中华消化杂志2024，Vol.44Issue(4) ：266-273.DOI:10.3760/cma.j.cn311367-20230830-00056

NORAD诱导的自噬促进食管胃结合部腺癌细胞对奥沙利铂耐药的研究

A study on the NORAD-induced autophagy promotes oxaliplatin resistance in adenocarcinoma of the esophagogastric junction

李守淼 刘志强 曹恒 聂志勇 李慧 李保中
中华消化杂志2024，Vol.44Issue(4) ：266-273.DOI:10.3760/cma.j.cn311367-20230830-00056
✕
  • NETL
  • NSTL
  • 万方数据

NORAD诱导的自噬促进食管胃结合部腺癌细胞对奥沙利铂耐药的研究

A study on the NORAD-induced autophagy promotes oxaliplatin resistance in adenocarcinoma of the esophagogastric junction

李守淼 1刘志强 1曹恒 2聂志勇 2李慧 3李保中1
扫码查看

作者信息

  • 1. 安阳市肿瘤医院腹部肿瘤外科暨安阳市胃贲门癌整合转化研究重点实验室,安阳 455001
  • 2. 安阳市肿瘤医院消化肿瘤内科暨安阳市胃贲门癌整合转化研究重点实验室,安阳 455001
  • 3. 河南省职工医院肿瘤多学科联合科,郑州 450002
  • 折叠

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(IC50).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细胞(4.040±0.336),且在PDC细胞中相对表过量低于 PDC-R 细胞,差异均有统计学意义(t=-14.94、-37.21、-19.43,均P＜0.001);在 shNORAD PDC-R细胞中的相对表达量(0.290±0.165)则低于PDC-R细胞,差异有统计学意义(t=-49.05,P＜0.001).miR-433-3p在GES-1细胞中的相对表达量(1.017±0.248)高于PDC细胞(0.470±0.156)和PDC-R细胞(0.203±0.045),且PDC细胞中的相对表达量高于PDC-R细胞,差异均有统计学意义(1=9.15、15.85、8.11,均 P＜0.001),在 shNORAD PDC-R 细胞中的相对表达量(0.699±0.256)也高于 PDC-R 细胞,差异有统计学意义(t=9.37,P＜0.001).蛋白质印迹法检测结果显示,PDC-R中LC3B-Ⅱ相对表达量高于PDC细胞(0.426±0.060比0.212±0.041),shNORAD PDC-R细胞中LC3B-Ⅱ的相对表达量(0.155±0.029)低于PDC细胞,差异均有统计学意义(t=8.70、-79.45,均P＜0.001);而p62在各细胞系中表现呈相反趋势,在PDC-R中相对表达量低于PDC(0.205±0.031比0.311±0.040),在shNORAD PDC-R中的相对表达量(0.504±0.084)高于PDC,差异均有统计学意义(t=-31.19、62.80,均P＜0.001).在OE19细胞系的原始细胞、耐药细胞和NORAD敲减细胞中NORAD和miR-433-3p,以及LC3B-Ⅱ和p62表达变化规律与PDC细胞系相似.CCK-8评估靶细胞活力发现,奥沙利铂对PDC、PDC-R 和 shNORAD PDC-R 细胞的 1C50值分别为 14.28、22.27 和 2.51 μg/mL,对 OE19、OE19-R 和shNORAD OE19-R细胞的1C50值分别为3.95、8.12和1.89 μg/mL.结论 NORAD在AEG组织及细胞中呈高表达;在奥沙利铂耐药的细胞呈过表达,而且增加了细胞的自噬活性.敲减NORAD后AEG细胞自噬活性受到抑制,而且AEG细胞对奥沙利铂的敏感性增加.

Abstract

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(IC50)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-luciferase reporter assay indicated that the relative luciferase activity of the NORAD-WT group was lower than that of NORAD-Mut group in PDC and PDC-R cells(0.441±0.104 vs.0.928±0.204,0.449±0.112 vs.0.947±0.201),and the differences were statistically significant(t=-14.74 and-14.94,both P＜0.001).The results of dual-luciferase reporter assay of OE19 and OE19-R cell lines were the same as those of PDC cell lines.The results of qRT-PCR showed that the expression of NORAD in GES-1 cells(1.016±0.213)was lower than that of PDC cells(2.194±0.322)and PDC-R cells(4.040±0.336),and the differences were statistically significant(t=-14.94 and-37.21,both P＜0.001).Furthermore,the expression level of NORAD in PDC was also found to be lower than that in PDC-R cells,and the difference was statistically significant(t=-19.43,P＜0.001).Additionally,shNORAD PDC-R cells exhibited lower expression level of NORAD(0.290±0.165)compared with PDC-R cells,and the difference was statistically significant(t=-49.05,P＜0.001).The expression level of miR-433-3p in GES-1 cells(1.017±0.248)was higher than that in PDC cells(0.470±0.156)and PDC-R cells(0.203±0.045),and the differences were statistically significant(t=9.15 and 15.85,both P＜0.001).Moreover,the expression level of miR-433-3p was found to be higher in PDC cells compared with PDC-R cells,and the difference was statistically significant(t=8.11,P＜0.001).Additionally,the expression level of miR-433-3p in shNORAD PDC-R cells(0.699±0.256)was also higher than that in PDC-R cells(t=9.37,P＜0.001).The results of Western blotting showed that the expression of LC3B-Ⅱ in PDC-R was higher than that in PDC cells(0.426±0.060 vs.0.212±0.041),the expression of LC3B-Ⅱ in shNORAD PDC-R cells(0.155±0.029)was lower than that in PDC cells,and the differences were statistically significant(t=8.70 and-79.45,both P＜0.001).However the expression of p62 protein in each cell line showed an opposite trend,with a lower relative expression in PDC-R than PDC(0.205±0.031 vs.0.311±0.400),and the expression in shNORAD PDC-R(0.504±0.084)was higher than that in PDC,and the differences were statistically significant(t=-31.19 and 62.80,both P＜0.001).The expression patterns of NORAD,miR-433-3p,LC3B-Ⅱ and p62 proteins in OE19,OE19-R and shNORAD OE19-R cells were similar to those in PDC.The results of CCK-8 assessment of target cell viability showed that the IC50 values of PDC,PDC-R and shNORAD PDC-R cell lines were 14.28,22.27 and 2.51 µg/mL,respectively;and the IC50 values of OE19,OE19-R and shNORAD PDC-R cell lines were 3.95,8.12 and 1.89 μg/mL,respectively.Conclusions NORAD is highly expressed in AEG tissues and cells.NORAD is overexpressed in oxaliplatin-resistant cell lines and increase the autophagy activity of cells.After NORAD is knockdown,autophagy activity is inhibited and the sensitivity of AEG cells to oxaliplatin is significantly enhanced.

关键词

食管胃结合部腺癌/长链非编码RNA/NORAD/微RNA-433-3p/奥沙利铂/耐药性/自噬活性

Key words

Adenocarcinoma of esophagogastric junction/Long non-coding RNA/NORAD/MicroRNA-433-3p/Oxaliplatin/Drug resistance/Autophagy activity

引用本文复制引用

基金项目

河南省科技发展计划(科技攻关)项目(232102310090)

河南省科技发展计划(科技攻关)项目(242102310125)

河南省医学科技攻关计划省部共建项目(SBGJ202002129)

出版年

2024
中华消化杂志
中华医学会

中华消化杂志

CSTPCD北大核心
影响因子：1.726
ISSN：0254-1432
参考文献量2
段落导航相关论文