Overexpression of hsa_circ_0001715 is a Potential Diagnostic and Prognostic Biomarker in Lung Adenocarcinoma

Abstract

Background: Circular RNAs (circRNAs) play important roles in tumorigenesis, including lung cancer. However, the expression profile and clinical value of circRNAs in lung adenocarcinoma remain unclear. The purpose of this study was to establish the circRNAs expression profile of lung adenocarcinoma and determine its potential diagnostic and prognostic value.

Materials and methods: The global expression profile of circRNAs in lung adenocarcinoma tissue was determined from five paired lung adenocarcinoma tissues and adjacent normal tissues. The expression levels of selected candidate circRNA were validated by qRT-PCR. Sequence analysis was used to confirm the specificity of amplified circRNA. The candidate circRNA level was further detected in plasma samples from lung adenocarcinoma patients and healthy controls. The relationships between their levels and clinicopathological factors were explored. Receiver operating characteristic (ROC) curve was constructed to differentiate lung adenocarcinoma from healthy controls. Kaplan-Meier was performed to show survival curves and survival characteristics. The significance of different prognostic factors for overall survival (OS) was analyzed using Cox proportional hazards model.

Results: CircRNA microarray showed 394 circRNAs were differentially expressed, including 215 up-regulated and 179 down-regulated circRNAs. Hsa_circ_0001715 was the most up-regulated circRNA in lung adenocarcinoma tissues. Plasma hsa_circ_0001715 levels were significantly higher in lung adenocarcinoma patients versus healthy controls (P < 0.001). We further found that high plasma hsa_circ_0001715 was significantly correlated with TNM stage (P = 0.039) and distant metastasis (P = 0.030). Furthermore, ROC curve analysis showed that hsa_circ_0001715 had high diagnostic value, and the area under the curve (AUC) was 0.871. Lung adenocarcinoma patients with plasma hsa_circ_0001715 levels over 0.417 had significantly shorter OS than those with lower levels (P = 0.004). Univariate and multivariate survival analysis showed that plasma hsa_circ_0001715 level was an independent prognostic factor for the OS.

Conclusion: Our study revealed an aberrant circRNA expression profile in lung adenocarcinoma, and hsa_circ_0001715 is up-regulated and could act as a novel diagnostic and prognostic biomarker for lung adenocarcinoma.

Keywords: biomarker; circular RNA; hsa_circ_0001715; lung adenocarcinoma tissues; prognosis.

Figure 1

Analysis of differentially expressed circRNAs in five paired lung adenocarcinoma tissues and adjacent normal tissues. (A) Hierarchical clustering results of circRNA expression profiles between the lung adenocarcinoma tissues and matched normal tissues. The result from unsupervised hierarchical clustering analysis reveals distinguishable circRNAs expression profiling among specimens. Red: higher expression level, green: lower expression level. (B) CircRNAs in the scatter plot above the top green line and below the bottom green line indicate change of circRNAs between the two groups (P< 0.05). (C) Volcano plot of circRNA expression profile. The vertical lines correspond to 2.0-fold upregulation and downregulation, and the horizontal line represents a P-value of 0.05. The red point in the plot represents the differentially expressed circRNAs with statistical significance.

Figure 2

The specificity of amplified hsa_circ_0001715 with divergent primers. (A) Electrophoresis of qRT-PCR product of hsa_circ_0001715. (B) Melting curve analysis of qRT-PCR product of hsa_circ_0001715. (C) Sequencing the amplified product of hsa_circ_0001715.

Figure 3

Relative hsa_circ_0001715 expression in lung adenocarcinoma tissues and plasma. (A) The expression levels of hsa_circ_0001715 in each comparison between lung adenocarcinoma and matched-adjacent normal tissue (n = 43). (B) The expression levels of hsa_circ_0001715 in lung adenocarcinoma tissues relative to matched-adjacent normal tissues. (C) The plasma hsa_circ_0001715 levels were significantly higher compared to healthy controls. (***P < 0.001).

Figure 4

ROC curve of plasma hsa_circ_0001715. Plasma hsa_circ_0001715 had an AUC value of 0.871 (95% CI: 0.807–0.936) to discriminate patients with lung adenocarcinoma from healthy controls. With a cutoff of 7.58, the sensitivity, specificity and accuracy were 87.72%, 71.67% and 79.49%, respectively.

Figure 5

Kaplan–Meier survival curves for OS of lung adenocarcinoma patients with low- and high-concentration of plasma hsa_circ_0001715. Lung adenocarcinoma patients with high plasma hsa_circ_0001715 expression had a shorter OS than those with low-level of hsa_circ_0001715 (24.88 months vs 40.56 months, P = 0.004).