Ascites-derived circulating microRNAs as potential diagnostic biomarkers of gastric cancer-associated malignant ascites

Peritoneal carcinomatosis with malignant ascites is associated with dismal prognosis in gastric cancer. Malignant ascites is the most relevant body fluid in which to seek diagnostic biomarkers for peritoneal carcinomatosis. We aimed to identify and validate ascites-derived circulating microRNAs (miRNAs) that are differentially expressed between liver cirrhosis-associated benign ascites (LC-ascites) and gastric cancer-associated malignant ascites (GC-ascites). MiRNA expression levels were investigated in three independent cohorts. Overall, 165 ascites samples (73 LC-ascites and 92 GC-ascites) were obtained from the National Biobank of Korea. Initially, microarrays were used to screen the expression levels of 2,006 miRNAs in the discovery cohort (n = 22). Subsequently, quantitative reverse transcriptase-polymerase chain reaction (qRT-PCR) analyses were used to validate the expression levels of selected miRNAs in the training (n = 70) and validation (n= 73) cohorts. In addition, the levels of carcinoembryonic antigen (CEA), a commonly used tumor marker, were determined in the ascites samples. Expression levels of miR-574-3p, miR-181b-5p, miR-4481, and miR-181d were significantly lower in the GC-ascites samples than in the LC-ascites samples, and miR-181b-5p showed the best diagnostic performance for GC-ascites (area under the curve [AUC] = 0.798 and 0.846 for the training and validation cohorts, respectively). The diagnostic performance of CEA for GC-ascites was improved if CEA and miR-181b-5p were analyzed together (AUC = 0.981 and 0.946 for the training and validation cohorts, respectively). Overall, we identified ascites-derived circulating miRNAs capable of differentiating non-malignant ascites and GC-ascites, and demonstrated that the combined use of miR-181b-5p and CEA produces the optimal diagnostic yield. All ascites samples were transported to the National Biobank of Korea within 1 hour of collection and then centrifuged at 3,134 g for 10 min to remove large cell particles and cell debris. The supernatant from each sample was aliquoted into microcentrifuge tubes and stored at -80°C prior to miRNA measurements. Exosomal RNA was extracted from the 22 ascites samples in the discovery cohort (10 liver cirrhosis [LC]-ascites and 12 gastric cancer [GC]-ascites). The exosomes in each ascites sample (500 μL of supernatant) were precipitated using the miRCURY Exosome Isolation Kit (Exiqon, Woburn, MA, USA). Exosomal RNA was isolated using Trizol LS Reagent (Molecular Research Center, Cincinnati, OH, USA) according to the manufacturer’s instructions, and then quantified by measuring absorbance at 260 nm. RNA integrity was confirmed using a Bioanalyzer 2100 instrument (Agilent Technologies, San Carlos, CA, USA). MiRNA profiling was performed using the Agilent Human miRNA Microarray Release 19.0 platform.