Wnt/β-catenin信号转导通路与肺腺癌顺铂耐药作用机制研究

Mechanism of Wnt/β-catenin signal transduction pathway mediating drug resistanc in lung adenocarcinoma

目的:探讨Wnt/β-catenin信号转导通路在肺腺癌顺铂耐药中的分子作用机制。方法:体外常规培养人肺腺癌A549细胞和其顺铂耐药A549/DDP细胞,取对数生长期的细胞用于实验。采用MTT法检测A549和A549/DDP细胞对顺铂的IC50;采用AnnexinⅤ/PI法和Hoechst 33342染色法检测顺铂处理后2种细胞的凋亡率;采用蛋白质印迹法检测2种细胞中β-catenin和Survivin的表达;采用siRNA干扰沉默β-catenin的表达,检测A549/DDP细胞的IC50值、细胞凋亡率及β-catenin和Survivin的表达。结果:顺铂对A549/DDP细胞的IC50值为(28.984±1.404)μmol/L高于A549细胞的(5.888±0.338)μmol/L,t=27.696,P<0.001;20μmol/L顺铂诱导A549/DDP细胞凋亡率为(21.75±0.96)%,显著低于A549细胞的(39.38±0.88)%,t=23.474,P<0.001。A549/DDP细胞中β-catenin蛋白的表达为1.890±0.060,显著高于A549细胞的1.063±0.035,t=20.595,P<0.001;在A549/DDP细胞中Survivin蛋白表达量为1.107±0.061,明显高于A549细胞的0.503±0.025,t=15.814,P<0.001。RNAi技术沉默β-catenin蛋白耐药性(14.615±0.939)μmol/L,显著低于未沉默β-catenin A549/DDP细胞的(28.984±1.404)μmol/L,t=14.732,P<0.001;RNAi技术沉默β-catenin蛋白细胞凋亡率为(37.57±0.64)%,显著高于未沉默β-catenin A549/DDP细胞的(21.75±0.96)%,t=23.699,P<0.001;RNAi技术沉默β-catenin蛋白下游靶基因Survivin蛋白的表达为0.527±0.065,显著低于A549/DDP组的1.027±0.025和瞬时转染阴性对照siRNA组的1.033±0.040,F=116.944,P<0.001。结论:抑制Wnt/β-catenin信号转导通路的活性可以降低A549/DDP细胞对顺铂的耐药性。

OBJECTIVE：To investigate molecule mechanism on the Wnt/β-catenin signal transduction pathway mediating drug resistanc in lung adenocarcinoma. METHODS： Human lung adenocarcinoma A549 cells and its cisplatin resistance A549/DDP ceils were cultured in vitro and the cells in logarithm growth period were used. MTT assay was used to determine IC50 values of cisplatin in A549 and A549/DDP cells. Annexing analysis using flow cytometry and Hoechst 33342 staining were used to observe apoptosis of A549 and A549/DDP cells treated with cisplatin compared to untreated cells. Western blot analysis was used to detect the expressions of β-catenin and survivin in A549 and A549/DDP cells. Transient interference of β-catenin by siRNA was used to measure the IC50 values,apoptosis and the expressions of β-catenin and survivin in A549/DDP cells. RESULTS： ICs0 values of cisplatin in A549/DDP cells（28. 984±1. 404）were higher than those in A549 cells（5. 888＋0. 338） ,t=27. 696 ,P〈0. 001. The 20μmol/L cisplatin induced apoptosis in A549/DDP cells（21.75 ± 0. 96） ; was significantly lower than that of A549 cells （ 39. 38 ± 0. 88）%, t= 23. 474, P〈0. 001. The expression of β-catenin protein（1. 890±0. 060）in A549/DDP cells was significantly higher than that in A549 cells （1. 063±0. 035） ,t= 20. 595 ,P〈0. 001. The expression of survivin protein （1. 107±0. 061）in A549/DDP cells was significantly higher than that in A549 cells （0. 503 ±0. 025） ,t=15. 814, P〈0. 001. In A549/DDP cells, the IC50 of cisplatin after knockdown of β-catenin was （14. 615± 0. 939）μmol/L, significantly lower than that before knockdown of β-catenin （28. 984 ± 1. 404） μmol/L, t = 14. 732, P〈0.001. The proportion of early apoptotic cells after knockdown of β-catenin was （37. 57±0.64）% significantly higher than that before knockdown of β-eatenin（21. 75±0.96）%, t=23. 699, P〈0. 001. The expression of survivin protein after knockdown of β-catenin was（0. 527 ±0. 065）remarkably lower than that before knockdown of β-catenin（1. 027 ± 0. 025） and that of positive group of transient transfection （1. 033 ± 0. 040）, F= 116. 944, P〈0. 001. CONCLUSION： Inhibition of Wnt/ β-catenin signal transduction pathway can reduce the resistance to cisplatin in A549/DDP cells.