5-氟尿嘧啶联合奥沙利铂通过微小RNA-92a/Wnt/β-连环蛋白通路影响非小细胞肺癌细胞的顺铂耐药机制研究

The mechanism of 5-fluorouracil combined with oxaliplatin on cisplatin resistance in non-small cell lung cancer through microRNA-92a/Wnt/β-catenin pathway

目的探讨5-氟尿嘧啶联合奥沙利铂通过微小RNA-92a(miR-92a)/Wnt/β-连环蛋白(β-catenin)通路影响非小细胞肺癌细胞的顺铂耐药机制。方法体外培养人非小细胞肺癌细胞(A549),构建顺铂耐药模型,实验分组如下:A549组为正常A549细胞;A549/顺铂组为顺铂耐药模型细胞;A549+顺铂组为A549组给予顺铂共培养;A549/顺铂+顺铂组为A549/顺铂组给予顺铂共培养;A549/顺铂+miR-92a inhibitor组为A549/顺铂组转染miR-92a inhibitor;5-氟尿嘧啶+奥沙利铂组为A549/顺铂组给予5-氟尿嘧啶+奥沙利铂共培养;5-氟尿嘧啶+奥沙利铂+miR-92a mimics组为5-氟尿嘧啶+奥沙利铂组转染miR-92a mimics;5-氟尿嘧啶+奥沙利铂+miR-92a mimics+Wnt/β-catenin inhibitor组为5-氟尿嘧啶+奥沙利铂+miR-92a mimics组转染Wnt/β-catenin inhibitor。采用5-乙炔基-2′-脱氧尿嘧啶核苷实验检测细胞增殖率;细胞划痕实验检测细胞迁移率;Transwell小室实验检测细胞侵袭能力;荧光原位杂交法检测miR-92a的表达情况;实时荧光定量聚合酶链反应法检测miR-92a、Wnt4和β-catenin的表达;蛋白质印迹法检测Wnt4、β-catenin和CD44的蛋白表达。结果与A549组相比,A549/顺铂组miR-92a的表达升高(P<0.05)。与A549/顺铂组相比,A549/顺铂+miR-92a inhibitor组细胞迁移率和侵袭个数均降低(均P<0.05)。与A549/顺铂组相比,5-氟尿嘧啶+奥沙利铂组细胞增殖率和侵袭个数以及miR-92a、Wnt4、β-catenin和CD44表达水平均降低(均P<0.05)。与5-氟尿嘧啶+奥沙利铂组相比,5-氟尿嘧啶+奥沙利铂+miR-92a mimics组细胞增殖率和侵袭个数以及miR-92a、Wnt4和β-catenin的表达水平均上升(均P<0.05)。与5-氟尿嘧啶+奥沙利铂+miR-92a mimics组相比,5-氟尿嘧啶+奥沙利铂+miR-92a mimics+Wnt/β-catenin inhibitor组细胞增殖率和侵袭个数以及miR-92a[(0.49±0.07)比(0.97±0.14)]、Wnt4[(0.37±0.05)比(1.19±0.16)]和β-catenin[(0.27±0.04)比(1.54±0.20)]的表达水平均下降(均P<0.05)。结论5-氟尿嘧啶联合奥沙利铂能够有效改善A549细胞的顺铂耐药性,抑制细胞增殖和侵袭,可能与下调miR-92a/Wnt/β-catenin信号通路相关蛋白的表达有关。

Objective To explore the mechanism of 5-fluorouracil(5-FU)combined with oxaliplatin(L-OHP)on cisplatin(DDP)resistance in non-small cell lung cancer through microRNA-92a(miR-92a)/Wnt/β-catenin pathway.Methods Cultivate human non-small cell lung cancer cells(A549)in vitro and construct the DDP resistance model.The experimental groups are as follows:A549 group was the normal A549 cells,A549/DDP group was the DDP resistant model cells,A549+DDP group was the A549 group co-cultured with DDP,A549/DDP+DDP group was the A549/DDP group co-cultured with DDP,A549/DDP+miR-92a inhibitor group was the A549/DDP group transfected with miR-92a inhibitor,5-FU+L-OHP group was the A549/DDP group co-cultured with 5-FU+L-OHP,5-FU+L-OHP+miR-92a mimics group was the 5-FU+L-OHP group transfected with miR-92a mimics,5-FU+L-OHP+miR-92a mimics+Wnt/β-catenin inhibitor group was the 5-FU+L-OHP+miR-92a mimics group transfected with Wnt/β-catenin inhibitor.5-Ethynyl-2′-deoxyuridine experiment was used to detect cell proliferation rate,wounding healing assay was used to detect cell migration rate,Transwell experiment was used to detect cell invasion ability,fluorescence in situ hybridization was used to detect the expression of miR-92a;quantitative real-time polymerase chain reaction was used to detect the expression of miR-92a,Wnt4,andβ-catenin;Western Blot was used to detect protein expression of Wnt4,β-catenin and CD44.Results Compared with A549 group,the expression level of miR-92a in A549/DDP group was significantly increased(P<0.05).Compared with A549/DDP group,the migration rate and cells invasion number in A549/DDP+miR-92a inhibitor group were significantly reduced(both P<0.05).Compared with A549/DDP group,the cell proliferation rate,cell invasion number and expression levels of miR-92a,Wnt4,β-catenin,and CD44 proteins in 5-FU+L-OHP group were significantly reduced(all P<0.05).Compared with 5-FU+L-OHP group,5-FU+L-OHP+miR-92a mimics group showed a significant increase(all P<0.05).Compared with 5-FU+L-OHP+miR-92a mimics group,5-FU+L-OHP+miR-92a mimics+Wnt/β-catenin inhibitor group showed a significant decrease[miR-92a:(0.49±0.07)vs(0.97±0.14),Wnt4:(0.37±0.05)vs(1.19±0.16),β-catenin:(0.27±0.04)vs(1.54±0.20)](all P<0.05).Conclusion The combination of 5-FU and L-OHP can effectively improve the DDP resistance of A549 cells,inhibit cell proliferation and invasion,which may be related to downregulating the expression of proteins related to the miR-92a/Wnt/β-catenin signaling pathway.