Wnt／β-catenin信号转导通路与肺腺癌顺铂耐药作用机制研究

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Hoechst33342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pcatenin的表达，检测A549／DDP细胞的Ic5。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）umol／L，t=27．696，P〈0．001；20umol／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士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）gmol／L，显著低于未沉默β-cateninA549／DDP细胞的（28．984±1．404）μmol／L，t=14．732，P〈0．001；RNAi技术沉默pcatenin蛋白细胞凋亡率为（37．57±0．64）％，显著高于未沉默β-cateninA549／DDP细胞的（21．75±0．96）％，t=23．699，P〈0．001；RNAi技术沉默伊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。

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

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.