Abstract
BackgroundExosomes are essential for tumor growth, metastasis, and are used as novel signaling molecules in targeted therapies. Therefore, exosomal miRNAs can be used in new diagnostic and therapeutic approaches due to their involvement in the development of cancers. However, the detailed biological function, potential molecular mechanism and clinical application of exo-miR-15b-3p in gastric cancer (GC) remains unclear.MethodsmiR-15b-3p mRNA levels in tissues, serum, cells and exosomes were analyzed using qRT-PCR assays. qRT-PCR, immunohistochemical and western blotting analyses were utilized for the determination of DYNLT1 expression. The interrelationship connecting miR-15b-3p with DYNLT1 was verified using Dual-luciferase report, western blotting and qRT-PCR assays. Fluorescent PKH-26 or GFP-Lv-CD63 labeled exosomes, as well as Cy3-miR-15b-3p, were utilized to determine the efficacy of the transfer of exo-miR-15b-3p between BGC-823 and recipient cells. Several in vitro assays and xenograft tumor models were conducted to determine exo-miR-15b-3p impact on GC progression.ResultsThis is the first study to confirm high miR-15b-3p expression in GC cell lines, tissues and serum. Exosomes obtained from 108 GC patient serum samples and GC cell-conditioned medium were found to show upregulation of exo-miR-15b-3p, with the area under the ROC curve (AUC) being 0.820 [0.763–0.876], which is superior to the AUC of tissues and serum miR-15b-3p (0.674 [0.600–0.748] and 0.642 [0.499–0.786], respectively). In addition, high exo-miR-15b-3p expression in serum was found to accurately predict worse overall survival. SGC-7901 and GES-1 cells are capable of internalizing BGC-823 cell-derived exosomes, allowing the transfer of miR-15b-3p. Migration, invasion, proliferation and inhibition of apoptosis in vitro and in vivo were enhanced by exo-miR-15b-3p, by restraining DYNLT1, Cleaved Caspase-9 and Caspase-3 expression.ConclusionsThis study identified a previously unknown regulatory pathway, exo-miR-15b-3p/DYNLT1/Caspase-3/Caspase-9, which promotes GC development and GES-1 cell malignant transformation. Therefore, serum exo-miR-15b-3p may be a potential GC diagnosis and prognosis biomarker, which can be used in precise targeted GC therapy.
Highlights
Worldwide, gastric cancer (GC) frequency is fourth highest among malignancies and the second most-likely cause of cancer-related death [1], as well as the second most frequent cancer in China [2]
Exosomes can carry numerous miRNAs that act locally or enter into circulation to act at distal sites, since internal miRNAs are protected from being digested by RNase, as a result of the protection offered by the lipid membrane of the exosomes [12, 13]
The cells were cultured at 37 °C, in Roswell Park Memorial Institute (RPMI) 1640 medium supplemented with 1% penicillin/streptomycin, 10% fetal bovine serum (FBS) and 5% CO2
Summary
Gastric cancer (GC) frequency is fourth highest among malignancies and the second most-likely cause of cancer-related death [1], as well as the second most frequent cancer in China [2]. Exosomes are extracellular vesicles with an average diameter of 30–200 nm that have the same topology as the cell and contain a specific composition of proteins, lipids, nucleic acids and glycoconjugates [9] They are derived from endocytic membranes and serve as vehicles for cell-to-cell communication, remodeling the extracellular environment or transmitting signals and molecules to neighboring recipient cells [9, 10]. Due to their potential use in numerous pathological and physiological processes of various diseases, differences in exosome function between healthy and diseased individuals has attracted much attention from researchers [9,10,11]. The detailed biological function, potential molecular mechanism and clinical application of exo-miR-15b-3p in gastric cancer (GC) remains unclear
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