摘要
目的探讨DNA损伤激活的非编码RNA(NORAD)诱导细胞自噬对食管胃结合部腺癌(AEG)奥沙利铂耐药的影响及分子机制。方法收集2023年1月至6月安阳市肿瘤医院诊治的进展期AEG患者AEG及其癌旁正常组织手术标本4对,应用长链非编码RNA微阵列芯片分析AEG及其癌旁组织中NORAD的表达情况。使用新鲜AEG组织标本制备肿瘤组织来源的AEG细胞系(PDC),构建PDC和AEG细胞系OE19的奥沙利铂耐药细胞系(PDC-R、OE19-R),并通过转染shNORAD制备敲减NORAD的PDC-R和OE19细胞系(shNORAD PDC-R、shNORAD OE19-R)。应用生物信息学工具Starbase v3.0和DIANA-lncBase v3.0预测NORAD潜在靶点及其与微RNA-433-3p(miR-433-3p)的相互作用。PDC、PDC-R,OE19、OE19-R细胞分别共转染miR-144-3p和野生型NORAD(NORAD-WT)或突变型NORAD(NORAD-Mut)质粒,应用双萤光素酶报告实验验证NORAD与miR-433-3p的相关性。通过定量反转录聚合酶链反应(qRT-PCR)检测胃正常黏膜细胞系GES-1及AEG细胞系PDC、PDC-R、shNORAD PDC-R、OE19、OE19-R、shNORAD OE19-R中NORAD和miR-433-3p表达水平,蛋白质印迹法检测上述细胞p62和微管相关蛋白1轻链3 B-Ⅱ(LC3B-Ⅱ)的表达。采用细胞计数试剂盒-8(CCK-8)测定奥沙利铂对PDC、PDC-R、shNORAD PDC-R、OE19、OE19-R和shNORAD OE19-R细胞的细胞半数抑制浓度(IC_(50))。统计学方法采用独立样本t检验。结果微阵列芯片分析发现与癌旁正常组织相比,AEG中NORAD表达显著上调(差异表达倍数≥2.0,P<0.05)。生物信息学研究发现miR-433-3p是NORAD的潜在靶点。双萤光素酶报告实验结果显示,在PDC和PDC-R细胞中,NORAD-WT组的相对萤光素酶活性低于NORAD-Mut组(0.441±0.104比0.928±0.204、0.449±0.112比0.947±0.201),差异均有统计学意义(t=-14.74、-14.94,均P<0.001);OE19和OE19-R细胞中双萤光素酶报告实验结果与PDC细胞系相同。qRT-PCR检测结果显示,NORAD在GES-1细胞中的相对表达量(1.016±0.213)低于PDC细胞(2.194±0.322)和PDC-R细
Objective To investigate the effects and molecular mechanism of non-coding RNA-activated DNA damage(NORAD)induced autophagy on oxaliplatin resistance in adenocarcinoma of esophagogastric junction(AEG).Methods Four pairs of surgical samples of AEG and para-carcinoma normal tissues from patients with advance AEG treated in Anyang Tumor Hospital from January to June 2023 were collected.The expression of NORAD in AEG and para-carcinoma tissues was analyzed by long non-coding RNA microarray chip.The primary tumor cell line of AEG(PDC)was derived from fresh AEG tissues.Oxaliplatin-resistant cell lines of PDC and AEG cell line OE19(PDC-R and OE19-R)were established.NORAD expression knockdown PDC-R and OE19 cell lines(shNORAD PDC-R and shNORAD OE19-R)were prepared by transfection.The target of NORAD,the correlation and interaction between microRNA-433-3p(miR-433-3p)and NORAD were predicted using Starbase v3.0 and DIANA-lncBase v3.0.PDC,PDC-R,OE19 and OE19-R cells were co-transfected with miR-144-3p and wild-type NORAD(NORAD-WT)or mutant NORAD(NORAD-Mut)plasmid,respectively.Dual-luciferase reporter assay was used to verify the correlation between NORAD and miR-433-3p.The expression levels of NORAD and miR-433-3p in normal gastric mucosal cell line GES-1 and AEG cell lines PDC,PDC-R,shNORAD PDC-R,OE19,OE19-R and shNORAD OE19-R were detected by real-time quantitative reverse transcription polymerase chain reaction(qRT-PCR).The expression of p62 protein and microtubule-associated protein 1 light chain 3B-Ⅱ(LC3B-Ⅱ)was determined by Western blotting.The half inhibitory concentration(IC_(50))of PDC,PDC-R,shNORAD PDC-R,OE19,OE19-R and shNORAD OE19-R cells was measured by cell counting kit-8(CCK-8)assay.Independent sample t-test was used for statistical analysis.Results The results of microarray analysis showed that NORAD was significantly up-regulated in AEG compared with that in para-carcinoma tissues(fold change≥2.0,P<0.05).Bioinformatics studies found that miR-433-3p was the potential target of NORAD.The results of dual
作者
李守淼
刘志强
曹恒
聂志勇
李慧
李保中
Li Shoumiao;Liu Zhiqiang;Cao Heng;Nie Zhiyong;Li Hui;Li Baozhong(Department of Abdominal Tumor Surgery,Anyang Key Laboratory of Gastric Cancer and Cardiac Cancer Integrated Transformation Research,Anyang Tumor Hospital,Anyang 455001,China;Department of Gastrointestinal Oncology,Anyang Key Laboratory of Gastric Cancer and Cardiac Cancer Integrated Transformation Research,Anyang Tumor Hospital,Anyang 455001,China;Department of Multidisciplinary Oncology,Henan Provincial Worker′s Hospital,Zhengzhou 450002,China)
出处
《中华消化杂志》
CAS
CSCD
北大核心
2024年第4期266-273,共8页
Chinese Journal of Digestion
基金
河南省科技发展计划(科技攻关)项目(232102310090、242102310125)
河南省医学科技攻关计划省部共建项目(SBGJ202002129)。
