SIRT3/FOXO3通路在肺癌A549细胞放疗抵抗中的作用机制研究

The role of SIRT3/FOXO3 pathway in radiotherapy resistance of lung cancer A549 cells

摘要:

目的 探讨沉默信息调节因子3（SIRT3）/叉头转录因子O3（FOXO3）通路在肺癌A549细胞放疗抵抗中的作用。方法 体外培养肺癌A549细胞，设置对照组（A549细胞）、放疗组（A549细胞+X射线照射）、SIRT3抑制剂（3-TYP）组（A549细胞+50 μmol/L 3-TYP）、X射线+3-TYP组（A549细胞+X射线照射+50 μmol/L 3-TYP）。采用CCK-8法检测各组A549细胞增殖情况；流式细胞分析仪检测各组A549细胞凋亡情况和细胞周期分布；Western blotting检测A549细胞中SIRT3、FOXO3、Bcl-2、Bax蛋白相对表达量。结果 各组A549细胞培养24 h、48 h、72 h的OD值比较，经重复测量设计的方差分析：①不同时间点的A549细胞OD值比较，差异有统计学意义（P <0.05）；②不同组间的A549细胞OD值比较，差异有统计学意义（P <0.05）；③各组A549细胞OD值变化趋势比较，差异无统计学意义（P >0.05）。放疗组、X射线+3-TYP组A549细胞凋亡率较对照组升高（P <0.05），3-TYP组较对照组降低（P <0.05），3-TYP组、X射线+3-TYP组较放疗组降低（P <0.05），X射线+3-TYP组较3-TYP组升高（P <0.05）。放疗组、X射线+3-TYP组G0/G1期A549细胞较对照组增加（P <0.05），S、G2/M期A549细胞较对照组减少（P <0.05）；3-TYP组G0/G1期A549细胞较对照组降低（P <0.05），S、G2/M期A549细胞较对照组增加（P <0.05）；3-TYP组、X射线+3-TYP组G0/G1期A549细胞较放疗组减少（P <0.05），S、G2/M期A549细胞较放疗组增加（P <0.05）；X射线+3-TYP组G0/G1期A549细胞较3-TYP组增加（P <0.05），S、G2/M期A549细胞较3-TYP组减少（P <0.05）。放疗组、X射线+3-TYP组A549细胞SIRT3、FOXO3、Bax蛋白相对表达量较对照组升高（P <0.05），Bcl-2蛋白相对表达量较对照组降低（P <0.05）；3-TYP组A549细胞SIRT3、FOXO3、Bax蛋白相对表达量较对照组降低（P <0.05），Bcl-2蛋白相对表达量较对照组升高（P <0.05）；3-TYP组、X射线+3-TYP组A549细胞SIRT3、FOXO3、Bax蛋白相对表达量较放疗组降低（P <0.05），Bcl-2蛋白相对表达量较放疗组升高（P <0.05）；X射线+3-TYP组A549细胞中SIRT3、FOXO3、Bax蛋白相对表达量较3-TYP组升高（P <0.05），Bcl-2蛋白相对表达量较3-TYP组降低（P <0.05）。结论 肺癌A549细胞发生放疗抵抗可能与SIRT3/FOXO3通路受抑制有关。

Abstract:

Objective To investigate the role of sirtuin-3/forkhead box O3 (SIRT3/FOXO3) pathway in radiotherapy resistance of lung cancer A549 cells.Methods The lung cancer A549 cells were cultured in vitro, and the control group (A549 cells), radiotherapy group (A549 cells + X-ray irradiation), SIRT3 inhibitor (3-TYP) group (A549 cells + 50 μmol/L 3-TYP), and X-ray + 3-TYP group (A549 cells + X-ray irradiation + 50 μmol/L 3-TYP) were set up. The proliferation of A549 cells was detected by cell counting kit-8 (CCK-8); the apoptosis and cell cycle distribution of A549 cells were detected by flow cytometry; and the expressions of SIRT3, FOXO3, B-cell lymphoma 2 (Bcl-2) and Bcl-2-associated X protein (Bax) proteins in A549 cells were detected by Western blotting.Results Compared with the control group, the optical density (OD) value of A549 cells, the proportions of A549 cells in S, G₂/M phases and the expression level of Bcl-2 protein in the radiotherapy group and X-ray + 3-TYP group were lower (P < 0.05), while the apoptosis rate, the proportion of A549 cells in G0/G1 phase, and the expression levels of SIRT3, FOXO3, and Bax proteins were higher (P < 0.05). Compared with the radiotherapy group, the OD value of A549 cells, the proportions of A549 cells in S, G2/M phases and the expression level of Bcl-2 protein in the 3-TYP group and X-ray + 3-TYP group were higher (P < 0.05), while the apoptosis rate, the proportion of A549 cells in G0/G1 phase, and the expression levels of SIRT3, FOXO3, and Bax proteins were lower (P < 0.05). Compared with the 3-TYP group, the OD value of A549 cells, the proportions of A549 cells in S, G₂/M phases and the expression level of Bcl-2 protein in the X-ray + 3-TYP group were lower (P < 0.05), while the apoptosis rate, the proportion of A549 cells in G₀/G₁ phase, and the expression levels of SIRT3, FOXO3, and Bax proteins were higher (P < 0.05).Conclusions The resistance of A549 cells to radiotherapy may be related to the inhibition of SIRT3/FOXO3 pathway.