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

Tokuhisa M, Ichikawa Y, Kosaka N, Ochiya T, Yashiro M, Hirakawa K, Kosaka T, Makino H, Akiyama H, Kunisaki C, Endo I - PLoS ONE (2015)

Bottom Line: In the six MA fluids, miR-21 showed the highest signal intensity.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.

View Article: PubMed Central - PubMed

Affiliation: Department of Gastroenterological Surgery, Yokohama City University Graduate School of Medicine, Yokohama, Japan.

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.

No MeSH data available.


Related in: MedlinePlus

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.
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pone.0130472.g005: 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.

Mentions: To date, no data have been available on the expression profiles of exosomal miRNAs associated with peritoneal dissemination, because of the unavailability of normal ascitic fluids against which to compare the MA expression profiles. We selected miRNAs related to peritoneal dissemination using a step-wise approach. The number of miRNAs commonly captured in the six MA samples was 327. In Steps 1 and 2, 140 and 118 metastasis-specific miRNAs were selected in the CM and PLF groups, respectively. From these 327, 140 and 118 miRNAs, five miRNAs were selected as candidates related to peritoneal dissemination (Fig 5). Two of them (miR-1225-5p and miR-320c) and miR-21 with the highest signal intensity among the MA-specific miRNAs were validated as being differentially expressed in 18 PLF samples, using qPCR (Table 3).


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

Tokuhisa M, Ichikawa Y, Kosaka N, Ochiya T, Yashiro M, Hirakawa K, Kosaka T, Makino H, Akiyama H, Kunisaki C, Endo I - PLoS ONE (2015)

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.
© Copyright Policy
Related In: Results  -  Collection

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Show All Figures
getmorefigures.php?uid=PMC4514651&req=5

pone.0130472.g005: 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.
Mentions: To date, no data have been available on the expression profiles of exosomal miRNAs associated with peritoneal dissemination, because of the unavailability of normal ascitic fluids against which to compare the MA expression profiles. We selected miRNAs related to peritoneal dissemination using a step-wise approach. The number of miRNAs commonly captured in the six MA samples was 327. In Steps 1 and 2, 140 and 118 metastasis-specific miRNAs were selected in the CM and PLF groups, respectively. From these 327, 140 and 118 miRNAs, five miRNAs were selected as candidates related to peritoneal dissemination (Fig 5). Two of them (miR-1225-5p and miR-320c) and miR-21 with the highest signal intensity among the MA-specific miRNAs were validated as being differentially expressed in 18 PLF samples, using qPCR (Table 3).

Bottom Line: In the six MA fluids, miR-21 showed the highest signal intensity.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.

View Article: PubMed Central - PubMed

Affiliation: Department of Gastroenterological Surgery, Yokohama City University Graduate School of Medicine, Yokohama, Japan.

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.

No MeSH data available.


Related in: MedlinePlus