Circular RNA_0001073 (circ_0001073) Suppresses The Progression of Non-Small Cell Lung Cancer via miR-582-3p/RGMB Axis

Abstract

Objective: Reportedly, circular RNAs (circRNAs) exert a crucial regulatory role in cancer. Circ_0001073 is derived from exons 3-5 of ACVR2A gene, which inhibits cancer progression. However, the role and mechanism of circ_0001073 in non-small cell lung cancer (NSCLC) are unclear. This study aimed to explore the role and mechanism of circ_0001073 in the development of NSCLC.

Materials and methods: In this experimental study, microarray analysis was employed to filter differential expressed circRNAs in NSCLC tissues. Also, circ_0001073, microRNA-582-3p (miR-582-3p), and repulsive guidance molecule B (RGMB) mRNA expressions were examined by quantitative real-time polymerase chain reaction (qRT-PCR). NSCLC cell multiplication was measured by the cell counting kit-8 (CCK-8) assay. Scratch healing experiment and Transwell experiment were performed to assess cell migration and invasion, respectively. Flow cytometry was applied to analyze the apoptosis of NSCLC cells. Western blot was employed to assess RGMB protein expression. Additionally, dualluciferase reporter gene experiment and RNA immunoprecipitation (RIP) experiment were applied to probe the binding sites between miR-582-3p and circ_0001073 or RGMB.

Results: circ_0001073 was remarkably under-expressed in NSCLC tissues and cells. circ_0001073 overexpression impeded the multiplication, migration, and invasion and enhanced the apoptosis of NSCLC cells in vitro. circ_0001073 directly bound to miR-582-3p and acted as a miRNA sponge to regulate RGMB expression. Besides, miR-582-3p overexpression or knockdown of RGMB remarkably reversed the malignant phenotypes of NSCLC cells induced by the up-regulation of circ_0001073 expression.

Conclusion: Circ_0001073 up-regulates RGMB expression through adsorbing miR-582-3p to inhibit NSCLC progression, suggesting its potential as a novel therapeutic target in NSCLC.

Keywords: Circular RNA; MicroRNA; Non-Small Cell Lung Cancer; Repulsive Guidance Molecule B.

Fig.1

Circ_0001073 expression in NSCLC tissues and cells. A. In dataset GSE112214, circ_0001073 expression was significantly down-regulated in NSCLC tissues compared with paracancerous tissues. B. Circ_0001073 expression in NSCLC tissues and paracancerous tissues was detected by qRT-PCR. C. Circ_0001073 expression in NSCLC cells and 16HBE cells was detected by qRT-PCR analysis. D. The schematic diagram of the formation of circ_0001073 from ACVR2A exons. E. The extracted RNA was treated with RNase R, and then the expression levels of circ_0001073 and GAPDH mRNA were determined by qRT-PCR to determine the circular structure of circ_0001073. F. The expressions of circ_0001073, U6 mRNA and GAPDH mRNA in the nuclei and cytoplasm of NSCLC cells were detected by qRT-PCR. G. Kaplan-Meier method was used to compare the survival time of the patients with high or low expression of circ_0001073. All experiments were performed in triplicate. *; P<0.05, NSCLC; Non-small cell lung cancer, and qRT-PCR; Quantitative real-time polymerase chain reaction.

Fig.2

Circ_0001073 overexpression inhibited the multiplication, migration, and invasion of NSCLC cells, and induced apoptosis. A. Using qRT-PCR analysis, circ_0001073 expression was detected in the A549 and H460 cells after circ_0001073 overexpression. B. After the transfection, cell multiplication was assessed using the CCK-8 method. C, D. Cell migration was assessed by wound-healing experiments. E-H. Transwell experiments were used to detect cell migration and invasion of NSCLC cells. I, J. Flow cytometry was used to detect apoptosis of NSCLC cells. All experiments were performed in triplicate. *; P<0.05, NC; Negative control, NSCLC; Non-small cell lung cancer, and qRT-PCR; Quantitative real-time polymerase chain reaction.

Fig.3

MiR-582-3p was the target of circ_0001073. A. Bioinformatics analysis predicted the binding sequence between miR-582-3p and circ_0001073. B. Dual-luciferase reporter gene experiments were used to verify the binding relationship between miR-582-3p and circ_0001073. C. The enrichment of circ_0001073 and miR-582-3p in Ago2 or IgG immunoprecipitate was determined using the RIP method. D. MiR-582- 3p expression in the circ_0001073 overexpression plasmid transfected cells was detected by qRT-PCR analysis. E, F. Using qRT-PCR, MiR-582- 3p expression was detected in the NSCLC tissues and cells. G. Pearson’s correlation analysis was employed to analyze the correlation between miR-582-3p expression and circ_0001073 expression in the NSCLC tissues. All experiments were performed in triplicate. *; P<0.05, NC; Negative control, NSCLC; Non-small cell lung cancer, and qRT-PCR; Quantitative real-time polymerase chain reaction.

Fig.4

RGMB was a downstream target of miR-582-3p in NSCLC cells. A. Bioinformatics analysis predicted the binding sequence between miR-582-3p and RGMB 3′UTR. B. The binding relationship between miR-582-3p and RGMB was detected using the dual-luciferase reporter gene assay. C. qRT-PCR analysis was used to detect miR-582-3p expression in A549 and H460 cells after the transfection with miR-582-3p mimics. D, E. The RGMB expressions (mRNA and protein) in A549 and H460 cells transfected with miR-582-3p mimics were detected using qRT-PCR and Western blot. F, G. Using qRT-PCR, mRNA expression of RGMB was detected in NSCLC tissues and cells. H, I. Pearson’s correlation analysis was used to analyze the correlation between RGMB mRNA and miR-582-3p or circ_0001073 expression in NSCLC tissues. J, K. RGMB expression (mRNA and protein) in si-RGMB transfected cells (A549 and H460) was detected by qRT-PCR analysis and Western blot. All experiments were performed in triplicate. *; P<0.05, NSCLC; Non-small cell lung cancer, and qRT-PCR; Quantitative real-time polymerase chain reaction.

Fig.5

Circ_0001073 modulated NSCLC progression via the miR-582-3p/RGMB axis. A, B. RGMB mRNA and protein expression was determined by qRT-PCR and Western blot. C. Cell multiplication was assessed using the CCK-8 method. D. Cell migration was assessed by wound-healing experiment. E, F. Transwell experiments were used to detect cell migration and invasion. G. Flow cytometry was used to detect the apoptosis of NSCLC cells. All experiments were performed in triplicate. *; P<0.05, NC; Negative control, NSCLC; Non-small cell lung cancer, and qRT-PCR; Quantitative real-time polymerase chain reaction.