Construction of circRNA-miRNA-mRNA network and identification of novel potential biomarkers for non-small cell lung cancer

Background The underlying circular RNAs (circRNAs)-related competitive endogenous RNA (ceRNA) mechanisms of pathogenesis and prognosis in non-small cell lung cancer (NSCLC) remain unclear. Methods Differentially expressed circRNAs (DECs) in two Gene Expression Omnibus datasets (GSE101684 and GSE112214) were identified by utilizing R package (Limma). Circinteractome and StarBase databases were used to predict circRNA associated-miRNAs and mRNAs, respectively. Then, protein-protein interaction (PPI) network of hub genes and ceRNA network were constructed by STRING and Cytoscape. Also, analyses of functional enrichment, genomic mutation and diagnostic ROC were performed. TIMER database was used to analyze the association between immune infiltration and target genes. Kaplan-Meier analysis, cox regression and the nomogram prediction model were used to evaluate the prognostic value of target genes. Finally, the expression of potential circRNAs and target genes was validated in cell lines and tissues by quantitative real-time PCR (qRT-PCR) and Human Protein Atlas (HPA) database. Results In this study, 15 DECs were identified between NSCLC tissues and adjacent-normal tissues in two GEO datasets. Following the qRT-PCR corroboration, 7 DECs (hsa_circ_0002017, hsa_circ_0069244, hsa_circ_026337, hsa_circ_0002346, hsa_circ_0007386, hsa_circ_0008234, hsa_circ_0006857) were dramatically downregulated in A549 and SK-MES-1 compared with HFL-1 cells. Then, 12 circRNA-sponged miRNAs were screened by Circinteractome and StarBase, especially, hsa-miR-767-3p and hsa-miR-767-5p were significantly up-regulated and relevant to the prognosis. Utilizing the miRDB and Cytoscape, 12 miRNA-target genes were found. Functional enrichment, genomic mutation and diagnostic analyses were also performed. Among them, FNBP1, AKT3, HERC1, COL4A1, TOLLIP, ARRB1, FZD4 and PIK3R1 were related to the immune infiltration via TIMER database. The expression of ARRB1, FNBP1, FZD4, and HERC1 was correlated with poor overall survival (OS) in NSCLC patients by cox regression and nomogram. Furthermore, the hub-mRNAs were validated in cell lines and tissues. Conclusion We constructed the circRNA-miRNA-mRNA network that might provide novel insights into the pathogenesis of NSCLC and reveal promising immune infiltration and prognostic biomarkers.

Automated summary

This study identified 15 differentially expressed circRNAs between NSCLC tissues and adjacent-normal tissues in two GEO datasets. Through validation by qRT-PCR, 7 circRNAs were found significantly downregulated in A549 and SK-MES-1. By utilizing various databases and analysis methods, miRNAs and mRNAs associated with the pathogenesis and prognosis of NSCLC were identified.