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