补中益气汤协同Siβ-catenin调控Wnt/β-catenin信号通路干预肺腺癌顺铂耐药性的分子机制研究

Molecular mechanism of Buzhong Yiqi Decoction in coordination with Siβ-catenin in intervention against cisplatin resistance in lung adenocarcinoma by regulating the Wnt/β-catenin signaling pathway

摘要:

目的 探讨补中益气汤含药血清协同Siβ-catenin调控Wnt/β-catenin信号通路干预A549/DDP细胞顺铂耐药性的分子机制研究。方法 采用siRNA干扰沉默β-catenin的表达，Western blot检测正常血清组、顺铂组、siCTNNB1-235组、siCTNNB1-510组、siCTNNB1-547组A549/DDP细胞的β-catenin蛋白的相对表达量；干扰β-catenin后，Western blot检测各组A549/DDP细胞β-catenin和Survivin的蛋白表达量，MTT法检测各组A549/DDP细胞对顺铂的IC₅₀，Annexin V/PI染色法检测各组A549/DDP细胞顺铂诱导的凋亡率。结果 相较于正常血清组（1.00），siCTNNB1-235组（0.323±0.021）对β-catenin表达抑制率达67%（P=0.000）。RNAi技术沉默β-catenin后，与正常血清组（1.00）相比，补中益气汤联合顺铂组的Survivin（0.247±0.015）和β-catenin（0.257±0.015）蛋白的表达下调更为明显（P=0.000，P=0.000）。IC50和正常血清组（28.330±1.029） μmol/L相比，单独补中益气汤含药血清（20.350±1.155） μmol/L或单独瞬时转染siCTNNB1-235组（15.577±1.535） μmol/L均可降低A549/DDP细胞顺铂的IC₅₀（P=0.000，P=0.000），2者联合（10.453±0.999） μmol/L使用可进一步降低顺铂的IC₅₀（P=0.000）。与正常血清组（9.130±0.384）%相比，瞬时转染siCTNNB1-235联合顺铂组（64.393±0.244）%和补中益气汤联合顺铂组（70.120±0.400）%的总凋亡率均升高（P=0.000，P=0.000）。结论 补中益气汤含药血清和瞬时转染siβ-catenin在抑制Wnt/β-catenin信号通路改善肺腺癌顺铂耐药性方面具有协同效应，且补中益气汤具有siβ-catenin瞬时转染样效应。

Abstract:

Objective To investigate the molecular mechanism of Buzhong Yiqi Decoction in coordination with Siβ-catenin in intervention against cisplatin resistance in A549/DDP cells by regulating the Wnt/β-catenin signaling pathway.Methods The expression of β-catenin was silenced by siRNA interference，and Western blot was used to measure the relative protein expression of β-catenin in A549/DDP cells in the normal serum group，the cisplatin group，the siCTNNB1-235 group，the siCTNNB1-510 group，and the siCTNNB1-547 group. After β-catenin interference，Western blot was used to measure the protein expression levels of β-catenin and Survivin in A549/DDP cells of each group； MTT assay was used to determine the IC₅₀ of cisplatin in A549/DDP cells of each group； Annexin V/PI staining was used to measure the cisplatin-induced apoptosis rate of A549/DDP cells in each group.Results Compared with the normal serum group（1.00），the siCTNBN1-235 group（0.323±0.021） had an inhibition rate of 67% on β-catenin expression（P=0.000）. After β-catenin was silenced by RNAi technique，compared with the normal serum group（1.00），the Buzhong Yiqi Decoction+cisplatin group had significantly greater reductions in the protein expression of Survivin（0.247±0.015） and β-catenin（0.257±0.015）（P=0.000，P=0.000）. As for the IC₅₀ of cisplatin in A549/DDP cells，compared with the normal serum group（28.330±1.029） μmol/L，serum containing Buzhong Yiqi decoction alone（20.350±1.155） μmol/L or siCTNNB1-235 transient transfection alone（15.577±1.535） μmol/L reduced the IC₅₀ of cisplatin in A549/DDP cells（P=0.000，P=0.000），and the combination of the two methods（10.453±0.999） μmol/L further reduced the IC₅₀ of cisplatin（P=0.000）. Compared with the normal serum group（9.130±0.384）%，the siCTNBN1-235 transient transfection+cisplatin group（64.393±0.244）% and the Buzhong Yiqi Decoction+cisplatin group（70.120±0.400）% had a significant increase in overall apoptosis rate（P=0.000，P=0.000）.Conclusion Serum containing Buzhong Yiqi Decoction and siβ-catenin transient transfection have a synergistic effect on inhibiting the Wnt/β-catenin signaling pathway and improving cisplatin resistance in lung adenocarcinoma，and Buzhong Yiqi Decoction has a siβ-catenin transient transfection-like effect.