缓激肽通过上调Snail促进非小细胞肺癌放疗耐受

Bradykinin promotes non-small cell lung cancer radioresistance development by upregulating Snail

目的探讨缓激肽(BK)促进非小细胞肺癌(NSCLC)放疗耐受的作用机制。方法实时定量反转录聚合酶链反应(RT-qPCR)检测细胞株中信使RNA(mRNA)的表达;瞬时转染技术在细胞株中转染小干扰RNA(siRNA);蛋白质印迹法(Western blot)检测细胞中Snail的表达;噻唑蓝(MTT)实验检测放疗后细胞存活情况;彗星实验检测放疗后细胞DNA修复能力变化。组间比较采用t检验。结果 BK在耐放射人肺癌细胞株中的表达显著高于亲代细胞(A549R26比A549:58.582±3.986比1.000±0.249, t=14.418, P<0.01;H157R24比H157:23.979±0.791比1.000±0.101, t=28.817, P<0.01)。在放疗4 h后亲代细胞中BK即明显升高(A549:6.530±0.603比1.000±0.214, t=8.642, P<0.01;H157:1.384±0.031比1.000±0.125, t=2.982, P<0.05)。用BK处理肺癌亲代细胞株后, 神经内分泌标志物NSE(A549:1.551±0.071比1.000±0.027, t=7.254, P<0.01;H157:1.343±0.064比1.000±0.037, t=4.640, P<0.01)、表达上升, 而用BK的拮抗剂处理肺癌耐放射细胞株后, NSE(A549R26:0.703±0.036比1.000±0.045, t=5.154, P<0.01;H157R24:0.780±0.017比1.000±0.046, t=4.486, P<0.05)表达下降。亲代细胞在过表达BK后, 细胞放射后的存活率增加, DNA修复能力增强;而耐放射细胞在抑制BK表达后, 这些耐放射特性减弱。Snail在耐放射细胞中表达高于亲代细胞(A549R26比A549:5.223±0.216比1.000±0.061, t=18.815, P<0.01;H157R24比H157:3.698±0.360比1.000±0.057, t=7.402, P<0.01)。BK处理亲代细胞后, Snail的蛋白表达升高;用BK拮抗剂处理耐放射细胞后, Snail蛋白表达下降。抑制肺癌耐放射细胞的Snail表达后, 细胞的耐放射特性减弱。结论 BK通过激活Snail通路促进NSCLC的放疗耐受。

Objective To investigate the mechanism of bradykinin(BK)in promoting radiotherapy tolerance of non-small cell lung cancer(NSCLC).Methods Real-time quantitative reverse transcription polymerase chain reaction(RT-qPCR)was used to detect mRNA expression in cell lines.Transient transfection technique was used to transfect small interfering RNA(siRNA)into cell lines.The expression of Snail in cells was detected by Western blotting.Methyl thiazolyl tetrazolium(MTT)assay was used to detect cell survival after radiotherapy.Comet assay was used to detect the changes of DNA repair ability after radiotherapy.Comparison between groups was performed by t test.Results The expression of BK in radioresistant lung cancer cell lines was significantly higher than that in parental cells(A549R26 vs.A549:58.582±3.986 vs.1.000±0.249,t=14.418,P<0.01;H157R24 vs.H157:23.979±0.791 vs.1.000±0.101,t=28.817,P<0.01).BK was significantly increased in parental cells at 4 h after radiotherapy(A549:6.530±0.603 vs.1.000±0.214,t=8.642,P<0.01;H157:1.384±0.031 vs.1.000±0.125,t=2.982,P<0.05).The expression of neuroendocrine markers NSE(A549:1.551±0.071 vs.1.000±0.027,t=7.254,P<0.01;H157:1.343±0.064 vs.1.000±0.037,t=4.640,P<0.01)increased after treating lung cancer parental cell lines with BK.The expression of NSE(A549R26:0.703±0.036 vs.1.000±0.045,t=5.154,P<0.01;H157R24:0.780±0.017 vs.1.000±0.046,t=4.486,P<0.05)decreased after treating lung cancer radioresistant cell lines with BK antagonist.After overexpression of BK,the survival rate and DNA repair ability of parental cells increased after radiation.Inhibition of BK expression weakened the radiation tolerance of radiotolerant cells.Snail expression in radioresistant cells was higher than that in parental cells(A549R26 vs.A549:5.223±0.216 vs.1.000±0.061,t=18.815,P<0.01;H157R24 vs.H157:3.698±0.360 vs.1.000±0.057,t=7.402,P<0.01).The protein expression of Snail in parental cells was increased after treatment with BK.The protein expression of Snail in radioresistant cell lines was decreased after treatment with BK antagonist.Inhibition of Snail expression in lung cancer cells weakened the resistance to radioactivity.Conclusion BK promotes NSCLC radiotherapy tolerance through activation of Snail pathway.