Dysregulated circulating miR-4429 serves as a novel non-invasive biomarker and is correlated with EGFR mutation in patients with non-small cell lung cancer

Abstract

This study aimed to investigate the correlation between microRNA (miR)-4429 and epidermal growth factor receptor (EGFR), the expression and clinical significance of miR-4429 in patients with non-small cell lung cancer (NSCLC), and the relationship between miR-4429 and EGFR mutation in NSCLC patients. Blood samples were collected from 122 NSCLC patients and 72 healthy volunteers. miR-4429 expression and EGFR mRNA expression were detected by real-time quantitative PCR. Correlation between miR-4429 and EGFR was evaluated by dual‑luciferase reporter assay and the Pearson correlation analysis. The ability of serum miR‑4429 to discriminate between NSCLC patients and healthy controls, and to discriminate between EGFR wild-type (EGFR-W) and EGFR mutant-type (EGFR-M) patients was assessed using receiver operating characteristic analysis. The relationship between miR-4429 and NSCLC patients' survival was identified by Kaplan-Meier survival curves and log-rank test. The prognostic value of miR-4429 in NSCLC patients was evaluated by Cox regression analysis. miR-4429 could directly bind to EGFR. Serum miR-4429, decreased in NSCLC patients, was negatively correlated with serum EGFR mRNA expression in NSCLC patients. Additionally, miR-4429 had a high diagnostic value for screening NSCLC patients from healthy controls, and was independently correlated with survival prognosis of NSCLC patients. Moreover, miR‑4429 was decreased in EGFR-M patients, which had a certain screening ability for EGFR‑M patients. Our findings indicate that miR-4429 is negatively correlated with EGFR in NSCLC, and may function as a diagnostic and prognostic biomarker for NSCLC patients. Additionally, miR-4429 is associated with EGFR mutation in NSCLC patients.

FIGURE 1

Correlation of miR-4429 with EGFR in NSCLC patients. (A) The binding sites of miR-4429 to EGFR was predicted by starBase v2.0 platform. (B) Relative luciferase activity in WT-EGFR group was decreased by miR-4429 mimic in HEK293T cells. (C) Serum miR-4429 expression in healthy controls and NSCLC patients. (D) Relative mRNA levels of EGFR in healthy controls and NSCLC patients. (E) Relative EGFR mRNA levels were negatively correlated with relative miR-4429 levels in the serum of NSCLC patients (r = -0.641, p < 0.001). *p < 0.05, ***p < 0.001 vs. mimic NC or healthy controls. EGFR, Epidermal growth factor receptor; WT, Wide-type; MUT, Mutant-type; NC, Negative control; NSCLC, Non-small cell lung cancer.

FIGURE 2

ROC analysis results indicated that serum miR-4429 had a high diagnostic value to screen NSCLC patients from healthy controls with an AUC of 0.918. AUC, Area under the ROC curve; ROC, Receiver operating characteristic; NSCLC, Non-small cell lung cancer.

FIGURE 3

NSCLC patients with low levels of miR-4429 had lower overall survival than patients with high miR-4429 levels (log-rank p = 0.0157). NSCLC, Non-small cell lung cancer.

FIGURE 4

Relationship between miR-4429 and EGFR mutation in NSCLC patients. (A) Serum miR-4429 expression in patients with EGFR-M and patients with EGFR-W. (B) Serum miR4429 expression in patients with different types of EGFR mutations. (C) ROC analysis indicated the ability of serum miR-4429 to discriminate between EGFR-M patients and EGFRW patients. **p < 0.01 vs. EGFR-W; EGFR, Epidermal growth factor receptor; EGFRM, EGFR mutant-type; EGFR-W, EGFR wild-type; AUC, Area under the ROC curve; ROC, Receiver operating characteristic; NSCLC, Non-small cell lung cancer.