lncRNA-NORAD表达对食管癌Eca-109细胞生物学行为的影响及其机制

doi:10.13481/j.1671-587X.20220105

摘要/Abstract

摘要： 目的

研究长链非编码RNA （lncRNA）-DNA损伤诱导的非编码RNA（NORAD）在食管癌Eca-109细胞中的表达，分析沉默NORAD通过miR-26a-5p/Unc-51样自噬激活激酶1（ULK1）对食管癌Eca-109细胞生物学行为的影响。

方法

收集45例食管癌患者癌组织和40例癌旁正常组织标本，培养正常人食管鳞状上皮Het-1A细胞和食管癌Eca-109细胞，采用实时荧光定量PCR（RT-qPCR）法检测癌组织、癌旁正常组织、Het-1A细胞和Eca-109细胞中NORAD mRNA、miR-26a-5p和ULK1 mRNA表达水平。采用miRanda数据库检测NORAD与miR-26a-5p的结合位点，双荧光素酶报告基因实验检测NORAD与miR-26a-5p的关系。根据实验目的和转染质粒的不同，Eca-109细胞分为siRNA NC组和NORAD siRNA组，inhibitor NC组和miR-26a-5p inhibitor组，pcDNA-3.1（+）+mimics NC组、pcDNA-NORAD+ mimics NC组、pcDNA-3.1（+）+miR-26a-5p mimics组和pcDNA-NORAD+ miR-26a-5p mimics组，采用MTT法检测各组细胞活性，Transwell法检测各组细胞中侵袭细胞数，Western blotting法检测各组细胞中E-钙黏蛋白（E-cadherin）和N-钙黏蛋白（N-cadherin）蛋白表达水平。采用TargetScan数据库检测miR-26a-5p与ULK1的结合位点，双荧光素酶报告基因实验检测miR-26a-5p与ULK1的关系。Eca-109细胞分为siRNA NC组和ULK1 siRNA 组，采用MTT法检测各组细胞活性，Transwell法检测各组细胞中侵袭细胞数，Western blotting法检测各组细胞中E-cadherin和N-cadherin蛋白表达水平。Eca-109细胞分为siRNA NC+inhibitor NC组、NORAD siRNA+inhibitor NC组、siRNA NC+miR-26a-5p inhibitor组和NORAD siRNA+miR-26a-5p inhibitor组，采用RT-qPCR和Western blotting法检测各组细胞中ULK1 mRNA和蛋白表达水平。

结果

与癌旁组织或Het-1A细胞比较，食管癌组织或Eca-109细胞中NORAD和ULK1 mRNA表达水平明显升高（P<0.01），miR-26a-5p表达水平明显降低（P<0.01）。与siRNA NC组比较，NORAD siRNA组细胞活性明显降低（P<0.01），侵袭细胞数明显减少（P<0.01），细胞中E-cadherin蛋白表达水平明显升高（P<0.01），N-cadherin蛋白表达水平明显降低（P<0.01）。miRcode数据库和双荧光素酶报告基因检测，NORAD靶向miR-26a-5p。与inhibitor NC组比较，miR-26a-5p inhibitor组细胞活性明显升高（P<0.01），侵袭细胞数明显增加（P<0.01），细胞中E-cadherin蛋白表达水平明显降低（P<0.01），N-cadherin蛋白表达水平明显升高（P<0.01）。与pcDNA-3.1（+）+miR-26a-5p mimics组比较，pcDNA-NORAD+miR-26a-5p mimics组细胞活性明显升高（P<0.01），侵袭细胞数明显增加（P<0.01），细胞中E-cadherin蛋白表达水平降低（P<0.01），N-cadherin蛋白表达水平明显升高（P<0.01）。TargetScan数据库和双荧光素酶报告基因检测，miR-26a-5p靶向ULK1。与siRNA NC组比较，ULK1 siRNA组细胞活性明显降低（P<0.01），侵袭细胞数明显减少（P<0.01），细胞中E-cadherin蛋白表达水平明显升高（P<0.01），N-cadherin蛋白表达水平明显降低（P<0.01）。与siRNA NC+miR-26a-5p inhibitor组比较，NORAD siRNA+miR-26a-5p inhibitor组细胞中ULK1 mRNA和蛋白表达水平明显降低（P<0.01）。

结论

沉默lncRNA-NORAD可抑制Eca-109细胞增殖、侵袭和上皮-间质转化（EMT），其机制可能是通过调控miR-26a-5p/ULK1轴实现的。

关键词: 长链非编码RNA, DNA损伤诱导的非编码RNA, miR-26a-5p, Unc-51样自噬激活激酶1, 食管肿瘤, 细胞增殖

Abstract:

Objective To investigate the expression of long non-coding RNA （lncRNA）-non-coding activated by DNA damage （NORAD） in the esophageal cancer Eca-109 cells， and to analyze，the effect of NORAD silencing on the biological behaviors of Eca-109 cells through miR-26a-5p/UNC-51-like autophagy activated kinase 1（ULK1）. Methods The cancer tissue samples from 45 patients with esophageal cancer and 40 case of para-cancer normal tissue samples were collected；the normal esphageal squamous epithelial Het-1A cells and epophageal cancer Eca-109 cells were cultured. The expression levels of NORAD mRNA， miR-26a-5p and ULK1 mRNA in cancer tissue，adjacent normal tissue， Het-1A cells and Eca-109 cells were detected by real-time fluorescence quantitative PCR （RT-qPCR） method.The binding site of NORAD and miR-26a-5p was detected by miRanda database， and the relationship between NORAD and miR-26a-5p was detected by dual luciferase reporter assay.The Eca-109 cells were divided into siRNA NC group and NORAD siRNA group，inhibitor NC group and miR-26a-5p inhibitor group， pcDNA-3.1（+）+ mimics NC group and pcDNA-NORAD+mimics NC group， pcDNA-3.1（+）+miR-26a-5p mimics group and pcDNA-NORAD+miR-26a-5p mimics group according to different experimental purposes and transfected plasmids. The cell viabilities in various groups were detected by MTT assay， the number of invasion cells was detected by Transwell assay， and the expression levels of E-cadherin and N-cadherin proteins in various groups were detected by Western blotting method. The binding site of miR-26a-5p and ULK1 was detected by TargetScan database， and the relationship between miR-26a-5p and ULK1 was detected by double luciferase reporter gene assay.The Eca-109 cells were divided into siRNA NC group and ULK1 siRNA group. MTT assay was used to detect the cell viabilities in various groups， and Transwell assay was used to detect the number of invasion cells in various groups. The expression levels of E-cadherin and N-Cadherin proteins were detected by Western blotting method.The Eca-109 cells were divided into siRNA NC+inhibitor NC group， NORAD siRNA+inhibitor NC group， siRNA NC+miR-26a-5p inhibitor group and NORAD siRNA+miR-26a-5p inhibitor group；the expression levels of ULK1 mRNA and protein in the cells in various groujps were detected by RT-qPCR and Western blotting methods. Results Compared with adjacent normal tissue or Het-1A cells， the expression levels of NORAD and ULK1 mRNA in esophageal cancer tissue or Eca-109 cells were significantly increased（P<0.01），and the expression levels of miR-26a-5p were significantly decreased （P<0.01）.Compared with siRNA NC group，the cell viability in NORAD siRNA group was significantly decreased（P<0.01）， the number of invasion cells was decreased （P<0.01），the expression level of E-cadherin protein was significantly increased（P<0.01），and the expression level of N-cadherin protein was significantly decreased（P<0.01）.The results of miRcode database and dual luciferase reporter gene detection showed that NORAD was targeted miR-26a-5p.Compared with inhibitor NC group， the cell viability in miR-26a-5p inhibitor group was significantly increased （P<0.01）， the number of invasion cells was increased （P<0.01），the expression level of N-cadherin protein was significantly increased（P<0.01），and the expression level of E-cadherin protein was significantly decreased（P<0.01）. Compared with pcDNA-3.1（+）+miR-26a-5p mimics group， the cell viability in pcDNA-NORAD+ miR-26a-5p mimics group was significantly increased （P<0.01），the number of invasion cells was increased （P<0.01），the expression level of N-cadherin protein was significantly increased（P<0.01），and the expression level of E-cadherin protein was decreased（P<0.01）. The results of TargetScan database and dual luciferase reporter gene detection showed that miR-26a-5p was targeted ULK1. Compared with siRNA NC group，the cell viability in ULK1 siRNA group was significantly decreased （P<0.01），the number of invasion cells was decreased （P<0.01），the expression level of E-cadherin protein was significantly increased（P<0.01），and the expression level of N-cadherin protein was significantly decreased （P<0.01）. Compared with siRNA NC+miR-26a-5p inhibitor group， the expression levels of ULK1 mRNA and protein in NORAD siRNA+ miR-26a-5p inhibitor group were significantly decreased（P<0.01）.Conclusion Silencing lncRNA-NORAD can inhibited the proliferation， invasion and epithelial-mesenchymal transformation （EMT） of Eca-109 cells，and its mechanism may be achieved by regulating the miR-26a-5p/ULK1 axis.

Key words: Long non-coding RNA, Non-coding RNA activated by DNA damage, MiRNA-26a-5p, Unc-51-like autophagy activating kinase 1, Esophageal neoplasms, Cell proliferation

中图分类号:

R735.1

周超锋,周世繁,田青,王赛,李洪霖,马纯政. lncRNA-NORAD表达对食管癌Eca-109细胞生物学行为的影响及其机制[J]. 吉林大学学报(医学版), 2022, 48(1): 33-43.

Chaofeng ZHOU,Shifan ZHOU,Qing TIAN,Sai WANG,Honglin LI,Chunzheng MA. Effect of lncRNA-NORAD overexpression on biological behaviors of esophageal cancer Eca-109 cells and its mechanism[J]. Journal of Jilin University(Medicine Edition), 2022, 48(1): 33-43.

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Group	Cell viability (η/%)	Number of invasion
PcDNA-3.1(+)+mimics NC	45.36±5.63	60.82±6.72
PcDNA-NORAD+mimics NC	82.48±6.58^*	120.56±7.85^*
PcDNA-3.1(+)+miR-26a-5p mimics	23.46±6.36^*	30.58±7.83^*
PcDNA-NORAD+miR-26a-5p mimics	60.82±6.72^△	62.65±7.25^△

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