下调富含脯氨酸蛋白11表达对食管癌耐药细胞EC9706/DDP耐药性的影响及其机制

Effect of downregulating proline-rich protein 11 expression on drug resistance of esophageal cancer drug resistant cell EC9706/DDP and its mechanism

摘要

目的 探讨下调富含脯氨酸蛋白11（PRR11）表达对食管癌耐药细胞耐药性的影响，阐明其相关机制。 方法 采用顺铂（DDP）浓度递增间断刺激人食管癌EC9706细胞建立DDP耐药细胞株EC9706/DDP，MTT法检测EC9706/DDP细胞药敏性，实时荧光定量PCR（RT-qPCR）法和Western blotting法检测EC9706/DDP细胞及其亲本EC9706细胞中PRR11 mRNA和蛋白表达水平。将EC9706/DDP细胞分为对照组、sh-NC组（转染sh-NC）、sh-PRR11组（转染sh-PRR11）、sh-NC+DDP组（转染sh-NC后用4 mg?L^－1 DDP处理）和sh-PRR11+DDP组（转染sh-PRR11后用4 mg?L^－1 DDP处理），RT-qPCR法检测各组细胞中PRR11 mRNA表达水平，Western blotting 法检测各组细胞中PRR11、磷脂酰肌醇3-激酶（PI3K）p110α、蛋白激酶B（AKT）、磷酸化AKT（p-AKT）、P-糖蛋白（P-gp）和多药耐药相关蛋白1（MRP1）蛋白表达水平，流式细胞术检测各组细胞凋亡率。 结果 成功获得DDP耐药细胞株EC9706/DDP，耐药指数为7.23±0.86。与EC9706细胞比较，EC9706/DDP细胞中PRR11mRNA和蛋白表达水平升高（P<0.05）。分别与对照组和sh-NC组比较，sh-PRR11组细胞中PRR11 mRNA和蛋白表达水平降低（P<0.05），细胞的DDP半数抑制浓度（IC₅₀）降低（P<0.05）。与sh-NC组比较，sh-NC+DDP组和sh-PRR11组细胞中PI3K p110α、p-AKT、P-gp和MRP1蛋白表达水平降低（P<0.05），细胞凋亡率升高（P<0.05）；分别与sh-NC+DDP组和sh-PRR11组比较，sh-PRR11+DDP组细胞中PI3K p110α、p-AKT、P-gp和MRP1蛋白表达水平降低（P<0.05），细胞凋亡率升高（P<0.05）。 结论 下调EC9706/DDP耐药细胞中PRR11 基因的表达，可抑制耐药相关蛋白的表达，逆转对DDP耐药，并诱导细胞凋亡，其作用机制可能与抑制PI3K/AKT信号通路激活有关。

Abstract

Objective To discuss the effect of downregulating the proline-rich protein 11 （PRR11） expression on drug resistance of the esophageal cancer drug resistant cells， and to clarify the related mechanism. Methods The drug resistant cells EC9706/cisplatin（DDP） were established by incrementally stimulating the human esophageal cancer EC9706 cells with the increasing concentrations of DDP. The drug sensitivity of the EC9706/DDP cells was detected by MTT assay； the expression levels of PRR11 mRNA and protein in the EC9706/DDP cells and their parent EC9706 cells were detected by real-time fluorescence quantitative PCR （RT-qPCR） and Western blotting methods. The EC9706/DDP cells were divided into control group，sh-NC group （infected with sh-NC），sh-PRR11 group（infected with sh-PRR11）， sh-NC+DDP group （infected with sh-NC and treated with 4 mg·L^－1 DDP）， and sh-PRR11+DDP group （infected with sh-PRR11 and treated with 4 mg·L^－1 DDP）. The expression levels of PRR11 mRNA in the cells in various groups were detected by RT-qPCR method； the expression levels of PRR11， phosphoinositide 3-kinase （PI3K） p110α， protein kinase B （AKT）， phosphorylated AKT （p-AKT）， P-glycoprotein （P-gp）， and multidrug resistance-associated protein 1 （MRP1） proteins in the cells in various groups were detected by Western blotting method；the apoptotic rates of the cells in various groups were detected by flow cytometry. Results The DDP-resistant cell line EC9706/DDP was successfully obtained，and the drug resistance index was 7.23±0.86. Compared with the EC9706 cells， the expression levels of PRR11 mRNA and protein in the EC9706/DDP cells were increased （P<0.05）. Compared with control and sh-NC groups， the expression levels of PRR11 mRNA and protein in the cells in sh-PRR11 group were decreased （P<0.05），and the 50% inhibitory concentration （IC₅₀） of DDP was decreased （P<0.05）.Compared with sh-NC group，the expression levels of PI3K p110α， p-AKT， P-gp， and MRP1 proteins in the cells in sh-NC+DDP and sh-PRR11 groups were decreased （P<0.05），and the apoptotic rate of the cells was increased （P<0.05）. Compared with sh-NC+DDP group and sh-PRR11 group，the expression levels of PI3K p110α， p-AKT， P-gp， and MRP1 proteins in the cells in sh-PRR11+DDP group were increased（P<0.05），and the apoptotic rate of the cells was increased（P<0.05）. Conclusion Downregulating the expression of PRR11 gene in the drug resistant EC9706/DDP cells can inhibit the expressions of drug resistance-related proteins， reverse the resistance to DDP， and induce the apoptosis； its mechanism may be related to the inhibition of activation of the PI3K/AKT signaling pathway.

关键词

富含脯氨酸蛋白11 / 食管肿瘤 / 顺铂 / 耐药 / 磷脂酰肌醇3-激酶/蛋白激酶B信号通路

Key words

Proline-rich protein 11 / Esophageal neoplasm / Cisplatin / Drug resistance / Phosphoinositide 3-kinase/protein kinase B signaling pathway

Author summay

亢春彦（1977－），女，河南省焦作市人，副教授，医学硕士，主要从事肿瘤早期诊断方面的研究。