Abstract
Gastric cancer is one of the most common malignancies and remains the third leading cause of cancer-related death worldwide. We previously developed a method of DNA methylation analysis for early detection of gastric cancer in gastric washes rather than the highly acidic gastric juice. Exosomes in gastric juice may provide an alternative to gastric washes for the molecular detection of gastric cancer. We sought to determine whether exosomes can be purified from the gastric juice of gastric cancer patients and whether the exosomes contain significant amounts of tumor-related methylated DNA. Using a polymer-based reagent, we purified exosomes from the culture media of gastric cancer cell lines as well as gastric juice of patients with gastric cancer. Methylation levels of LINE1 and tumor-related SOX17 gene in exosomal DNA were analyzed by bisulfite pyrosequencing. The microvesicles were verified as exosomes by transmission electron microscopy and western blotting with the CD9 exosomal marker. LINE1 methylation were reduced in nuclear DNA and in the corresponding exosomal DNA in gastric cancer cell lines. Concordant methylation levels of SOX17 gene were observed in exosomal and nuclear DNA in gastric cancer cell lines, suggesting the methylated DNA is efficiently packaged in exosomes. Using exosomal DNA derived from gastric juice, we were also able to detect SOX17 DNA methylation, which reflects the nuclear DNA methylation status of the corresponding tumor. SOX17 methylation was detected in both early and advanced gastric cancer of intestinal and diffuse types. These findings expand the functional molecular content of tumor exosomes to include tumor-related methylated DNA and suggest a potential use for methylation analysis of exosomal DNA derived from gastric juice as a biomarker for gastric cancer.
Highlights
Gastric cancer (GC) is the third highest cause of global cancer mortality [1]
One alternative to gastric juice is the use of gastric washes, for which we have developed a method for early GC detection by DNA methylation analysis of genes such as MINT25 and sex determining region Y-Box 17 (SOX17) [13,14,15]
We demonstrated the feasibility of purifying exosomes from the gastric juices of patients with GC; we sought to determine whether exosomes contain significant amounts of methylated DNA and whether they reflect the DNA methylation status of the corresponding cancer
Summary
Gastric cancer (GC) is the third highest cause of global cancer mortality [1]. GC is a heterogeneous disease with multiple environmental etiologies and alternative carcinogenic pathways [26]. The development of noninvasive biomarkers to detect early cancer and/or reflect an individual’s cancer risk is essential to reducing GC mortality [7,8]. Molecular markers in the gastric juice may provide a noninvasive approach to detecting GC. The various cellular sources of miRNAs in gastric juice could make quantitative expression analysis difficult for the molecular detection of GC. The use of gastric juice DNA for molecular diagnostics has been deemed unfeasible because the DNA is degraded by gastric acidity [13]. One alternative to gastric juice is the use of gastric washes, for which we have developed a method for early GC detection by DNA methylation analysis of genes such as MINT25 and sex determining region Y-Box 17 (SOX17) [13,14,15]
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