Exosomal miRNAs as circulating biomarkers for prediction of metastasis in stage II/III gastric cancer

Abstract

Background: Metastasis is the fatal character of cancer, leading to mortality of gastric cancer. In recent years, exosomes have been reported as extracellular vesicles secreted by live cells. They contain various biological molecules including miRNAs from the original cells. Exosomal miRNAs are significant for pre-metastatic niche formation, as they can alter microenvironment and make it favorable for future metastasis. Therefore, the aim of this study was to identify dysregulated exosomal miRNAs as circulating biomarkers for prediction of development of haemetogenous metastasis after surgery for stage II/III gastric cancer. Materials and Methods: Pre-treatment serum samples of 90 patients with stage II/III gastric cancer were collected. Among these patients, 35 of them developed haemetogenous metastasis after surgery, while the other 55 did not develop haemetogenous metastasis after surgery. Exosomes were extracted and validated by western blot, transmission electron microscope and nanoparticle tracking analysis. MiRCURY LNATM miRNA miRNome PCR Array (containing 752 miRNAs) was performed in three pairs of serum exosomes. Each pair samples were with the same stage, race, gender and similar age. These three pairs of samples represented stage II, IIIA or IIIB gastric cancer, respectively. A panel of commonly dysregulated exosomal miRNAs was released from the PCR Array. Expressions of exosomal miRNAs were validated in the remaining 32 metastatic and 52 non-metastatic patients by RT-qPCR, as well as in gastric cancer cell culture medium and cell lines. Results: By comparing the expressions of exosomal miRNAs among three pairs of samples, 13 upregulated and 6 downregulated miRNAs were released after normalization with miR-16-5p and miR-93-5p. Among these miRNAs, 7 of them were selected for further validation according to their fold changes, p-values and association with cancer development. MiR-379-5p and miR-410-3p were validated to be significantly upregulated in metastatic patients (P<0.01). Sensitivity, specificity and AUC were 62.50%, 73.08% and 0.7073 for miR-379-5p, and 65.63%, 69.23% and 0.6923 for miR-410-3p. Higher expression of serum exosomal miR-379-5p or miR-410-3p showed shorter progress-free survival of the patients (P<0.05). It was also found that miR-379-5p and miR-410-3p expressed significantly higher in gastric cancer cell culture medium comparing with gastric cancer cells. Conclusions: Exosomal miRNAs are dysregulated in the serum of gastric cancer patients who will develop haemetogenous metastasis after surgery. Upregulation of serum exosomal miR-379-5p and miR-410-3p may serve as circulating biomarkers for the prediction of development of haemetogenous metastasis after surgery for stage II/III gastric cancer.