ARF4通过EGFR通路调控非小细胞肺癌细胞增殖、迁移、侵袭和凋亡

ARF4 regulates the proliferation,migration,invasion and apoptosis of non-small cell lung cancer cells through EGFR pathway

目的:探究ADP核糖基化因子4(ADP-ribosylation factor 4,ARF4)在非小细胞肺癌(non-small cell lung cancer,NSCLC)发生发展中的生物学作用及作用机制。方法:采用蛋白质印迹法(Western blot,WB)和免疫组化技术检测ARF4在临床NSCLC组织和癌旁组织中的表达情况;使用TCGA数据库分析ARF4与肺癌患者预后的相关性;使用siARF4敲降人NSCLC细胞系A549和H1975细胞中ARF4的表达水平,并用WB和实时定量PCR(real-time quantitative PCR,RT-qPCR)法进行验证;通过CCK-8实验检测敲低ARF4以及敲低ARF4与顺铂联用对NSCLC细胞增殖的影响;利用Transwell实验检测敲低ARF4以及敲低ARF4与顺铂联用对NSCLC细胞迁移及侵袭的影响;通过流式细胞术检测敲低ARF4以及敲低ARF4与顺铂联用对NSCLC细胞凋亡的影响;通过WB实验探究敲低ARF4后NSCLC中EGFR通路的变化。结果:ARF4在NSCLC组织中的表达较癌旁组织显著升高,且和患者的较差预后显著相关;在A549和H1975细胞中敲低ARF4能够抑制细胞的增殖、迁移、侵袭和促进细胞凋亡;敲低ARF4和顺铂联用可进一步抑制细胞的增殖、迁移、侵袭和增加细胞凋亡;敲低ARF4抑制了细胞内EGFR通路的激活。结论:ARF4通过EGFR通路调控NSCLC的生理功能;ARF4敲低和顺铂联用有效抑制NSCLC细胞增殖、迁移和侵袭,并促进细胞凋亡。

Objective:To investigate the effects and mechanisms of ADP-ribosylation factor 4(ARF4)in the development of non-small cell lung cancer(NSCLC).Methods:The expression level of ARF4 in clinical NSCLC tissues and paracarcinoma tissues was analyzed by Western blot(WB)and immunohistochemistry.The correlation of ARF4 and patient prognosis with NSCLC was verified based on TCGA database.The expression level of ARF4 in human NSCLC cell lines A549 and H1975 cells was knocked down by siARF4 and knockdown efficiency was detected by WB and real-time quantitative PCR(RT-qPCR)analysis.The effect of ARF4 knockdown or combination of ARF4 knockdown and cisplatin treatment on the proliferation of NSCLC cells was detected by CCK-8 assay.The effect of ARF4 knockdown or combination of ARF4 knockdown and cisplatin treatment on migration and invasion of NSCLC cells was verified by Transwell assay.The effect of ARF4 knockdown or combination of ARF4 knockdown and cisplatin treatment on apoptosis of NSCLC was detected by flow cytometry analysis.The effect of ARF4 knockdown on EGFR pathways in NSCLC was determined by WB analysis.Results:The expression of ARF4 in NSCLC tissues was significantly higher than that in paracarcinoma tissues,and was significantly correlated with poor prognosis of patients.Knockdown of ARF4 in A549 and H1975 cells inhibited cell proliferation,migration,invasion and promoted cell apoptosis.The combination of ARF4 knockdown and cisplatin treatment could further inhibit cell proliferation,migration,invasion,and increase cell apoptosis.The knockdown of ARF4 inhibited the activation of EGFR pathways in NSCLC cells.Conclusion:ARF4 may regulate the physiological function of NSCLC through EGFR pathways.The combination of ARF4 knockdown and cisplatin effectively inhibited cell proliferation,migration,invasion,and promoted cell apoptosis.