RESUMEN
Circular RNAs (circRNAs) have been reported to play essential roles in tumorigenesis and progression. This study aimed to identify dysregulated circRNAs in gastric cancer (GC) and investigate the functions and underlying mechanism of these circRNAs in GC development. Here, we identify circ_CEA, a circRNA derived from the back-splicing of CEA cell adhesion molecule 5 (CEA) gene, as a novel oncogenic driver of GC. Circ_CEA is significantly upregulated in GC tissues and cell lines. Circ_CEA knockdown suppresses GC progression, and enhances stress-induced apoptosis in vitro and in vivo. Mechanistically, circ_CEA interacts with p53 and cyclin-dependent kinases 1 (CDK1) proteins. It serves as a scaffold to enhance the association between p53 and CDK1. As a result, circ_CEA promotes CDK1-mediated p53 phosphorylation at Ser315, then decreases p53 nuclear retention and suppresses its activity, leading to the downregulation of p53 target genes associated with apoptosis. These findings suggest that circ_CEA protects GC cells from stress-induced apoptosis, via acting as a protein scaffold and interacting with p53 and CDK1 proteins. Combinational therapy of targeting circ_CEA and chemo-drug caused more cell apoptosis, decreased tumor volume and alleviated side effect induced by chemo-drug. Therefore, targeting circ_CEA might present a novel treatment strategy for GC.
Asunto(s)
MicroARNs , Neoplasias Gástricas , Apoptosis/genética , Antígeno Carcinoembrionario/genética , Antígeno Carcinoembrionario/metabolismo , Línea Celular Tumoral , Proliferación Celular , Regulación Neoplásica de la Expresión Génica , Humanos , MicroARNs/genética , ARN Circular/genética , Neoplasias Gástricas/patología , Proteína p53 Supresora de Tumor/genética , Proteína p53 Supresora de Tumor/metabolismoRESUMEN
Peptidylarginine deiminase 4 (PAD4), a Ca2+-dependent enzyme, catalyzes the conversion of arginine to citrulline and has been strongly associated with many malignant tumors. However, the molecular mechanisms of PAD4 in the development and progression of colorectal cancer (CRC) remain unclearly defined. In our study, PAD4 expression was increased in CRC tissues and cells, and was closely related to tumor size, lymph node metastasis. Moreover, the transcription factor KLF9 directly bound to PADI4 gene promoter, leading to overexpression of PAD4 in CRC cells, which augmented cell growth and migration. We revealed that PAD4 interacted with and citrullinated glycogen synthase kinase-3ß (GSK3ß) in CRC cells, and GSK3ß Arg-344 was the dominating PAD4-citrullination site. Furthermore, IgL2 and catalytic domains of PAD4 directly bound to the kinase domain of GSK3ß in CRC cells. Mechanistically, PAD4 promoted the transport of GSK3ß from the cytoplasm to the nucleus, thereby increasing the ubiquitin-dependent proteasome degradation of nuclear cyclin-dependent kinase inhibitor 1 (CDKN1A). Our study is the first to reveal the details of a critical PAD4/GSK3ß/CDKN1A signaling axis for CRC progression, and provides evidence that PAD4 is a potential diagnosis biomarker and therapeutic target in CRC.
Asunto(s)
Citrulinación , Neoplasias Colorrectales , Arginina/genética , Biomarcadores/metabolismo , Citrulina/genética , Citrulina/metabolismo , Neoplasias Colorrectales/genética , Inhibidor p21 de las Quinasas Dependientes de la Ciclina , Quinasas Ciclina-Dependientes/genética , Quinasas Ciclina-Dependientes/metabolismo , Glucógeno Sintasa Quinasa 3 beta/genética , Glucógeno Sintasa Quinasa 3 beta/metabolismo , Humanos , Hidrolasas/genética , Hidrolasas/metabolismo , Factores de Transcripción de Tipo Kruppel/genética , Complejo de la Endopetidasa Proteasomal/metabolismo , Arginina Deiminasa Proteína-Tipo 4 , Desiminasas de la Arginina Proteica/genética , Desiminasas de la Arginina Proteica/metabolismo , Factores de Transcripción/genética , Ubiquitinas/genéticaRESUMEN
Circular RNAs (circRNAs) are covalently closed, endogenous molecules that are widespread in eukaryotes. Recent evidence indicates that circRNAs play important roles in carcinogenesis. Several circRNAs have been reported to comprise translatable RNA; however, whether circRNAs encode functional proteins remains unknown. In our study, circRNA sequencing was carried out using five pathologically diagnosed gastric carcinoma (GC) samples and their paired adjacent normal tissues, we characterized the circRNA GSPT1 (circGSPT1), which is expressed at low levels in GC. Antibody detections, and mass spectrometry were used to validate active circRNA translation. The spanning junction open reading frame in circGSPT1, driven by an internal ribosome entry site (IRES), encodes a functional peptide, termed GSPT1-238aa. Interestingly, GSPT1-238aa tends to select the start codon used to initiate translation. This is the first finding of selective translation driven by IRES. CircGSPT1 and GSPT1-238aa halted the proliferation, migration, and invasion in GC cells in vitro. We also confirmed that the vimentin/Beclin1/14-3-3 complex interacts with GSPT1-238aa and modulates autophagy via the PI3K/AKT/mTOR signaling pathway in GC cells. Our study reveals that GSPT1-238aa, a novel protein encoded by circGSPT1, halts GC tumorigenesis. We also provide insights into the function and underlying molecular mechanisms of GSPT1-238aa in GC and suggest that this protein represents a novel target for GC treatment.
Asunto(s)
Carcinoma , Neoplasias Gástricas , Autofagia/genética , Carcinogénesis/genética , Carcinoma/genética , Regulación Neoplásica de la Expresión Génica , Humanos , Sitios Internos de Entrada al Ribosoma , Factores de Terminación de Péptidos , Fosfatidilinositol 3-Quinasas/genética , ARN Circular/genética , Neoplasias Gástricas/genética , Neoplasias Gástricas/patología , Proteínas Supresoras de Tumor/genética , Vimentina/genéticaRESUMEN
BACKGROUND: Circular RNA (circRNA), a subclass of non-coding RNA, plays a critical role in cancer tumorigenesis and metastasis. It has been suggested that circRNA acts as a microRNA sponge or a scaffold to interact with protein complexes; however, its full range of functions remains elusive. Recently, some circRNAs have been found to have coding potential. METHODS: To investigate the role of circRNAs in gastric cancer (GC), parallel sequencing was performed using five paired GC samples. Differentially expressed circAXIN1 was proposed to encode a novel protein. FLAG-tagged circRNA overexpression plasmid construction, immunoblotting, mass spectrometry, and luciferase reporter analyses were applied to confirm the coding potential of circAXIN1. Gain- and loss-of-function studies were conducted to study the oncogenic role of circAXIN1 and AXIN1-295aa on the proliferation, migration, invasion, and metastasis of GC cells in vitro and in vivo. The competitive interaction between AXIN1-295aa and adenomatous polyposis coli (APC) was investigated by immunoprecipitation analyses. Wnt signaling activity was observed using a Top/Fopflash assay, real-time quantitative RT-PCR, immunoblotting, immunofluorescence staining, and chromatin immunoprecipitation. RESULTS: CircAXIN1 is highly expressed in GC tissues compared with its expression in paired adjacent normal gastric tissues. CircAXIN1 encodes a 295 amino acid (aa) novel protein, which was named AXIN1-295aa. CircAXIN1 overexpression enhances the cell proliferation, migration, and invasion of GC cells, while the knockdown of circAXIN1 inhibits the malignant behaviors of GC cells in vitro and in vivo. Mechanistically, AXIN1-295aa competitively interacts with APC, leading to dysfunction of the "destruction complex" of the Wnt pathway. Released ß-catenin translocates to the nucleus and binds to the TCF consensus site on the promoter, inducing downstream gene expression. CONCLUSION: CircAXIN1 encodes a novel protein, AXIN1-295aa. AXIN1-295aa functions as an oncogenic protein, activating the Wnt signaling pathway to promote GC tumorigenesis and progression, suggesting a potential therapeutic target for GC.
Asunto(s)
Proteína Axina/genética , Regulación Neoplásica de la Expresión Génica , ARN Circular/genética , Neoplasias Gástricas/genética , Neoplasias Gástricas/metabolismo , Vía de Señalización Wnt , Secuencia de Aminoácidos , Animales , Proteína Axina/química , Proteína Axina/metabolismo , Carcinogénesis/genética , Línea Celular Tumoral , Biología Computacional , Modelos Animales de Enfermedad , Progresión de la Enfermedad , Femenino , Perfilación de la Expresión Génica , Humanos , Metástasis Linfática , Ratones , Modelos Biológicos , Estadificación de Neoplasias , Conformación Proteica , Neoplasias Gástricas/patologíaRESUMEN
ATF3 is an essential transcription activator in regulating cancer-related genetic expression. To identify the role of ATF3 in ovarian tumor, we investigated the correlation between ATF3 expression and the clinicopathological properties using multiple database. The cBioPortal and GEPIA database displayed the clinical information of ovarian patients harboring or without harboring ATF3 mutation. Furthermore, we assessed the relationship between survival and ATF3 expression level using Kaplan-Meier plotter, which reveals that the ovarian patients with higher expression of ATF3 suffered the worse overall survival and progression-free survival. The differentially expressed genes were analyzed using gene ontology, protein-protein interaction network, and gene set enrichment analysis to identify the hub gene and critical pathways, significantly affecting the tumorigenesis of ovarian tumor. Finally, we assessed the correlation between ATF3 and immune cell infiltration using Tumor Immunoassay Resource (TIMER) database. The results demonstrated that higher expression has a positive correlation with macrophage infiltration, expression for M1- and M2-type macrophages. Our study suggests that ATF3 can regulate the cell cycle and heme-related oxidative phosphorylation process, and it may be a critical factor to regulate the macrophage cell to be infiltrated into ovarian cancer. ATF3 can be used as a biomarker for diagnosis and therapy of ovarian tumor.
Asunto(s)
Factor de Transcripción Activador 3/inmunología , Biomarcadores de Tumor/inmunología , Carcinogénesis/inmunología , Biología Computacional/métodos , Neoplasias Ováricas/inmunología , Factor de Transcripción Activador 3/biosíntesis , Factor de Transcripción Activador 3/genética , Adulto , Anciano , Anciano de 80 o más Años , Biomarcadores de Tumor/biosíntesis , Biomarcadores de Tumor/genética , Carcinogénesis/metabolismo , Bases de Datos Genéticas/tendencias , Femenino , Humanos , Macrófagos/inmunología , Macrófagos/metabolismo , Persona de Mediana Edad , Neoplasias Ováricas/genética , Neoplasias Ováricas/metabolismo , Mapas de Interacción de Proteínas/fisiologíaRESUMEN
Previous research has found that miRNA-20b is highly expressed in gastric cancer (GC), however, its function and underlying mechanism are not clear. Wnt signaling pathway, implicated in tumorigeneisis, is activated in more than 30% of GC. We would like to characterize the biological behavior of miRNA-20b in terms of modulating Wnt/ß-catenin signaling and EMT. We showed that miRNA-20b inhibitors suppressed Topflash/Fopflash dependent luciferase activity and the ß-catenin nuclear translocation, resulting in inhibition of Wnt pathway activity and EMT. SUFU, negatively regulating Wnt and Hedgehog signaling pathway, was proved to be targeted by miRNA-20b. Moreover, additional knockdown of SUFU alleviated the inhibitory effect on Wnt pathway activity, EMT, cell proliferation/migration and colony formation caused by miRNA-20b inhibition. In summary, miRNA-20b is an oncogenic miRNA and promoted cell proliferation, migration and EMT in GC partially by activating Wnt pathway via targeting SUFU.
RESUMEN
Mounting evidence has shown that miRNA-150 expression is upregulated in gastric cancer (GC) and is associated with gastric carcinogenesis, but the underlying oncogenic mechanism remains elusive. Here, we discovered that miRNA-150 targets the tumor suppressor SUFU to promote cell proliferation, migration, and the epithelial-mesenchymal transition (EMT) via the dual activation of Hedgehog (Hh) and Wnt signaling. MiRNA-150 was highly expressed in GC tissues and cell lines, and the level of this miRNA was negatively related to that of SUFU. In addition, both the miRNA-150 and SUFU levels were associated with tumor differentiation. Furthermore, miRNA-150 activated GC cell proliferation and migration in vitro. We found that miRNA-150 inhibitors repressed not only Wnt signaling by promoting cytoplasmic ß-catenin localization, but also repressed Hh signaling and EMT. MiRNA-150 inhibition also resulted in significant tumor volume reductions in vivo, suggesting the potential application of miRNA-150 inhibitors in GC therapy. The expression of genes downstream of Hh and Wnt signaling was also reduced in tumors treated with miRNA-150 inhibitors. Notably, anti-SUFU siRNAs rescued the inhibitory effects of miRNA-150 inhibitors on Wnt signaling, Hh activation, EMT, cell proliferation, cell migration, and colony formation. Taken together, these findings indicate that miRNA-150 is oncogenic and promotes GC cell proliferation, migration, and EMT by activating Wnt and Hh signaling via the suppression of SUFU expression.
Asunto(s)
Proteínas Hedgehog/genética , MicroARNs/genética , Proteínas Represoras/genética , Neoplasias Gástricas/genética , Vía de Señalización Wnt/fisiología , Animales , Carcinogénesis/genética , Línea Celular Tumoral , Movimiento Celular/genética , Proliferación Celular/genética , Regulación hacia Abajo , Transición Epitelial-Mesenquimal/genética , Regulación Neoplásica de la Expresión Génica , Proteínas Hedgehog/metabolismo , Humanos , Ratones , MicroARNs/metabolismo , Proteínas Represoras/metabolismo , Neoplasias Gástricas/metabolismo , Neoplasias Gástricas/patologíaRESUMEN
Gastric cancer (GC) is the most common cancer throughout the world. Despite advances of the treatments, detailed oncogenic mechanisms are largely unknown. In our previous study, we investigated microRNA (miR) expression profiles in human GC using miR microarrays. We found miR-192/215 were upregulated in GC tissues. Then gene microarray was implemented to discover the targets of miR-192/215. We compared the expression profile of BGC823 cells transfected with miR-192/215 inhibitors, and HFE145 cells transfected with miR-192/-215 mimics, respectively. SET8 was identified as a proposed target based on the expression change of more than twofold. SET8 belongs to the SET domain-containing methyltransferase family and specifically catalyzes monomethylation of H4K20me. It is involved in diverse functions in tumorigenesis and metastasis. Therefore, we focused on the contributions of miR-192/215/SET8 axis to the development of GC. In this study, we observe that functionally, SET8 regulated by miR-192/215 is involved in GC-related biological activities. SET8 is also found to trigger oncogene-induced senescence (OIS) in GC in vivo and in vitro, which is dependent on the DDR (DNA damage response) and p53. Our findings reveal that SET8 functions as a negative regulator of metastasis via the OIS-signaling pathway. Taken together, we investigated the functional significance, molecular mechanisms, and clinical impact of miR-192/215/SET8/p53 in GC.
Asunto(s)
Daño del ADN , N-Metiltransferasa de Histona-Lisina/metabolismo , MicroARNs/metabolismo , Neoplasias Gástricas/metabolismo , Proteína p53 Supresora de Tumor/metabolismo , Animales , Apoptosis/fisiología , Movimiento Celular/fisiología , Femenino , Xenoinjertos , N-Metiltransferasa de Histona-Lisina/genética , Humanos , Estimación de Kaplan-Meier , Ratones , Ratones Endogámicos BALB C , Ratones Desnudos , MicroARNs/genética , Oncogenes , Neoplasias Gástricas/genética , Neoplasias Gástricas/patología , Análisis de Supervivencia , Microambiente Tumoral , Proteína p53 Supresora de Tumor/genéticaRESUMEN
The poor prognosis of late gastric carcinomas (GC) underscores the necessity to identify novel biomarkers for earlier diagnosis and effective therapeutic targets. MiRNA-324-5p has been shown to be over-expressed in GC, however the biological function of miRNA-324-5p implicated in gastric cancer and its downstream targets were not well understood. Wnt/ß-catenin signaling pathway is aberrantly regulated in GC. We sought to explore if miRNA-324-5p promotes oncogenesis through modulating Wnt signaling and EMT. MiRNA-324-5p is highly expressed in GC based on qRT-PCR and TCGA data. In addition, in vitro cell proliferation, cell migration assays and in vivo animal exenograft were executed to show that miRNA-324-5p is an oncogenic miRNA in GC. MiRNA-324-5p activates Wnt signaling and induces EMT in GC. Further, SUFU was identified as a target of miRNA-324-5p confirmed by western blotting and luciferase assays. Spearson analysis and TCGA data indicate that the expression of SUFU is negatively associated with the expression of miRNA-324-5p. Rescue experiments were performed to determine if SUFU mediates the Wnt activation, EMT and oncogenic function of miRNA-324-5p. MiRNA-324-5p inhibitors plus SUFU siRNAs rescue partially the inhibitory effect on Wnt signaling and EMT caused by miRNA-324-5p inhibitors. Finally, the suppression of cell proliferation, migration, and colony formation ability induced by miRNA-324-5p inhibitors is alleviated by addition of SUFU siRNAs. In summary, miRNA-324-5p is overexpressed in vivo and exerts cell growth and migration-promoting effects through activating Wnt signaling and EMT by targeting SUFU in GC. It represents a potential miRNA with an oncogenic role in human gastric cancer.
Asunto(s)
MicroARNs/genética , Proteínas de Transporte de Catión Orgánico/genética , Proteínas Represoras/genética , Neoplasias Gástricas/genética , Vía de Señalización Wnt/genética , beta Catenina/genética , Animales , Carcinogénesis/genética , Carcinogénesis/patología , Línea Celular , Línea Celular Tumoral , Movimiento Celular/genética , Proliferación Celular/genética , Transición Epitelial-Mesenquimal/genética , Femenino , Regulación Neoplásica de la Expresión Génica/genética , Humanos , Ratones , Ratones Endogámicos BALB C , Ratones Desnudos , Ratones SCID , Oncogenes/genética , Neoplasias Gástricas/patologíaRESUMEN
Although great progress has been made in surgical techniques, traditional radiotherapy, and chemotherapy, gastric cancer (GC) is still the most common malignant tumor and has a high mortality, which highlights the importance of novel diagnostic markers. Emerging studies suggest that different microRNAs (miRNAs) are involved in tumorigenesis of GC. In this study, we found that miRNA-192 and -215 are significantly upregulated in GC and promote cell proliferation and migration. Adenomatous polyposis coli (APC), a well-known negative regulator in Wnt signaling, has been proved to be a target of miRNA-192 and -215. Inhibition of miRNA-192 or -215 reduced the Topflash activities and repressed the expression of Wnt signaling pathway proteins, while APC small interfering RNAs reversed the inhibitory effects, suggesting that miRNA-192 and -215 activate Wnt signaling via APC. In addition, APC mediates the cell proliferation and migration regulated by miRNA-192 and -215. Furthermore, APC is downregulated in GC tissues and negatively correlated with the expression of miRNA-192 and -215. In summary, miRNA-192 and -215 target APC and function as oncogenic miRNAs by activating Wnt signaling in GC, revealing to be potential therapeutic targets.
Asunto(s)
Proteína de la Poliposis Adenomatosa del Colon/genética , MicroARNs/genética , Neoplasias Gástricas/genética , Línea Celular Tumoral , Movimiento Celular/genética , Proliferación Celular/genética , Femenino , Regulación Neoplásica de la Expresión Génica/genética , Humanos , Masculino , Neoplasias Gástricas/patología , Vía de Señalización WntRESUMEN
OBJECTIVES: The present study aimed to clarify the role of the ERK1/2 pathway in simvastatin (SV)-loaded nanomicelles (SVNs)- and SV-mediated promotion of cell osteogenic differentiation and explore the molecular mechanisms by which SVNs exhibited a greater efficacy in promoting osteogenic differentiation than SV. MATERIALS AND METHODS: SVNs were synthesized using a dialysis method. MG63 cells were treated with 2.5, 0.25, and 0.025 µmol/L of the drug. The optimal drug dosage was determined by examining the proliferative activity and ALP activity of the MG63 cells. Subsequently, Western blot analysis was performed to analyze the levels of the phosphorylated ERK1/2 proteins in each experimental group at various time points. Finally, the inhibitor PD98059 was used to effectively inhibit the ERK1/2 pathway. The resulting changes in the proliferative activity of MG63 cells and the osteogenesis-related markers were analyzed. RESULTS: The SVNs synthesized in the present study had a mean diameter of 27 nm. The encapsulation and drug-loading efficiencies were 52.03% ± 4.05% and 9.42% ± 0.66%, respectively. SVNs and SV exhibited optimum osteogenesis-promoting effects when the drugs were administered at a concentration of 0.25 µmol/L. The drug-induced activation of the ERK1/2 pathway reached a peak at 15 minutes after administration and then declined rapidly. From 24 hours to 7 days, SVNs and SV exerted an inhibitory effect on the ERK1/2 pathway rather than an activating effect. Throughout the whole experimental process, the regulatory effect of SVNs on the ERK1/2 pathway was significantly greater than that of SV. Inhibition of the ERK1/2 pathway by PD98059 markedly reduced the proliferative activity of the cells in all experimental groups. In addition, the ALP activity and the expression levels of the osterix (OSX) and osteocalcin (OC) proteins were drastically increased. CONCLUSION: SVNs significantly increased the effect of SV-induced osteogenic differentiation by strongly inhibiting the ERK1/2 pathway.
Asunto(s)
Sistema de Señalización de MAP Quinasas/efectos de los fármacos , Micelas , Nanopartículas/química , Osteogénesis/efectos de los fármacos , Simvastatina/farmacología , Fosfatasa Alcalina/metabolismo , Apoptosis/efectos de los fármacos , Ciclo Celular/efectos de los fármacos , Diferenciación Celular/efectos de los fármacos , Línea Celular Tumoral , Proliferación Celular/efectos de los fármacos , Humanos , Nanopartículas/ultraestructura , Osteocalcina/metabolismo , Fosforilación/efectos de los fármacosRESUMEN
A mounting body of evidence has revealed that microRNAs (miRs) serve pivotal roles in various developmental processes, and in tumourigenesis, by binding to target genes and subsequently regulating gene expression. Continued activation of the Wnt/ßcatenin signalling is positively associated with human malignancy. In addition, miR194 dysregulation has been implicated in gastric cancer (GC); however, the molecular mechanisms underlying the effects of miR194 on GC carcinogenesis remain to be elucidated. The present study demonstrated that miR194 was upregulated in GC tissues and SUFU negative regulator of Ηedgehog signaling (SUFU) was downregulated in GC cell lines. Subsequently, inhibition of miR194 attenuated nuclear accumulation of ßcatenin, which consequently blocked Wnt/ßcatenin signalling. In addition, the cytoplasmic translocation of ßcatenin induced by miR194 inhibition was mediated by SUFU. Furthermore, genes associated with the Wnt/ßcatenin signalling pathway were revealed to be downregulated following inhibition of the Wnt signalling pathway by miR194 suppression. Finally, the results indicated that cell apoptosis was markedly increased in response to miR194 inhibition, strongly suggesting the carcinogenic effects of miR194 in GC. Taken together, these findings demonstrated that miR194 may promote gastric carcinogenesis through activation of the Wnt/ßcatenin signalling pathway, making it a potential therapeutic target for GC.
Asunto(s)
Regulación Neoplásica de la Expresión Génica , MicroARNs/metabolismo , Proteínas Represoras/metabolismo , Neoplasias Gástricas/genética , Vía de Señalización Wnt/genética , Adulto , Anciano , Anciano de 80 o más Años , Apoptosis/genética , Carcinogénesis/genética , Línea Celular Tumoral , Proliferación Celular/genética , Regulación hacia Abajo , Femenino , Humanos , Masculino , MicroARNs/genética , Persona de Mediana Edad , Proteínas Represoras/genética , Estómago/patología , Neoplasias Gástricas/patología , Proteínas Supresoras de Tumor/genética , Proteínas Supresoras de Tumor/metabolismo , Regulación hacia ArribaRESUMEN
Less than a century ago, gastric cancer (GC) was the most common cancer throughout the world. Despite advances in surgical, chemotherapeutic, and radiotherapeutic treatment, GC remains the number 3 cancer killer worldwide. This fact highlights the need for better diagnostic biomarkers and more effective therapeutic targets. RAB11-FIP2, a member of the Rab11 family of interacting proteins, exhibits potential tumor suppressor function. However, involvement of RAB11-FIP2 in gastric carcinogenesis is yet to be elucidated. In this study, we demonstrated that RAB11-FIP2 was downregulated in GC tissues and constituted a target of the known onco-miRs, miR-192/215. We also showed that functionally, Rab11-FIP2 regulation by miR-192/215 is involved in GC-related biological activities. Finally, RAB11-FIP2 inhibition by miR-192/215 affected the establishment of cell polarity and tight junction formation in GC cells. In summary, this miR-192/215-Rab11-FIP2 axis appears to represent a new molecular mechanism underlying GC progression, while supplying a promising avenue of further research into diagnosis and therapy of GC.
Asunto(s)
Proteínas Portadoras/metabolismo , Progresión de la Enfermedad , Proteínas de la Membrana/metabolismo , MicroARNs/metabolismo , Neoplasias Gástricas/metabolismo , Neoplasias Gástricas/patología , Uniones Adherentes/metabolismo , Animales , Secuencia de Bases , Proteínas Portadoras/genética , Línea Celular Tumoral , Proliferación Celular , Transición Epitelial-Mesenquimal/genética , Regulación Neoplásica de la Expresión Génica , Humanos , Proteínas de la Membrana/genética , Ratones , MicroARNs/genética , Invasividad Neoplásica , Metástasis de la Neoplasia , Neoplasias Gástricas/genética , Proteínas de Unión al GTP rabRESUMEN
SMG-1,a member of the phosphoinositide kinase-like kinase family, functioned as a tumor suppressor gene. However, the role of SMG-1 in GC remain uncharacterized. In this study, regulation of SMG-1 by miR-192 and-215, along with the biological effects of this modulation, were studied in GC. We used gene microarrays to screening and luciferase reporter assays were to verify the potential targets of miR-192 and-215. Tissue microarrays analyses were applied to measure the levels of SMG-1 in GC tissues. Western blot assays were used to assess the signaling pathway of SMG-1 regulated by miR-192 and-215 in GC. SMG-1 was significantly downregulated in GC tissues.The proliferative and invasive properties of GC cells were decreased by inhibition of miR-192 and-215, whereas an SMG-1siRNA rescued the inhibitory effects. Finally, SMG-1 inhibition by miR-192 and-215 primed Wnt signaling and induced EMT. Wnt signaling pathway proteins were decreased markedly by inhibitors of miR-192 and-215, while SMG-1 siRNA reversed the inhibition apparently. Meanwhile, miR-192 and-215 inhitibtors increased E-cadherin expression and decreased N-cadherin and cotransfection of SMG-1 siRNA reversed these effects. In summary, these findings illustrate that SMG-1 is suppressed by miR-192 and-215 and functions as a tumor suppressor in GC by inactivating Wnt signaling and suppressing EMT.
Asunto(s)
MicroARNs/metabolismo , Fosfatidilinositol 3-Quinasas/genética , Neoplasias Gástricas/genética , Vía de Señalización Wnt/genética , Animales , Carcinogénesis/efectos de los fármacos , Carcinogénesis/genética , Línea Celular Tumoral , Transición Epitelial-Mesenquimal/efectos de los fármacos , Transición Epitelial-Mesenquimal/genética , Femenino , Regulación Neoplásica de la Expresión Génica/efectos de los fármacos , Humanos , Masculino , Ratones , Ratones Endogámicos BALB C , Ratones Desnudos , MicroARNs/antagonistas & inhibidores , MicroARNs/genética , Persona de Mediana Edad , Análisis de Secuencia por Matrices de Oligonucleótidos , Fosfatidilinositol 3-Quinasas/metabolismo , Proteínas Serina-Treonina Quinasas , Estómago/patología , Neoplasias Gástricas/mortalidad , Neoplasias Gástricas/patología , Análisis de Supervivencia , Análisis de Matrices Tisulares , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
Mounting evidence has indicated microRNA (miR) dysregulation and the Wnt/ß-catenin signaling pathway jointly drive carcinogenesis, cancer metastasis, and drug-resistance. The current review will focus on the role of the crosstalk between miRs and the Wnt/ß-catenin signaling pathway in cancer development. MiRs were found to activate or inhibit the canonical Wnt pathway at various steps. On the other hand, Wnt activation increases expression of miR by directly binding to its promoter and activating transcription. Moreover, there are mutual feedback loops between some miRs and the Wnt/ß-catenin signaling pathway. Clinical trials of miR-based therapeutic agents are investigated for solid and hematological tumors, however, challenges concerning low bioavailability and possible side effects must be overcome before the final clinical application. This review will describe current understanding of miR crosstalk with the Wnt/ß-catenin signaling cascade. Better understanding of the regulatory network will provide insight into miR-based therapeutic development.
Asunto(s)
MicroARNs/metabolismo , Neoplasias/patología , Receptor Cross-Talk/fisiología , Vía de Señalización Wnt/fisiología , Animales , Regulación Neoplásica de la Expresión Génica/fisiología , Humanos , Neoplasias/genética , Neoplasias/metabolismoRESUMEN
Emerging evidence has shown that miRNA-194 is aberrantly upregulated in gastric cancer (GC); however, the biological mechanisms underlying its involvement are largely unknown. Wnt/ß-catenin signaling has been implicated in gastric tumorigenesis; we therefore hypothesized that miRNA-194 promotes gastric carcinogenesis by activating Wnt/ß-catenin signaling. MiRNA-194 was found to be overexpressed in GC cell lines and 43 paired GC tissues. Overexpression of miRNA-194 promoted cell proliferation and migration, while inhibition of miRNA-194 blocked these processes. Inhibition of miRNA-194 decreased tumor volumes in nude mice. Furthermore, miRNA-194 inhibitors promoted cytoplasmic localization of ß-catenin, leading to repression of Wnt signaling. We also discovered that SUFU, a known negative regulator of Hedgehog and Wnt signaling, was a target of miRNA-194. Anti-SUFU siRNAs rescued the inhibitory effects of miRNA-194 antagonists on cell proliferation and migration and on colony formation. We also found that SUFU expression was downregulated in GC tissues and cell lines and negatively correlated with miRNA-194 expression in primary GC tissues. Moreover, SUFU expression was negatively correlated with tumor stage, supporting its potential as a diagnostic or prognostic marker in GC. Taken together, these findings suggest that miRNA-194 is oncogenic and promotes GC cell proliferation and migration by activating Wnt signaling, at least in part, via suppression of SUFU.
Asunto(s)
MicroARNs/metabolismo , Proteínas Represoras/metabolismo , Neoplasias Gástricas/metabolismo , Vía de Señalización Wnt , Animales , Línea Celular Tumoral , Movimiento Celular , Proliferación Celular , Regulación hacia Abajo , Femenino , Regulación Neoplásica de la Expresión Génica , Humanos , Ratones Endogámicos BALB C , Ratones Desnudos , MicroARNs/genética , Invasividad Neoplásica , Estadificación de Neoplasias , Oncogenes , Interferencia de ARN , Proteínas Represoras/genética , Neoplasias Gástricas/genética , Neoplasias Gástricas/patología , Factores de Tiempo , TransfecciónRESUMEN
Gastric cancer (GC) is one of the leading causes of cancer-related deaths throughout China and worldwide. The discovery of microRNAs (miRNAs) has provided a new opportunity for developing diagnostic biomarkers and effective therapeutic targets in GC. By performing microarray analyses of benign and malignant gastric epithelial cell lines (HFE145, NCI-N87, MKN28, RF1, KATO III and RF48), 16 significantly dysregulated miRNAs were found. 11 of these were validated by real-time qRT-PCR. Based on miRWalk online database scans, 703 potential mRNA targets of the 16 miRNAs were identified. Bioinformatic analyses suggested that these dysregulated miRNAs and their predicted targets were principally involved in tumor pathogenesis, MAPK signaling, and apoptosis. Finally, miRNA-gene network analyses identified miRNA-125b as a crucial miRNA in GC development. Taken together, these results develop a comprehensive expression and functional profile of differentially expressed miRNAs related to gastric oncogenesis. This profile may serve as a potential tool for biomarker and therapeutic target identification in GC patients.
Asunto(s)
Adenocarcinoma/genética , Biomarcadores de Tumor/genética , Biología Computacional , Perfilación de la Expresión Génica/métodos , MicroARNs/genética , Neoplasias Gástricas/genética , Adenocarcinoma/patología , Regulación Neoplásica de la Expresión Génica , Redes Reguladoras de Genes , Predisposición Genética a la Enfermedad , Humanos , Fenotipo , Valor Predictivo de las Pruebas , Reacción en Cadena en Tiempo Real de la Polimerasa , Reproducibilidad de los Resultados , Transducción de Señal/genética , Neoplasias Gástricas/patología , Neoplasias Gástricas/terapiaRESUMEN
Propidium iodide (PI) reacts with both DNA and RNA and is a commonly used fluorescent reagent for nucleic acid staining. The aim of the study was to compare the cellular staining patterns of PI with that of Nissl staining in rat nervous tissues and to report a modified staining method that selectively labels Nissl bodies in neurons. Cryosections and paraffin sections of different tissues of normal Sprague-Dawley rats, including trigeminal ganglia, dorsal root ganglia, spinal cord, liver, and small intestine, were stained by either PI or the hematoxylin and eosin method. Some sections were treated with RNase or DNase before the above staining, and some were double stained with PI and a Nissl stain. The sections were observed by light, fluorescence or confocal microscopy. Results showed strong PI signals detected as patterns of granules in the neuronal cytoplasm of all nervous tissues, whereas the staining of neuronal nuclei was weaker. In contrast, nuclei of neuroglial cells were strongly stained by PI, while the cytoplasm was not obviously stained. Pretreatment of the neural tissue with RNase abolished the PI signals. Furthermore, the PI positive granules in neuronal cytoplasm co-localized with Nissl bodies stained by the fluorescent Nissl stain. When the tissue was pretreated with DNase, PI only stained the cytoplasmic granules of neurons, but not that of glial cells. Our results show that PI stains Nissl bodies and may serve as an economical and convenient neuron marker for neuronal cell counting when specific neural markers such as antibodies are not readily available.
Asunto(s)
Neuronas/citología , Cuerpos de Nissl , Propidio/química , Coloración y Etiquetado/métodos , Animales , Biomarcadores , Recuento de Células/métodos , Femenino , Ratas , Ratas Sprague-DawleyRESUMEN
BACKGROUND: Esophageal cancer ranks eighth among frequent cancers worldwide. Our aim was to investigate whether and at which neoplastic stage promoter hypermethylation of CAV1 is involved in human esophageal carcinogenesis. METHODS: Using real-time quantitative methylation-specific PCR (qMSP), we examined CAV1 promoter hypermethylation in 260 human esophageal tissue specimens. Real-time RT-PCR and qMSP were also performed on OE33 esophageal cancer cells before and after treatment with the demethylating agent, 5-aza-2'-deoxycytidine (5-Aza-dC). RESULTS: CAV1 hypermethylation showed highly discriminative ROC curve profiles, clearly distinguishing esophageal adenocarcinomas (EAC) and esophageal squamous cell carcinomas (ESCC) from normal esophagus (NE) (EAC vs. NE, AUROC = 0.839 and p < 0.0001; ESCC vs. NE, AUROC = 0.920 and p < 0.0001). Both CAV1 methylation frequency and normalized methylation value (NMV) were significantly higher in Barrett's metaplasia (BE), low-grade and high-grade dysplasia occurring in BE (D), EAC, and ESCC than in NE (all p < 0.01, respectively). Meanwhile, among 41 cases with matched NE and EAC or ESCC, CAV1 NMVs in EAC and ESCC (mean = 0.273) were significantly higher than in corresponding NE (mean = 0.146; p < 0.01, Student's paired t-test). Treatment of OE33 EAC cells with 5-Aza-dC reduced CAV1 methylation and increased CAV1 mRNA expression. CONCLUSIONS: CAV1 promoter hypermethylation is a frequent event in human esophageal carcinomas and is associated with early neoplastic progression in Barrett's esophagus.
Asunto(s)
Adenocarcinoma/metabolismo , Esófago de Barrett/metabolismo , Carcinoma de Células Escamosas/metabolismo , Caveolina 1/metabolismo , Metilación de ADN , Neoplasias Esofágicas/metabolismo , Adenocarcinoma/genética , Adenocarcinoma/patología , Área Bajo la Curva , Azacitidina/análogos & derivados , Azacitidina/farmacología , Esófago de Barrett/genética , Esófago de Barrett/patología , Carcinoma de Células Escamosas/genética , Carcinoma de Células Escamosas/patología , Caveolina 1/genética , Línea Celular Tumoral , Metilasas de Modificación del ADN/antagonistas & inhibidores , Metilasas de Modificación del ADN/metabolismo , Decitabina , Progresión de la Enfermedad , Inhibidores Enzimáticos/farmacología , Neoplasias Esofágicas/genética , Neoplasias Esofágicas/patología , Regulación Neoplásica de la Expresión Génica , Humanos , Regiones Promotoras Genéticas , ARN Mensajero/metabolismo , Curva ROC , Factores de TiempoRESUMEN
BACKGROUND/AIM: Ras-related associated with diabetes (RRAD), a member of the Ras-related GTPase superfamily, is frequently methylated in several human cancers, though its methylation profile remains unclear in esophageal cancer. MATERIALS AND METHODS: We examined RRAD promoter hypermethylation using real-time quantitative methylation-specific PCR in 229 primary human esophageal tissues of contrasting histological types. RESULTS: RRAD hypermethylation showed highly discriminative receiver-operator characteristic curve profiles, clearly distinguishing esophageal squamous cell carcinoma (ESCC) from esophageal adenocarcinoma (EAC) or normal esophagus (NE) (p<0.01 and p<0.01, respectively). RRAD normalized methylation values were significantly higher in ESCC (0.0242) than in NE (0.0057, p<0.05) or EAC (0.0139, p<0.01). RRAD hypermethylation frequency was also significantly higher in ESCC (23.1%) than in NE (0%, p<0.05) or EAC (5.4%, p<0.05). CONCLUSION: Promoter hypermethylation of RRAD is a frequent, tissue-specific event in ESCC, and is uncommon in EAC. The aberrant methylation of RRAD may be involved in the pathogenesis of a subset of ESCC, but not in EAC.