| Objective: To clarify the radioresistance of A549-SC and to explore the role and mechanism of autophagy in A549-SC radioresistance.Methods:1.A549-SC was enriched by serum-free suspension culture method,and the percentage of CD133+ and CD44+ labeled cells was detected by FCM,the expression of stemness-related genes and autophagy-related genes was detected by WB,and the SF and cell viability of A549-SC and A549 after 0,2,4,6 and 8Gy X-ray irradiation were detected by clone formation assay and CCK8 assay to identify their difference in resistance to radiotherapy.2.A549-SC-sh Beclin-1,A549-SC-NC,A549-NC,A549-oe Beclin-1 cell models were constructed by lentiviral transfection.CCK8 and clone formation assay were used to detect the SF and cell viability of the above four cells after 4Gy X-ray irradiation to detect the effect of regulatory Beclin-1 on A549-SC and A549 radioresistance.3.WB was used to detect the differences in expression of γ-H2 AX protein,RAD51 protein,KU80 protein,BAX protein and Bcl-2 protein in A549-SC and A549 after4 Gy X-ray irradiation,and to detect the expression of γ-H2 AX protein,RAD51 protein,KU80 protein,BAX protein,and Bcl-2 protein after A549-SC-sh Beclin-1,A549-SC-NC,A549-NC,and A549-oe Beclin-1 irradiated by 4Gy X-rays.Results:1.The FCM results showed that the rate of CD133+ and CD44+ labeled cells was significantly higher in A549-SC(33.16±2.46%)compared with A549(0.65±0.17%),which was significantly different by statistical analysis(P<0.05).WB results showed that the protein expression of LC3 B,Beclin-1 and Bmi-1,Nanog in A549-SC was increased compared with A549;the protein expression of p62 was decreased compared with A549,which was significantly different by statistical analysis(P<0.05).The results of clone formation experiments showed that after 0,2,4,6 and8 Gy X-ray irradiation,the SF of A549-SC(100%,74.61±5.94%,61.23±2.69%,35.49±1.68%,11.58±17.4%)was higher than that of A549(100%,49.08±07.01%,16.06±4.2%,7.91±1.2%,4.44±1.2%).CCK8 results showed that the cell viability of A549-SC(100%,90.28±1.2%,83.23±2.1%,71.36±7.2%,52.77±2.77%)was significantly higher after 0,2,4,6,and 8Gy X-ray irradiation compared to A549(100%,90.28±1.2%,83.23±2.1%,71.36±7.2%,52.77±2.77%).(100%,76.91±2.36%,62.39±9.16%,54.99±5.55%,30.72±7.09%)was significantly higher than that of A549(100%,76.91±2.36%,62.39±9.16%,54.99±5.55%,30.72±7.09%),which was significantly different by statistical analysis(P<0.05).2.The results of clone formation assay showed that the number of clone formation of A549-SC-sh Beclin-1(85.33±10.02)was reduced after 4 Gy X-ray irradiation compared to A549-SC-NC(36.67±11.72),and the number of clone formation of A549-oe Beclin-1(74.67±7.23)was increased compared to A549-NC(34±8.72)were increased,which were significantly different by statistical analysis(P<0.05).CCK8 results showed that after 4Gy X-ray irradiation,the cell viability of A549-SCsh Beclin-1(36.67±11.72%)was reduced compared to A549-SC-NC(78.9±3.93%),and A549-oe Beclin-1 cell viability(80.34±3.47%)was increased compared to A549-NC(43.33±8.93%),which was significantly different by statistical analysis(P<0.05).3.WB results showed that after 4Gy X-ray irradiation,γ-H2 AX and BAX protein expressions were decreased in A549-SC compared with A549,while RAD51 and Bcl-2 were increased compared with A549,which were significantly different by statistical analysis(P<0.05),and the expression of DNA repair protein(KU80)was not significantly different in each group.γ-H2 AX and BAX protein expressions were decreased in A549-oe Beclin-1 compared with A549,while RAD51 and Bcl-2 were increased compared with A549.H2 AX,BAX protein expression was decreased in A549-NC compared to A549-NC,while RAD51 and Bcl-2 were increased compared to A549-NC.γ-H2 AX,BAX protein expression was increased in A549-SC-sh Beclin-1compared to A549-SC-NC,while RAD51 and Bcl-2 were decreased compared to A549-SC-NC,and by statistical analysis there were There was no significant difference in the expression of KU80 in A549-NC,A549-oe Beclin-1,A549-SCsh Beclin-1,and A549-SC-NC after 4Gy X-ray irradiation.Conclusion:1.A549-SC enriched from serum-free suspension culture was radioresistance and high expression of stemness and autophagy related genes.2.The key autophagy gene Beclin-1 induced radioresistance in A549-SC by promoting DNA repair and inhibiting apoptosis. |
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