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

Abstract

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. IntroductionLung cancer, mainly non-small cell lung cancer (NSCLC), is the leading cause of cancer-associated mortality globally. More than 133,000 new diagnoses and over 77,000 associated mortalities are expected to occur in 2015 in Japan (1). In spite of progress in NSCLC therapy, the prognosis for patients with NSCLC remains poor (2). Therefore, clarification of the biomarkers most likely to predict the recurrence of NSCLC after curative surgery is required to improve the outcome of NSCLC patients.MicroRNAs (miRNAs/miRs) are one of the most compelling molecular markers in the diagnosis and prognosis of tumors (3,4). miRNAs are short (20-24 nt) non-coding RNAs that take part in the post-transcriptional regulation of gene expression in multicellular organisms by impacting mRNA stability and translation. miRNAs target protein-coding mRNAs at the post-transcriptional level by directly c...

Usefulness of Plasma Exosomal MicroRNA-451a as a Noninvasive Biomarker for Early Prediction of Recurrence and Prognosis of Non-Small Cell Lung Cancer

“…Another study reported that elevated levels of plasma exosomal miR-23b-3p, miR-10b-5p, and miR-21-5p were prognostic biomarkers for NSCLC patients [20]. Our group also demonstrated that miR-21 and miR-4257 levels of plasma exosomes are useful biomarkers for diseasefree survival (DFS) of NSCLC patients [21]. However, no published reports have evaluated the prognostic value of exosomal miR-451a, which we selected by miRNA array analysis of early-stage NSCLC (stage IA) patients.…”

“…Plasma and blood cells were separated by centrifugation at 1,200 g for 15 min at 4 ° C; 1.0 mL of plasma was used for miRNA array analysis, and 500 μL for qRT-PCR. The exosomes of the plasma were purified by the ultracentrifugation method described previously [21,22]. In brief, exosomes were separated by ultracentrifugation at 15,000 g for 70 min at 4 ° C, and the pellets were washed with phosphate-buffered saline and stored at -80 ° C for microarray and qRT-PCR analysis.…”

“…Relative quantification of miRNA expression was calculated using the 2 -ΔΔC T method as described previously [21][22][23]. In the exosome samples, ΔC T was calculated by subtracting the C T values of miR-16a from the C T values of miR-451a.…”

“…Recently, exosomal transport of miRNAs has gained attention as a carrier of genetic information [18]. Regarding NSCLC patients, only a few papers have reported on the usefulness of exosomal miRNA as a prognostic biomarker [19][20][21]. It was shown that miR-146a-5p levels in serum exosomes predict progression-free survival of NSCLC patients [19].…”

The functional role of exosome microRNAs in lung cancer

“…In addition, they found that exosomal miR-21 was significantly associated with tumor size and tumor-node-metastasis (TNM) stage and exosomal miR-4257 showed an obvious association with lymphatic invasion, histological type and TNM stage of lung cancer. Additionally, the disease-free survival (DFS) rates of high expression exosomal miR-21 patients were significantly worse than those of low exosomal miR-21 patients, and the DFS rates of patients with high exosomal miR-4257 levels were significantly worse than those with low exosomal miR-4257 levels [29].…”