CXCL4对非小细胞肺癌H460细胞顺铂耐药性的影响及其机制探讨

The effect of CXCL4 on cisplatin resistance in non-small cell lung cancer H460 cells and its mechanism

目的:探讨血小板因子4(CXCL4)对人非小细胞肺癌H460细胞顺铂耐药性的影响及其作用机制。方法:体外诱导法建立顺铂耐药的非小细胞肺癌细胞株H460/DDP,并鉴定其生物学特性。采用CCK-8法分别检测H460及H460/DDP对顺铂的耐药性。qRT-PCR及Western Blot检测亲本细胞株H460及顺铂耐药株H460/DDP中CXCL4的mRNA及蛋白表达水平。通过CCK-8法检测过表达或者敲低CXCL4后亲本细胞株H460或顺铂耐药株H460/DDP对顺铂的敏感性。最后利用Western Blot及qRT-PCR法探讨CXCL4抵抗非小细胞肺癌化疗敏感性的调控机制。结果:成功构建了非小细胞肺癌顺铂耐药株H460/DDP(H460/DDP IC_(50)=55.005μg/ml),CCK-8结果提示顺铂耐药株H460/DDP与亲本细胞株H460细胞的增殖速度没有明显差异(P>0.05);顺铂耐药株H460/DDP中,CXCL4 mRNA及蛋白水平均显著增高(P<0.000 1);在亲本细胞株H460中稳定过表达CXCL4能显著降低其对顺铂的敏感性(P<0.000 1),而于顺铂耐药株H460/DDP中敲低CXCL4能显著增加H460/DDP对顺铂的敏感性(P<0.000 1);Western Blot及qRT-PCR结果显示,CXCL4能激活Wnt/β-catenin信号通路,促进下游基因MYC、CyclinD1、MMP-7、CDK4的表达。结论:CXCL4通过调控Wnt/β-catenin信号通路促进非小细胞肺癌对顺铂的耐药性。

Objective： To explore the effect of CXCL4 on cisplatin resistance in non-small cell lung cancer H460 cells and its underlying mechanism.Methods： Cisplatin resistance non-small cell lung cancer cell line H460/DDP was constructed and its biological characteristics were indetified successfully.The mRNA and protein expression of CXCL4 were evaluated both in parental H460 and H460/DDP cells.Gain-of function and loss-of function assays were further used to investigate the effect of CXCL4 on the cisplatin resistance of H460 and H460/DDP cells.Western Blot and RT-PCR assays were further performed to explore the underlying mechanisms of cisplatin resistance of CXCL4 on non-small cell lung cancer.Results： The cisplatin resistance non-small cell lung cancer cell line H460/DDP was established successfully and its drug-resistanct ability was evaluated（ H460/DDP IC_（50）= 55.005 μg/ml）.No significant growth ability difference was observed between parental H460 and H460/DDP cells（ P 0.05）.Both mRNA and protein expression of CXCL4 were significantly up-regulated in H460/DDP cells（ P 0.000 1）,as detected by qRT-PCR and Western Blot.Overexpression of CXCL4 obviously reduced the cisplatin sensitivity on H460 cells（ P 0.000 1）.On the contrary,silencing CXCL4 in H460/DDP cells markedly increased its sensitivity on cisplatin treatment（ P 0.000 1）.Western Blot and qRT-PCR assays showed CXCL4 activated the Wnt/β-catenin signaling and increased its downstreams including MYC,CyclinD1,MMP-7,CDK4.Conclusion： CXCL4 promotes cisplatin resistance in non-small cell lung cancer via Wnt/β-catenin signaling.