Exosomal microRNA in plasma as a non-invasive biomarker for the recurrence of non-small cell lung cancer

Abstract

Predictive biomarkers for the recurrence of non-small cell lung cancer (NSCLC) in patients who have received curative resection are important for cancer treatment. The functional microRNAs (miRNAs/miRs) in the exosomes of plasma and serum samples are of interest as stable and non-invasive biomarkers for recurrence in cancer patients. The aim of the present study was to clarify the usefulness of plasma exosomal miRNAs as biomarkers for the prediction of recurrence in NSCLC following curative resection. First, microarray-based expression profiling of miRNAs derived from exosomes in the plasma of 6 patients was employed to identify a biomarker that distinguishes between patients with and without NSCLC recurrence. In the miRNA microarray analyses, the exosomal miR-21 and miR-4257 levels of the NSCLC patients showed marked upregulation in those individuals with recurrence compared with those without recurrence and healthy individuals. These two miRNAs were thus selected as recurrence-specific biomarkers and their potential was evaluated in a separate cohort of 195 NSCLC patients. In comparison to the levels in 30 healthy individuals, exosomal miR-21 and miR-4257 levels showed a significant increase in the NSCLC patients (P<0.01). When evaluating the clinicopathological significance of these miRNAs, exosomal miR-21 showed a significant association with tumor size and tumor-node-metastasis (TNM) stage (P<0.05). Exosomal miR-4257 showed a significant association with histological type, lymphatic invasion and TNM stage (P<0.05). The disease-free survival (DFS) rates of high exosomal miR-21 patients were significantly worse than those of low exosomal miR-21 patients (P<0.05), and the DFS rates of patients with high exosomal miR-4257 levels were significantly worse than those with low exosomal miR-4257 levels (P<0.01). In the Cox multivariate analysis, plasma exosomal miR-21 and miR-4257 expression showed a significance association with DFS (P<0.05). These results suggest that plasma exosomal miR-21 and mir-4257 expression has potential as a predictive biomarker for recurrence in NSCLC patients who have received curative resection.

Keywords: biomarker; exosome; microRNA-21; microRNA-4257; non-small cell lung cancer.

Figure 1.

Exosome images captured by transmission electron microscope. Original magnification, ×25,000.

Figure 2.

Expression of miR-21 and miR-4257 in NSCLC patients and healthy individuals. To investigate the miRNA expression of exosome samples, reverse transcription-quantitative polymerase chain reaction using Taqman miRNA assays was performed. (A) Comparison of exosomal miR-21 levels between the 30 healthy volunteers and 195 patients with NSCLC. Exosomal miR-21 levels showed a significant increase in the NSCLC patients (*P<0.01). (B) Comparison of exosomal miR-4257 levels between the 30 healthy volunteers and 195 patients with NSCLC. Exosomal miR-4257 levels showed a significant increase in the NSCLC patients (*P<0.01). miR/miRNA, microRNA; NSCLC, non-small cell lung cancer.

Figure 3.

Kaplan-Meier survival curves of DFS based on exosomal miR-21 expression in non-small cell lung cancer patients. Patients were divided into two groups: those with low and those with high miR-21. The significance of these groups with regard to DFS was demonstrated. miR, microRNA; DFS, disease-free survival.

Figure 4.

Kaplan-Meier survival curves of DFS based on exosomal miR-4257 expression in non-small cell lung cancer patients. Patients were divided into two groups: those with low and those with high miR-4257. The significance of these groups for DFS was demonstrated. miR, microRNA; DFS, disease-free survival.