Abstract 5279: Expression of exosomal microRNAs in malignant ascites, peritoneal lavage fluid and cell culture supernatant of gastric cancer.

Abstract

Abstract Background. A microRNA (miRNA) is non-coding RNA molecules of 21-25nt. miRNAs are post-transcriptional regulators that bind to messenger RNA transcripts, resulting in translational repression of target degradation and gene silencing. miRNAs are wrapped in exosome and secreted stably in body fluid, which can prevent RNase from degrading the miRNAs. Therefore several methods for miRNA-based early cancer detection using serum, plasma, and urine are reported. However there is no report to detect peritoneal dissemination of gastric cancer using malignant ascitis. Aim. To detect predictive factors for peritoneal dissemination of gastric cancer, global analysis of exosomal miRNAs in gastric malignant ascites, intraoperative peritoneal lavage fluid and cell culture supernatant were performed. Materials and Methods. A total of 6 gastric malignant ascites, 20 peritoneal lavage fluids and 2 human gastric carcinoma cell culture supernatants (OCUM-2M and its sub line with high potential for metastasis to the peritoneal cavity named OCUM-2MD3) were included in this study. The amount of 10 ml samples underwent a final ultracentrifugation at 120,000g twice to pellet the exosomes. miRNA was extracted and analyzed using Bioanalyzer. Exosomal miRNA expression profiling was performed using the Agilent Human miRNA Microarrays. To quantitatively detect mature miRNAs, TaqMan MicroRNA Assays was used. Statistical tests by spearman's rank-correlation coefficient were performed to investigate correlation of exosomal miRNAs that were expressed between malignant ascites and cell culture supernatant. Results. miRNAs were detected in each gastric malignant ascites at concentrations ranging from 61.7 ng to 220 ng, however no or very low amounts of ribosomal RNA (18S and 28S). Microarray analysis detected 327 miRNAs in malignant ascites, 389 in OCUM-2M and 414 in OCUM-2MD3. miRNA expression of Malignant ascites correlated that of cell culture supernatants; OCUM-2M(r=0.774, p<0.01) and OCUM-2MD3(r=0.776, r<0.001). Total of 140 miRNAs in OCUM-2MD3 showed a higher expression level (>2-fold change) than in OCUM-2M. Among these 140 miRNAs, 7 miRNAs detected at high expression in malignant ascites were selected for confirmation by qRT-PCR. All 7 miRNA was detectable both malignant ascites and intraoperative peritoneal lavage fluids by qRT-PCR. Conclusions. Exosomal miRNAs were highly detectable in malignant ascites and peritoneal lavage fluids. In this study 7 miRNAs related to peritoneal dissemination were identified. This result may provide a new diagnostic approach of peritoneal dissemination. Citation Format: Motohiko Tokuhisa, Yasushi Ichikawa, Takashi Kosaka, Satoshi Hasegawa, Hirotoshi Akiyama, Takashi Ishikawa, Nobuyoshi Kosaka, Takahiro Ochiya, Masakazu Yashiro, Kousei Hirakawa, Chikara Kunisaki, Ayumu Goto, Noritoshi Kobayashi, Itaru Endo. Expression of exosomal microRNAs in malignant ascites, peritoneal lavage fluid and cell culture supernatant of gastric cancer. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 5279. doi:10.1158/1538-7445.AM2013-5279 Note: This abstract was not presented at the AACR Annual Meeting 2013 because the presenter was unable to attend.