NORAD regulates epithelial‑mesenchymal transition of non‑small cell lung cancer cells via miR‑422a

Abstract

The poor prognosis of non‑small cell lung cancer (NSCLC) is related to epithelial‑mesenchymal transition (EMT). Recent studies demonstrated that non‑coding RNA activated by DNA damage (NORAD) displays a carcinogenic effect and targets microRNA (miR)‑422a, which may be involved in tumor cell migration and invasion. The aim of the present study was to investigate the effect of NORAD on NSCLC cell EMT and the underlying mechanism. Reverse transcription‑quantitative PCR and western blotting were performed to detect the expression levels of long non‑coding RNAs, miRNAs and mRNAs. Cell viability, migration and invasion were detected by conducting Cell Counting Kit‑8, wound healing and Transwell assays, respectively. The target of NORAD was predicted using starBase and further confirmed by conducting a dual‑luciferase reporter assay. The results indicated that NORAD expression was significantly increased in lung cancer tissues and cells compared with adjacent healthy tissues and cells. Compared with the control groups, NORAD overexpression promoted SK‑MES‑1 cell viability, migration and invasion, whereas NORAD knockdown resulted in the opposite effects in A549 cells. Moreover, miR‑422a, which was predicted to be a target of NORAD, displayed lower expression levels in lung cancer tissues compared with adjacent healthy tissues. In addition, miR‑422a overexpression partially reversed NORAD overexpression‑induced increases in SK‑MES‑1 cell viability, migration, invasion and EMT. In addition, miR‑422a knockdown partially reversed the effects of NORAD knockdown. The present study suggested that NORAD regulated lung cancer cell EMT by regulating the expression of miR‑422a, providing a potential therapeutic target for the intervention of the development of NSCLC.

Keywords: non‑coding RNA activated by DNA damage; microRNA‑422a; NSCLC; EMT.

Figure 1.

Effects of NORAD in NSCLC tissues and on NSCLC cell viability. (A) NORAD expression in NSCLC tissues. **P<0.001 vs. Adjacent tissue. (B) NORAD expression in NSCLC cell lines. **P<0.001 vs. 16HBE. Transfection efficiency of (C) pc-NORAD in SK-MES-1 and (D) si-NORAD in A549 cells. **P<0.001 vs. pc-Control or si-Control. Effect of NORAD on (E) SK-MES-1 and (F) A549 cell viability. **P<0.001 vs. pc-Control or si-Control. Effect of NORAD on (G) SK-MES-1 and (H) A549 cell cycle distribution. *P<0.05 and **P<0.001 vs. pc-Control or si-Control. NORAD, non-coding RNA activated by DNA damage; NSCLC, non-small cell lung cancer; si, small interfering RNA.

Figure 2.

Effects of NORAD on NSCLC cell biological behaviors, such as migration and invasion. Effect of NORAD on (A) SK-MES-1 and (B) A549 cell migration. Effect of NORAD on (C) SK-MES-1 and (D) A549 cell invasion. **P<0.001 vs. pc-Control or si-Control. NORAD, non-coding RNA activated by DNA damage; NSCLC, non-small cell lung cancer; si, small interfering RNA.

Figure 3.

Interaction between NORAD and miR-422a in NSCLC tissues. (A) Binding site between miR-422a and NORAD. Effect of miR-422a mimic on the luciferase activity of NORAD-wt and NORAD-mut in (B) SK-MES-1 and (C) A549 cells. **P<0.001 vs. Blank. (D) miR-422a expression levels in NSCLC tissues. **P<0.001 vs. adjacent tissue. (E) miR-422a expression levels in NSCLC cell lines. **P<0.001 vs. 16HBE. (F) miR-422a expression was negatively correlated with NORAD expression in NSCLC tissues. NORAD, non-coding RNA activated by DNA damage; miR, microRNA; NSCLC, non-small cell lung cancer; wt, wild-type; mut, mutant.

Figure 4.

NORAD regulates NSCLC cell viability, migration and invasion via miR-422a. Effect of miR-422a and NORAD on (A) SK-MES-1 and (B) A549 cell viability. Effect of miR-422a and NORAD on (C) SK-MES-1 and (D) A549 cell migration. Effect of miR-422a and NORAD on (E) SK-MES-1 and (F) A549 cell invasion. *P<0.05 and **P<0.001 vs. mimic-Control or inhibitor-Control; ^#P<0.05 and ^##P<0.001 vs. mimic or inhibitor; ^{^}P<0.05 and ^{^^}P<0.001 vs. mimic + pc-NORAD or inhibitor + si-NORAD. NORAD, non-coding RNA activated by DNA damage; NSCLC, non-small cell lung cancer; miR, microRNA; si, small interfering RNA.

Figure 5.

NORAD regulates EMT by regulating miR-422a. Effects of miR422a and NORAD on miR422a expression in (A) SK-MES-1 and (B) A549 cells. EMT-related protein expression levels in (C) SK-MES-1 and (D) A549 cells. **P<0.001 vs. mimic-Control or inhibitor-Control; ^##P<0.001 vs. mimic or inhibitor; ^{^}P<0.05 and ^{^^}P<0.001 vs. mimic + pc-NORAD or inhibitor + si-NORAD. NORAD, non-coding RNA activated by DNA damage; EMT, epithelial-mesenchymal transition; miR, microRNA; si, small interfering RNA; MMP, matrix metallopeptidase.