RESUMEN
MicroRNAs (miRNAs), mRNA, and proteins in/on extracellular vesicles (EVs) represent potential cancer biomarkers. Concurrent detection of multiple biomarkers at a single-EV level would greatly improve prognosis and/or diagnosis and understanding of EV phenotypes, biogenesis, and functions. Here, we introduced a High-throughput Nano-bio Chip Integrated System for Liquid Biopsy (HNCIB) system for simultaneous detection of proteins and mRNA/miRNA in a single EV. Validated through systematic control experiments, HNCIB showed high reliability, sensitivity, and specificity. In a panel of 34 patients with lung adenocarcinoma (LUAD) and 35 healthy donors, HNCIB detected an up-regulated expression of programmed death-ligand 1 mRNA and protein and miR-21 in EVs derived from patients with LUAD compared to those from healthy donors. HNCIB has low sample requirement (~90 µl), fast assay time (~6 hours), and high throughput (up to 384 samples per assay) and would have great potential in the study of EVs and their clinical applications.
Asunto(s)
Adenocarcinoma del Pulmón , Vesículas Extracelulares , Neoplasias Pulmonares , MicroARNs , Adenocarcinoma del Pulmón/diagnóstico , Adenocarcinoma del Pulmón/genética , Vesículas Extracelulares/metabolismo , Humanos , Biopsia Líquida , Neoplasias Pulmonares/diagnóstico , Neoplasias Pulmonares/genética , Neoplasias Pulmonares/metabolismo , MicroARNs/genética , MicroARNs/metabolismo , ARN Mensajero/genética , ARN Mensajero/metabolismo , Reproducibilidad de los ResultadosRESUMEN
BACKGROUND: Drug resistance is common in cancer chemotherapy. This study investigates the role of Glycerol kinase 5 (GK5) in mediating gefitinib resistance in NSCLC. METHODS: The exosomal mRNA of GK5 was detected using a tethered cationic lipoplex nanoparticle (TCLN) biochip. Real-time PCR and Western blot were used to examine the expression of GK5 mRNA and protein in gefitinib-sensitive and -resistant human lung adenocarcinoma cells. The cell counting kit-8, EdU assay, flow cytometry, and JC-1 dye were used to measure cell proliferation, cell cycle, and the mitochondrial membrane potential. RESULTS: We found that the exosomal mRNA of GK5 in the plasma of patients with gefitinib-resistant adenocarcinoma was significantly higher compared with that of gefitinib-sensitive patients. The mRNA and protein levels of GK5 were significantly upregulated in gefitinib-resistant human lung adenocarcinoma PC9R and H1975 cells compared with gefitinib-sensitive PC9 cells. Silencing GK5 in PC9R cells induced mitochondrial damage, caspase activation, cell cycle arrest, and apoptosis via SREBP1/SCD1 signaling pathway. CONCLUSIONS: We demonstrated that GK5 confers gefitinib resistance in lung cancer by inhibiting apoptosis and cell cycle arrest. GK5 could be a novel therapeutic target for treatment of NSCLC with resistance to EGFR tyrosine kinase inhibitors.
Asunto(s)
Resistencia a Antineoplásicos/genética , Gefitinib/farmacología , Glicerol Quinasa/genética , Transducción de Señal/genética , Estearoil-CoA Desaturasa/genética , Proteína 1 de Unión a los Elementos Reguladores de Esteroles/genética , Adenocarcinoma/tratamiento farmacológico , Adenocarcinoma/genética , Adenocarcinoma del Pulmón/dietoterapia , Adenocarcinoma del Pulmón/genética , Animales , Antineoplásicos/farmacología , Apoptosis/genética , Carcinoma de Pulmón de Células no Pequeñas/tratamiento farmacológico , Carcinoma de Pulmón de Células no Pequeñas/genética , Ciclo Celular/genética , Línea Celular Tumoral , Proliferación Celular/genética , Receptores ErbB/genética , Humanos , Neoplasias Pulmonares/tratamiento farmacológico , Neoplasias Pulmonares/genética , Masculino , Potencial de la Membrana Mitocondrial/genética , Ratones , Ratones Endogámicos BALB C , Inhibidores de Proteínas Quinasas/farmacología , Quinazolinas/farmacología , ARN Mensajero/genética , Regulación hacia Arriba/genéticaRESUMEN
BACKGROUND/AIMS: Tyrosine kinase inhibitor gefitinib significantly improves the survival of patients with non-small-cell lung cancer (NSCLC) by inhibiting epidermal growth factor receptor (EGFR) tyrosine kinase. However, patients eventually develop resistance to gefitinib through uncharacterized mechanisms. It is known that plasminogen activator urokinase receptor (PLAUR) plays an important role in cell proliferation, migration and apoptosis. However, the role of PLAUR, particularly exosomal PLAUR in gefitinib resistance in NSCLC has not been reported. The aim of this study is to determine the relationship between PLAUR and gefitinib resistance. METHODS: In this study, a tethered cationic lipoplex nanoparticle (TCLN) biochip containing molecular beacons was used as probes to detect PLAUR mRNA in plasma exosomes from patients with gefitinib-sensitive and -resistant NSCLC. In vitro, Real-time PCR was used to examine the expression of PLAUR mRNA and Western blot was applied to examine the expression of related proteins. The gene knockdown was achieved by Lentivirus based RNA silence technique. The cell counting kit-8 assay and EdU incorporation were used to examine cell proliferation. The flow cytometry was applied to determine cell apoptosis and cell cycle, while the mitochondrial membrane potential was measured by JC-1 dye assay. Signaling pathway affected by PLAUR knockdown was identified by cDNA Microarray. The effect of PLAUR knockdown on tumorigenesis was analyzed in vivo. RESULTS: We found that the exosomal PLAUR mRNA in the plasma of gefitinib-resistant NSCLC patients was significantly increased compared to that of gefitinib-sensitive NSCLC patients. The PLAUR mRNA and soluble PLAUR protein were also significantly increased in gefitinib-resistant human lung adenocarcinoma PC9R cells compared to gefitinib-sensitive PC9 cells. Silencing PLAUR in PC9R cells impaired mitochondrial membrane potential and increased cell apoptosis via EGFR/p-AKT/survivin signaling pathway. Furthermore, EGFR was upregulated in the geftinib-resistant PC9R cells, and knockdown of EGFR significantly increased cell apoptosis. CONCLUSIONS: Taken together, our results demonstrated that PLAUR induces geftinib-resistance through EGFR/p-AKT/survivin signaling pathway in gefitinib-resistant human lung adenocarcinoma cells. PLAUR could be a novel therapeutic target for gefitinib-resistant NSCLC patients.