.11, 1.00±0.11; 52.67±2.05 vs 85.00±2.45, 84.00±1.63; P<0.05), but the apoptotic rate and the expression of Cleaved-caspase3 protein were increased [(28.22±1.47)% vs (14.42±0.88)%, (14.61±1.02)%; 0.77±0.04 vs 0.40±0.03, 0.41±0.05; P<0.05]. circ_DONSON could target miR-671-5p, and the expression of miR-671-5p in A549 cells transfected with si-circ_DONSON was significantly higher than that in cells transfected with si-NC (4.66±0.21 vs 1.00±0.02, P<0.05). Compared with 4 Gy X-ray irradiated A549 cells transfected with si-circ_DONSON, the cell proliferation activity and the number of colony formation of 4 Gy X-ray irradiated A549 cells co-transfected with si-circ_DONSON and miR-671-5p inhibitor were increased (0.91±0.06 vs 0.50±0.04; 71.00±2.83 vs 40.67±2.49; P<0.05), but the apoptotic rate and the expression of Cleaved-caspase3 protein were decreased [(20.66±0.96)% vs (31.13±1.57)%; 0.57±0.03 vs 0.90±0.07; P<0.05]. Conclusion: Silencing circ_DONSON may enhance the radiosensitivity of lung cancer cell A549 by targeting up-regulation of miR-671-5p.
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