hnRNPA2B1通过抑制转铁蛋白受体增强胰腺癌细胞对铁死亡的抵抗  

hnRNPA2B1 enhances the resistance of pancreatic cancer cells to ferroptosis by inhibiting transferrin receptor

作　　者：李政 刘雅雯 陈家希 王慧之 于正悦 王舜宇 龚爱华[1] 徐岷[3] LI Zheng;LIU Yawen;CHEN Jiaxi;WANG Huizhi;YU Zhengyue;WANG Shunyu;GONG Aihua;XU Min(School of Medicine,Jiangsu University,Zhenjiang Jiangsu 212013;Department of Gastroenterology,Taixing People′s Hospital,Taixing Jiangsu 225400;Department of Gastroenterology,Affiliated Hospital of Jiangsu University,Zhenjiang Jiangsu 212001,China)

机构地区：[1]江苏大学医学院,江苏镇江212013 [2]泰兴市人民医院消化科,江苏泰兴225400 [3]江苏大学附属医院消化科,江苏镇江212001

出　　处：《江苏大学学报（医学版）》2024年第1期1-10,共10页Journal of Jiangsu University：Medicine Edition

基　　金：国家自然科学基金面上项目(82072754);江苏省卫生健康委员会面上项目(M2020011);江苏省重点研发计划社会发展项目(BE2018689);镇江市重点研发计划社会发展项目(SH2018033)。

摘　　要：目的:探讨异质核核糖核蛋白A2B1(heterogeneous nuclear ribonucleoprotein A2B1,hnRNPA2B1)对胰腺癌细胞铁死亡的影响及其潜在机制。方法:将3种胰腺癌PaTu8988、MIA-paca2、PANC1细胞用不同浓度Erastin处理3 d,CCK8法检测细胞活性,计算3种细胞Erastin半数抑制浓度(IC_(50))并比较其对Erastin的抵抗力;荧光实时定量PCR(qRT-PCR)和蛋白免疫印迹法分别检测3种胰腺癌细胞中hnRNPA2B1 mRNA和蛋白的相对表达。通过GEPIA平台分析hnRNPA2B1在胰腺癌组织和正常组织中的差异性表达,通过GEO数据库下载临床数据集分析差异性表达hnRNPA2B1患者的预后。在PaTu8988细胞中干扰hnRNPA2B1表达,分别转染sh-Control、sh-hnRNPA2B1质粒;在PANC1细胞中过表达hnRNPA2B1,分别转染Vector、Flag-hnRNPA2B1质粒;分别采用qRT-PCR和蛋白免疫印迹检测转染效率,通过Transwell实验检测hnRNPA2B1对胰腺癌细胞迁移和侵袭的影响,通过CCK8法检测hnRNPA2B1对胰腺癌细胞增殖的影响;对sh-Control组和sh-hnRNPA2B1组、Vector组和Flag-hnRNPA2B1组,分别予以对照(0μmol/L Erastin)、Erastin(IC_(50)浓度)处理,通过流式细胞术检测脂质过氧化物含量,比色法检测丙二醛和组织铁含量,此外sh-hnRNPA2B1组和Flag-hnRNPA2B1组加入Erastin的同时加入铁死亡挽救剂Ferrostatin-1,坏死挽救剂Necrostatin-1或凋亡挽救剂ZVAD-FMK,台酚蓝染色法检测细胞活性;qRT-PCR和蛋白免疫印迹法分别检测胰腺癌细胞转铁蛋白受体(transferrin receptor,TFRC)、铁蛋白重链、谷胱甘肽过氧化物酶4、溶质载体家族7成员11等mRNA和蛋白相对表达水平。结果:3种胰腺癌细胞对Erastin抗性由高到低依次是PaTu8988、MIA-paca2、PANC1细胞,其Erastin IC_(50)依次为18.020、15.760、2.947μmol/L;hnRNPA2B1表达量由高到低的趋势与Erastin IC_(50)趋势相同,于PaTu8988细胞中最高,MIA-paca2细胞中次之,PANC1细胞中最低(P均<0.05);胰腺癌组织中hnRNPA2B1表达水平明显高于正常组织,高表达hnObjective:To investigate the role of heterogeneous nuclear ribonucleoprotein A2B1(hnRNPA2B1)in ferroptosis of pancreatic cancer cells and its potential mechanisms.Methods:Three pancreatic cancer cell lines PaTu8988,MIA-paca2 and PANC1 were treated with different concentrations of Erastin for 3 days.The cell viability was detected by CCK8 method.The half inhibitory concentrations(IC_(50))of Erastin in three kinds of pancreatic cancer cell lines were calculated and their resistance capabilities to Erastin were compared;qRT-PCR and Western blotting were used to detect the relative expression levels of mRNA and protein of hnRNPA2B1 in pancreatic cancer cells.The differential expression of the hnRNPA2B1 in pancreatic cancer tissues and normal tissues was analyzed by GEPIA,and the survival prognosis of patients with differential expression of hnRNPA2B1 was analyzed from clinical data sets from GEO database.PaTu8988 cells was transfected with sh-Control and sh-hnRNPA2B1 plasmid,respectively,to knock down hnRNPA2B1;PANC1 cells was transfected with Vector and Flag-hnRNPA2B1 plasmid,respectively,to overexpress hnRNPA2B1;and the transfection efficiency was verfied by qRT-PCR and Western blotting,respectively;the effects of knockdown or overexpression of hnRNPA2B1 on the migration and invasion were detected by Transwell assay,and the effects on the proliferation of pancreatic cancer cells were detected by CCK8 assay.The cells in sh-Control,sh-hnRNPA2B1,Vector and Flag-hnRNPA2B1 group were treated with control(0μmol/L Erastin)and Erastin(IC_(50)concentration),respectively,then flow cytometry was used to determine the lipid peroxidation level,and colorimetry was used to determine the malindialdehyde(MDA)and tissue iron concentration;in addition to Erastin,the sh-hnRNPA2B1 and Flag-hnRNPA2B1 groups were added with Ferrostatin-1,Necrostatin-1,ZVAD-FMK,respectively,the cells activity was detected by trypan blue dye exclusion.The relative expression levels of RNA and protein of transferrin receptor(TFRC),ferritin heavy chain,glut

关 键 词：hnRNPA2B1 胰腺癌 铁死亡 铁代谢 转铁蛋白受体 

分 类 号：R735.9[医药卫生—肿瘤]