RESUMEN
BACKGROUND: Circular RNAs (CircRNAs) play essential roles in cancer occurrence as regulatory RNAs. However, circRNA-mediated regulation of gastric cancer (GC) remains poorly understood. AIM: The purpose of this study was to investigate the molecular mechanism of circSLC22A23 (hsa_circ_0075504) underlying GC occurrence. METHODS: CircSLC22A23 levels were first quantified by quantitative real-time reverse transcription-polymerase chain reaction in GC cell lines, 80 paired GC tissues and adjacent normal tissues, and 27 pairs of plasma samples from preoperative and postoperative patients with GC. Then circSLC22A23 was knocked-down with short hairpin RNA to analyze its oncogenic effects on the proliferation, migration, and invasion of GC cells. Finally, circRNA-binding proteins and their downstream target genes were identified by RNA pulldown, mass spectrometry, RNA immunoprecipitation, quantitative real-time reverse transcription-polymerase chain reaction, and Western blot assays. RESULTS: CircSLC22A23 was found to be highly expressed in GC cells, GC tissues, and plasma from GC patients. Knockdown of circSLC22A23 inhibited GC cell proliferation, migration and invasion. RNA pulldown and RNA immunoprecipitation assays verified the interaction between circSLC22A23 and heterogeneous nuclear ribonucleoprotein U (HNRNPU). Knockdown of circSLC22A23 decreased HNRNPU protein levels. Moreover, rescue assays showed that the tumor suppressive effect of circSLC22A23 knockdown was reversed by HNRNPU overexpression. Finally, epidermal growth factor receptor (EGFR) was found to be one of the downstream target genes of HNRNPU that was up regulated by circSLC22A23. CONCLUSION: CircSLC22A23 regulated the transcription of EGFR through activation of HNRNPU in GC cells, suggesting that circSLC22A23 may serve as a potential therapeutic target for the treatment of GC.
Asunto(s)
MicroARNs , ARN Circular , Neoplasias Gástricas , Humanos , Línea Celular Tumoral , Proliferación Celular/genética , Receptores ErbB/metabolismo , Regulación Neoplásica de la Expresión Génica , Ribonucleoproteína Heterogénea-Nuclear Grupo U/genética , Ribonucleoproteína Heterogénea-Nuclear Grupo U/metabolismo , MicroARNs/genética , ARN Circular/genética , ARN Circular/metabolismo , ARN Interferente Pequeño , Neoplasias Gástricas/patología , Transportadores de Anión Orgánico/genéticaRESUMEN
Numerous studies have shown that circular RNAs are associated with the occurrence and development of various cancers, but the biological functions and mechanisms of hsa_circ_0006847 (circASPHD1) in gastric cancer (GC) remain unclear. The expression of hsa_circ_0006847 in GC cell lines, tissue, and plasma from GC patients was assayed by quantitative real-time reverse transcription-polymerase chain reaction. Hsa_circ_0006847 expression in cells was downregulated or upregulated by transfected small interfering RNA (siRNA) or overexpression plasmid. The role of hsa_circ_0006847 in GC was investigated with Cell Counting Kit-8, EdU, Transwell, flow cytometry assays, and in a subcutaneous xenograft tumor model. In addition, the interaction of eukaryotic translation initiation factor 4A3 (EIF4A3) and hsa_circ_0006847 was determined with western blot, biotin-labeled RNA pull-down, and RNA immunoprecipitation assays. Co-immunoprecipitation and mass spectrometry were used to validate the combination of EIF4A3 and synaptopodin-2 (SYNPO2). The expression of hsa_circ_0006847 was decreased in GC tissues and cells and indicated poor survival and prognosis. Overexpression of hsa_circ_0006847 inhibited cell proliferation, migration, and invasion. Flow cytometry showed that upregulation of hsa_circ_0006847 resulted in promotion of apoptosis of GC cells and inhibited their progression through the G0/G1 phase. Downregulation of hsa_circ_0006847 expression had the opposite effects. Overexpression of hsa_circ_0006847 in subcutaneous tumor xenografts inhibited tumor growth. Mechanically, hsa_circ_0006847 promoted the binding of EIF4A3 to SYNPO2 by recruiting EIF4A3, which inhibited the growth of GC. The tumor suppressor activity of hsa_circ_0006847, inhibition of the occurrence and development of GC, was mediated by promotion of EIF4A3 and the binding of EIF4A3 to SYNPO2. The results support the study of hsa_circ_0006847 as a novel therapeutic target for the treatment of GC.
Asunto(s)
Proliferación Celular , Factor 4A Eucariótico de Iniciación , Ratones Desnudos , ARN Circular , Neoplasias Gástricas , Neoplasias Gástricas/genética , Neoplasias Gástricas/patología , Neoplasias Gástricas/metabolismo , Humanos , Factor 4A Eucariótico de Iniciación/metabolismo , Factor 4A Eucariótico de Iniciación/genética , ARN Circular/genética , ARN Circular/metabolismo , Animales , Proliferación Celular/genética , Línea Celular Tumoral , Ratones , Regulación Neoplásica de la Expresión Génica , Movimiento Celular/genética , Femenino , Masculino , Apoptosis/genética , Ratones Endogámicos BALB C , Persona de Mediana Edad , ARN Helicasas DEAD-boxRESUMEN
It has been demonstrated that tRNA-derived small RNAs (tsRNAs) perform essential functions in the pathophysiology of cancer. In this study, we focused on the possible mechanisms of tRF-33-P4R8YP9LON4VDP (tRF-33) underlying the development of gastric malignancy. In total, 454 tissue samples with different gastric mucosal lesions were collected. The tRF-33 expression level in different cohorts was determined, and its value for diagnostic efficiency and prognosis evaluation were assessed. Cell proliferation assays, Transwell assay, flow cytometry, and xenotransplantation model were used to evaluate its effect on gastric cancer cells. The molecular mechanism was verified by fluorescence in situ hybridization, dual luciferase assay, Western blot, and RNA binding protein immunoprecipitation. The results showed that the expression of tRF-33 exhibited a gradual modification from normal control samples to gastritis tissues, early and latent stage of gastric cancer tissues. Consequently, tRF-33 holds significant potential as a predictive and diagnostic biomarker for gastric malignancy. Over-expression of tRF-33 inhibited gastric cancer cell progression and metastatic viability, and induced cell apoptosis. Tumorigenicity in nude mice showed the suppressive characteristics of tRF-33. Mechanistic investigation revealed that tRF-33 exerted silencing on STAT3 mRNA via binding to AGO2. In conclusion, tRF-33 exhibited values in diagnosing gastric cancer and evaluating its prognosis, and suppressed tumor cell viability by inhibiting STAT3 signaling pathway. The schematic mechanisms underlying tRF-33 regulating gastric cancer occurrence. tRF-33 binds to AGO2 proteins and then negatively regulates STAT3 expression through targeting its 3'UTR. The downregulated expression of STAT3 results in the decrease of STAT3 and p-STAT3 and further blocks the transcription of the downstream genes and finally inhibits the gastric cancer occurrence. MMP-9, matrix metalloproteinase-9; Bcl-2, B-cell lymphoma-2; STAT3, signal transducer and activator of transcription 3; UTR, untranslated region.