Plasma microRNA profiles: identification of miR-25 as a novel diagnostic and monitoring biomarker in oesophageal squamous cell carcinoma

Abstract

Background: Recent studies have demonstrated that microRNAs are stably detectable in plasma/serum because of their binding to specific proteins or being packaged in secretory particles. This study was designed to detect novel microRNAs in plasma for cancer detection and monitoring using microRNA array-based approaches in oesophageal squamous cell carcinoma (ESCC) patients.

Methods: Through the integration of two Toray 3D-Gene microRNA array-based approaches to compare plasma microRNA levels between ESCC patients and healthy volunteers and between preoperative and postoperative ESCC patients, we identified a novel plasma biomarker in ESCC.

Results: (1) Eight upregulated and common microRNAs (miR-15b, 16, 17, 25, 19b, 20a, 20b, and 106a) were selected using two high-resolution microRNA array approaches. (2) Test-scale analyses by quantitative RT-PCR validated a significant higher levels of plasma miR-19b (P=0.0020) and miR-25 (P=0.0030) in ESCC patients than controls. However, a significant correlation was observed between plasma miR-19b levels and concentrations of red blood cells (P=0.0073) and haemoglobin (P=0.0072). (3) miR-25 expression was found to be significantly higher in ESCC tissues (P=0.0157) and ESCC cell lines (P=0.0093) than in normal tissues and fibroblasts. (4) In a large-scale validation analysis, plasma miR-25 levels were significantly higher in 105 preoperative (P<0.0001) ESCC patients who underwent curative oesophagectomy and 20 superficial ESCC patients who underwent endoscopic resection (P<0.0001) than in 50 healthy volunteers. (5) Plasma miR-25 levels were significantly reduced in postoperative samples than in preoperative samples (P<0.0005) and were significantly increased during ESCC recurrences (P=0.0145).

Conclusions: Plasma miR-25 might be a clinically useful biomarker for cancer detection and the monitoring of tumour dynamics in ESCC patients.

Figure 1

Study design to detect novel plasma miRNA biomarkers for ESCC.

Figure 2

Selection of plasma miRNA candidates through the integration of two comprehensive miRNA array-based approaches. While using two miRNA array-based approaches to compare plasma miRNA levels between (A) ESCC patients and healthy volunteers and (B) between preoperative and postoperative ESCC patients, novel miRNA candidates for cancer detection and tumour monitoring were selected. Of the 15 highly upregulated miRNAs from each group, eight common miRNAs were selected.

Figure 3

Test-scale analyses comparing plasma levels of eight common miRNAs in ESCC patients and healthy controls. For test-scale analyses, we investigated the plasma levels of eight selected miRNAs in 20 ESCC patients and 10 health volunteers by qRT–PCR. miR-25 (P=0.0030) and miR-19b (P=0.0020) were validated to be significantly upregulated in ESCC patients than in healthy volunteers.

Figure 4

Evaluation of the correlation between plasma miRNA levels and peripheral blood cells. The correlation between selected plasma miRNA levels and peripheral blood cells in 20 ESCC patients was determined. (A) A significant correlation was observed between plasma miR-19b levels and concentrations of red blood cells and haemoglobin in the peripheral blood, (B) whereas no significant correlation was observed between plasma miR-25 levels and any type of blood cells in the peripheral blood.

Figure 5

Evaluation of miR-25 levels in ESCC tissues, ESCC cell lines and plasma samples of patients with ESCC. (A) Difference in miR-25 expression between ESCC tissues and normal tissues determined by a waterfall plot. miR-25 expression levels were significantly higher in ESCC tissues than in normal oesophageal tissues (P=0.0157). The upper and lower limits of the boxes and lines inside the boxes indicate the 75th and 25th percentiles and the median, respectively. The upper and lower horizontal bars denote the 90th and 10th percentiles, respectively. (B) Difference in miR-25 expression between ESCC cell lines, a fibroblast cell line WI-38, and normal tissues. miR-25 expression levels were significantly higher in ESCC cell lines than in both a fibroblast cell line and normal oesophageal tissues (P=0.0093). (C) Plasma miR-25 levels in 105 consecutive ESCC patients and 50 healthy volunteers. Using a real-time RT–PCR assay, difference in plasma miR-25 expression between ESCC patients and normal healthy volunteers was determined by a waterfall plot. Plasma miR-25 levels were significantly higher in ESCC patients than in healthy volunteers (P<0.0001).

Figure 6

Plasma miR-25 levels in 20 consecutive superficial ESCC patients undergoing curative endoscopic resection and 50 healthy volunteers. (A) Plasma miR-25 levels were significantly higher in superficial ESCC patients than in healthy volunteers (P<0.0001). (B) The area under the receiver operating characteristic curve was 0.8555.

Figure 7

Evaluation of whether plasma miR-25 level could reflect tumor dynamics. (A) Comparison of plasma miR-25 concentrations between preoperative and postoperative samples from ESCC patients. Plasma miR-25 levels were significantly lower in postoperative samples than in preoperative samples (P=0.0001). (B) Comparison of plasma miR-25 levels between postoperative and recurrence state samples from ESCC patients. In some patients from whom paired samples were collected after oesophagectomy and after recurrences, a significant re-elevation in plasma miR-25 levels was found at recurrence (P=0.0145).