Circulating miR-200c as a diagnostic and prognostic biomarker for gastric cancer

Abstract

Background: MicroRNAs are aberrantly expressed and correlate with tumourigenesis and the progression of solid tumours. The miR-200 family determines the epithelial phenotype of cancer cells and regulates invasiveness and migration. Thus, we hypothesised that the quantitative detection of the miR-200 family as epithelial-specific microRNAs in the blood could be a useful clinical biomarker for gastric cancer (GC).

Methods: We initially validated the expression levels of miR-200a, 200b, 200c and 141 in GC cell lines (n = 2) and blood from healthy controls (n = 19) using real-time quantitative reverse transcription PCR (qRT-PCR). The microarray expression profiles of the miR-200 family in 160 paired samples of non-tumour gastric mucosae and GC were downloaded through ArrayExpress and analysed. MiR-200c was selected for clinical validation. The qRT-PCR prospective assessment of miR-200c was performed using 67 blood samples (52 stage I-IV GC patients and 15 controls); the area under the receiver operating characteristic curve (AUC-ROC) was estimated. The Kaplan-Meier and Breslow-Wilcoxon tests were used to assess the correlation of miR-200c with overall and progression-free survival (OS and PFS). Multivariate analyses were performed using the Cox model.

Results: The miR-200c blood expression levels in GC patients were significantly higher than in normal controls (p = 0.018). The AUC-ROC was 0.715 (p = 0.012). The sensitivity, specificity and accuracy rates of 65.4%, 100% and 73.1%, respectively, were observed. The levels of miR-200c in the blood above the cutoff defined by the ROC curve was found in 17.6% of stage I-II GC patients, 20.6% of stage III patients and 67.7% of stage IV patients (p < 0.001). The miR-200c expression levels were not associated with clinical or pathological characteristics or recent surgical procedures. There was a correlation (p = 0.016) with the number of lymph node metastases and the increased expression levels of miR-200c in blood were significantly associated with a poor OS (median OS, 9 vs 24 months; p = 0.016) and PFS (median PFS, 4 vs 11 months; p = 0.044). Multivariate analyses confirmed that the upregulation of miR-200c in the blood was associated with OS (HR = 2.24; p = 0.028) and PFS (HR = 2.27; p = 0.028), independent of clinical covariates.

Conclusions: These data suggest that increased miR-200c levels are detected in the blood of gastric cancer patients. MiR-200c has the potential to be a predictor of progression and survival.

Figure 1

Real-time PCR of the miR-200 family in control blood and gastric cancer cell lines. The raw quantification cycle (Cq) data for the miR-200 cluster in the control blood samples (n = 19) and gastric cancer cell lines (OE-19 and MKN-45) are depicted. In the blood, the mean Cq was lower for miR-141 (Cq = 28) compared with those for miR-200a (Cq = 35), 200b (Cq = 35) and 200c (Cq = 35; ANOVA, p < 0.001; Bonferroni post hoc test, p < 0.001). In the GC cell lines, the mean Cqs were 15.3, 16.7, 17.7 and 16.1 for miR-141, 200a, 200b and 200c, respectively, without significant differences (ANOVA, p = 0.133). The red boxes indicate control blood samples, while the light-blue boxes indicate the gastric cancer cell lines.

Figure 2

Real time PCR of miR-200c in blood samples. The graph depicts the increasing relative expression levels for the mean blood expression levels of miR-200c (Kruskal-Wallis test, p =0.018) from controls (n = 15) and gastric cancer samples (n = 52). Significant differences were observed between the blood expression levels of miR-200c in each TNM stage subgroup and the control group (Bonferroni post hoc test: stage I-III vs control, *p = 0.018; stage IV vs control, **p < 0.001). MiR-200c was measured in triplicate using qRT-PCR and normalised to U6 snRNA and 5S rRNA. The horizontal bar denotes the mean value for each group.

Figure 3

The role of blood miR-200c in gastric cancer diagnosis. The receiver-operating characteristic (ROC) curve analysis using blood miR-200c expression levels for discriminating gastric cancer (n = 52) and controls (n = 15) is shown. The area under the ROC curve is shown [AUC 0.715 (95% CI, 0.597–0.833); p = 0.012; cutoff value is 62.4; sensitivity, 65.4%; specificity, 100%].

Figure 4

miR-200c expression levels measured in the peripheral blood are associated with poor prognosis in gastric cancer patients. Kaplan-Meier curves showing (a) the overall survival (OS) and (b) the progression-free survival (PFS) of 52 subjects with high or low blood expression levels of miR-200c. Continuous miR-200c expression levels measured using qRT-PCR were converted to a dichotomous variable using the mean level of expression as a threshold. The p values were computed using the Breslow-Wilcoxon test.