A five-microRNA panel in plasma was identified as potential biomarker for early detection of gastric cancer

Abstract

Background: Circulating microRNAs (miRNAs) have been implicated as novel biomarkers for gastric cancer (GC) diagnosis. However, the mixture of GC subtypes may have led to the inconsistent circulating miRNA profiles, and the clinical performance of circulating miRNAs has not yet been evaluated independently on early detection of GC.

Methods: A four-phase study was designed with a total of 160 cancer-free controls, 124 patients with gastric non-cardia adenocarcinoma (GNCA) and 36 patients diagnosed gastric cardia adenocarcinoma (GCA). In the discovery phase, we screened the miRNA expression profile in plasma of 40 GNCA patients (stage I) and 40 matched controls by TaqMan low density array (TLDA) chips with pooled samples. Differentially expressed miRNAs were further validated in individual sample using quantitative reverse-transcriptase PCR (qRT-PCR) in the training phase. Subsequently, in an independent validation phase, the identified miRNAs were evaluated in 48 GNCA patients (stage I) and 102 matched controls. Finally, the identified miRNAs were further assessed in an external validation phase including advanced GNCA and GCA patients. Additionally, the expression levels of identified miRNAs were measured in the media of BGC823 and MGC803 cell lines.

Results: Five miRNAs (miR-16, miR-25, miR-92a, miR-451 and miR-486-5p) showed consistently elevated levels in plasma of the GC patients as compared with controls, and were identified to be potential markers for GNCA with area under the receiver operating characteristic (ROC) curves (AUCs) ranging from 0.850 to 0.925 and 0.694 to 0.790 in the training and validation phases, respectively. The five-miRNA panel presented a high diagnostic accuracy for the early-stage GNCA (AUCs=0.989 and 0.812 for the training and validation phases, respectively). Three miRNAs (miR-16, miR-25 and miR-92a) were excreted into the culture media of GC cell lines.

Conclusions: The five-miRNA panel in plasma may serve as a potential non-invasive biomarker in detecting the early-stage GC.

Figure 1

Overview of the study design. Abbreviations: GCA= gastric cardia adenocarcinoma; GNCA= gastric non-cardia adenocarcinoma; TLDA= TaqMan low density array; qRT–PCR= quantitative reverse-transcriptase PCR assay; ROC= receiver operating characteristic curve.

Figure 2

Expression levels of the five miRNAs in plasma in the training and validation phases. The y axis represents relative expression of miRNAs normalised to cel-miR-39. GNCA, gastric non-cardia adenocarcinoma.

Figure 3

The discriminative ability of the five-miRNA panel between the GNCA patients and cancer-free subjects by receiver operating characteristic curve (ROC) analysis. Red line, the training phase (AUC=0.989, sensitivity=97.5%, specificity=87.5%); green line, the validation phase (AUC=0.812, sensitivity=72.9%, specificity=89.2%); blue line, two phases combined (AUC=0.890, sensitivity=84.1%, specificity=90.8%).

Figure 4

Expression of the five identified miRNAs in culture media of gastric cancer (GC) cell lines (BGC823 and MGC803). MiR-16, miR-25 and miR-92a levels in media of both BGC823 and MGC803 increased with increased cell counts (0.5–2 × 10⁶ cells per well) and longer incubation intervals (24 and 48 h), whereas miR-451 and miR-486-5p levels did not change in either cell line. The y axis represents relative expression of miRNAs normalised to cel-miR-39 and cell-culture media at 0 h.