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. 2015 Jul 24;10(7):e0130472.
doi: 10.1371/journal.pone.0130472. eCollection 2015.

Exosomal miRNAs from Peritoneum Lavage Fluid as Potential Prognostic Biomarkers of Peritoneal Metastasis in Gastric Cancer

Motohiko Tokuhisa  1 , Yasushi Ichikawa  2 , Nobuyoshi Kosaka  3 , Takahiro Ochiya  3 , Masakazu Yashiro  4 , Kosei Hirakawa  4 , Takashi Kosaka  1 , Hirochika Makino  1 , Hirotoshi Akiyama  1 , Chikara Kunisaki  1 , Itaru Endo  1
Affiliations

Exosomal miRNAs from Peritoneum Lavage Fluid as Potential Prognostic Biomarkers of Peritoneal Metastasis in Gastric Cancer

Motohiko Tokuhisa et al. PLoS One. .

Abstract

Peritoneal metastasis is the most frequent type of recurrence in patients with gastric cancer (GC) and is associated with poor prognosis. Peritoneal lavage cytology, used to evaluate the risk of peritoneal metastasis, has low sensitivity. Here, we assessed the diagnostic potential of exosomal miRNA profiles in peritoneal fluid for the prediction of peritoneal dissemination in GC. Total RNA was extracted from exosomes isolated from six gastric malignant ascites (MA) samples, 24 peritoneal lavage fluid (PLF) samples, and culture supernatants (CM) of two human gastric carcinoma cell lines that differ in their potential for peritoneal metastasis. Expression of exosomal miRNAs was evaluated with Agilent Human miRNA microarrays and quantitative reverse transcription polymerase chain reaction (qRT-PCR). The microarray analysis indicated a low variability in the number and signal intensity of miRNAs detected among the samples. In the six MA fluids, miR-21 showed the highest signal intensity. We identified five miRNAs (miR-1225-5p, miR-320c, miR-1202, miR-1207-5p, and miR-4270) with high expression in MA samples, the PLF of serosa-invasive GC, and the CM of a highly metastatic GC cell line; these candidate miRNA species appear to be related to peritoneal dissemination. Differential expression of miR-21, miR-320c, and miR-1225-5p was validated in the PLF of serosa-invasive and non-invasive GC by qRT-PCR and miR-21 and miR-1225-5p were confirmed to be associated with serosal invasion in GC. PLF can be used to profile the expression of exosomal miRNAs. Our findings suggest that miR-21 and miR-1225-5p may serve as biomarkers of peritoneal recurrence after curative GC resection, thus providing a novel approach to early diagnosis of peritoneal dissemination of GC.

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Conflict of interest statement

Competing Interests: The authors have declared that no competing interests exist.

Figures

Fig 1
Fig 1. Exosomal miRNA expression in malignant ascites (MA), peritoneal lavage fluid (PLF), and cell culture media (CM).
Variation and commonality between the samples were examined. Evaluation of total exosomal RNA used a Bioanalyzer 2100. The electropherograms show the size distribution (nucleotides, nt) and fluorescence intensity (FU) of total exosomal RNA isolated from CM, MA, and PLF. The lowest spike (around 25 nt) in each sample is the marker of the Agilent RNA 6000 Pico kit.
Fig 2
Fig 2. The number of detected miRNA species in 14 samples.
Fig 3
Fig 3. Signal intensity distribution among MA (A), PLF (B), and CM (C) samples and among the groups (D).
Fig 4
Fig 4. The number of miRNA species detected in malignant ascites, peritoneal lavage fluids, and cell culture media groups; 196 miRNAs were common to all groups.
Fig 5
Fig 5. Identification of five exosomal miRNAs related to peritoneal dissemination.
Schematic representation of the step-wise approach to screening miRNAs related to peritoneal dissemination. Step 1: selection of miRNAs differentially expressed (fold-change, >2) in culture medium (CM) of the highly metastatic peritoneal cell line OCUM-2MD3 (OCUM-2M parental cell line was used as control). Step 2: six peritoneal lavage fluid (PLF) samples were divided into T4 (n = 4) and T1–T3 groups according to gastrointestinal cancer stage; miRNAs differentially expressed (fold-change, >2) in the T4 group were selected. Step 3: the selected miRNA species common for Steps 1 and 2 and expressed in malignant ascites (MA) with signal intensity > log2 10 in MA and PLF were considered as candidate miRNAs related to peritoneal metastases.
Fig 6
Fig 6. Relative expression levels of exosomal miRNAs in intraoperative peritoneal lavage fluid of T4 and T1–T3 gastric cancer patients.
An asterisk indicates P < 0.05.
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