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.
Highlights
MicroRNAs are small (19–23 nucleotides) non-coding RNAs that function as posttranscriptional regulators of gene expression and play important roles in the control of many biological processes, including cell differentiation, proliferation, and apoptosis [1]
All patients had previously been diagnosed with gastric cancer (GC) by histopathological analysis of biopsy samples taken from the primary lesions, and six malignant ascites (MA) patients had been diagnosed for the presence of ascites by abdominal computed tomography (CT) scan
To test the expression of exosomal miRNA microarrays in each group of samples, we examined the number of detected miRNAs, percentile of signal intensity, the number of commonly captured miRNAs, and the correlation of miRNA expression among the groups
Summary
MicroRNAs (miRNAs) are small (19–23 nucleotides) non-coding RNAs that function as posttranscriptional regulators of gene expression and play important roles in the control of many biological processes, including cell differentiation, proliferation, and apoptosis [1]. MiRNAs have been shown to have oncogenic or tumour-suppressing activity, and deregulated expression of many miRNA species has been detected in several cancers, suggesting potential application of miRNAs as biomarkers of cancer progression and metastasis [2,3,4,5]. Exosomes are small membranous vesicles that have been implicated in cellular immune responses; recently it has become evident that exosomes contain substantial amounts of RNA and may be involved in immune-independent regulatory mechanisms. Higher levels of miRNA-containing exosomes have been detected in the plasma of cancer patients than in that of healthy individuals [7], suggesting that exosomebased miRNA secretion is involved in cancer development. Analysis of aberrant miRNA expression in the serum, saliva, faeces, and urine has been employed for the early detection of B-cell lymphoma, and oral, intestinal, and bladder cancers [8,9,10,11]
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