YKT6 expression, exosome release, and survival in non-small cell lung cancer
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Marc Ruiz-Martinez1, Alfons Navarro1, Ramón M. Marrades2, Nuria Viñolas3, Sandra Santasusagna1, Carmen Muñoz1, Josep Ramírez4, Laureano Molins5, Mariano Monzo1
1Molecular Oncology and Embryology Laboratory, Department of Human Anatomy and Embryology, School of Medicine, University of Barcelona, IDIBAPS, Barcelona, Spain
2Department of Pneumology, Institut Clínic del Tórax (ICT), Hospital Clinic de Barcelona, University of Barcelona, IDIBAPS, CIBER de Enfermedades Respiratorias (CIBERES), Barcelona, Spain
3Department of Medical Oncology, Institut Clinic Malalties Hemato-Oncològiques (ICMHO), Hospital Clinic de Barcelona, University of Barcelona, IDIBAPS, Barcelona, Spain
4Department of Pathology, Centro de Diagnóstico Biomédico (CDB), Hospital Clinic de Barcelona, University of Barcelona, IDIBAPS, CIBERES, Barcelona, Spain
5Department of Thoracic Surgery, Institut Clínic del Tórax (ICT), Hospital Clinic de Barcelona, University of Barcelona, Barcelona, Spain
Mariano Monzo, email: [email protected]
Keywords: YKT6, exosomes, NSCLC, miR-134, miR-135b
Received: February 11, 2016 Accepted: May 19, 2016 Published: June 06, 2016
Background: Cancer-derived exosomes are involved in metastasis. YKT6 is a SNARE protein that participates in the regulation of exosome production and release, but its role in non-small cell lung cancer (NSCLC) has not been examined.
Materials and Methods: Ultracentrifugation-purified exosomes from the A549 cell line were studied by CRYO-TEM, nanoparticle tracking analysis and western blot (TSG101 marker). YKT6 was inhibited using a DsiRNA and selected pre-microRNAs. MicroRNAs targeting YKT6 were validated by Renilla/Luciferase assay and western blot. YKT6 expression and its prognostic impact were analyzed in 98 tissue specimens from resected NSCLC patients.
Results: Membranous nanosized vesicles (mode size: 128nm) with TSG101 protein were purified from A549 cells. YKT6 inhibition reduced exosome release by 80.9%. We validated miR-134 and miR-135b as miRNAs targeting YKT6, and transfection with the pre-miRNAs also produced a significant reduction in exosome release. The analysis of YKT6 in tumor samples showed that patients with high levels had shorter disease-free and overall survival.
Conclusions: YKT6 is a key molecule in the regulation of exosome release in lung cancer cells and is in turn precisely regulated by miR-134 and miR-135b. Moreover, YKT6 levels impact prognosis of resected NSCLC patients.
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