RESUMEN
Esophageal squamous cell carcinoma (ESCC) is the main subtype of esophageal cancer in China, and the prognosis of patients remains poor mainly due to the occurrence of lymph node and distant metastasis. The long noncoding RNA (lncRNA) maternally expressed gene 3 (MEG3) has been shown to have tumorsuppressive properties and to play an important role in epithelialtomesenchymal transition (EMT) in some solid tumors. However, whether MEG3 is involved in EMT in ESCC remains unclear. In the present study, the MEG3 expression level and its association with tumorigenesis were determined in 43 tumor tissues of patients with ESCC and in ESCC cells using reverse transcriptionquantitative PCR analysis. Gene microarray analysis was performed to detect differentially expressed genes (DEGs). Based on the functional annotation results, the effects of ectopic expression of MEG3 on cell growth, migration, invasion and EMT were assessed. MEG3 expression level was found to be markedly lower in tumor tissues and cells. Statistical analysis revealed that MEG3 expression was significantly negatively associated with lymph node metastasis and TNM stage in ESCC. Fluorescence in situ hybridization assay demonstrated that MEG3 was expressed mainly in the nucleus. Ectopic expression of MEG3 inhibited cell proliferation, migration, invasion and cell cycle progression in EC109 cells. Gene microarray results demonstrated that 177 genes were differentially expressed ≥2.0 fold in MEG3overexpressing cells, including 23 upregulated and 154 downregulated genes. Functional annotation revealed that the DEGs were mainly involved in amino acid biosynthetic process, mitogenactivated protein kinase signaling, and serine and glycine metabolism. Further experiments indicated that the ectopic expression of MEG3 significantly suppressed cell proliferation, migration, invasion and EMT by downregulating phosphoserine aminotransferase 1 (PSAT1). In pathological tissues, PSAT1 and MEG3 were significantly negatively correlated, and high expression of PSAT1 predicted poor survival. Taken together, these results suggest that MEG3 may be a useful prognostic biomarker and may suppress EMT by inhibiting the PSAT1dependent glycogen synthase kinase3ß/Snail signaling pathway in ESCC.
Asunto(s)
Transición Epitelial-Mesenquimal/genética , Neoplasias Esofágicas/metabolismo , Carcinoma de Células Escamosas de Esófago/metabolismo , Glucógeno Sintasa Quinasa 3 beta/antagonistas & inhibidores , ARN Largo no Codificante/metabolismo , Factores de Transcripción de la Familia Snail/antagonistas & inhibidores , Transaminasas/antagonistas & inhibidores , Adulto , Anciano , Animales , Línea Celular Tumoral , Movimiento Celular/fisiología , Proliferación Celular/fisiología , Regulación hacia Abajo , Transición Epitelial-Mesenquimal/fisiología , Neoplasias Esofágicas/genética , Neoplasias Esofágicas/patología , Carcinoma de Células Escamosas de Esófago/genética , Carcinoma de Células Escamosas de Esófago/patología , Femenino , Glucógeno Sintasa Quinasa 3 beta/metabolismo , Xenoinjertos , Humanos , Metástasis Linfática , Masculino , Ratones , Ratones Endogámicos BALB C , Ratones Desnudos , Persona de Mediana Edad , Pronóstico , ARN Largo no Codificante/genética , Transducción de Señal , Factores de Transcripción de la Familia Snail/metabolismo , Tasa de Supervivencia , Transaminasas/metabolismoRESUMEN
In cancer research, autophagy acts as a doubleedged sword: it increases cell viability or induces cell apoptosis depending upon the cell context and functional status. Recent studies have shown that adenosine (Ado) has cytotoxic effects in many tumors. However, the role of autophagy in Adoinduced apoptosis is still poorly understood. In the present study, Adoinduced apoptotic death and autophagy in hepatoblastoma HepG2 cells was investigated and the relationship between autophagy and apoptosis was identified. In the present study, it was demonstrated that Ado inhibited HepG2 cell growth in a time and concentrationdependent manner and activated endoplasmic reticulum (ER) stress, as indicated by G0/G1 cell cycle arrest, the increased mRNA and protein levels of GRP78/BiP, PERK, ATF4, CHOP, cleaved caspase3, cytochrome c and the loss of mitochon-drial membrane potential (ΔΨm). Ado also induced autophagic flux, revealed by the increased expression of the autophagy marker microtubuleassociated protein 1 light chain 3II (LC3II), Beclin1, autophagosomes, and the degradation of p62, as revealed by western blot analysis and macrophagederived chemokine (MDC) staining. Blocking autophagy using LY294002 notably entrenched Adoinduced growth inhibition and cell apoptosis, as demonstrated with the increased expression of cytochrome c and p62, and the decreased expression of LC3II. Conversely, the autophagy inducer rapamycin alleviated Adoinduced apoptosis and markedly increased the ΔΨm. Moreover, knockdown of AMPK with siAMPK partially abolished Adoinduced ULK1 activation and mTOR inhibition, and thus reinforced CHOP expression and Adoinduced apoptosis. These results indicated that Adoinduced ER stress resulted in apoptosis and autophagy concurrently. The AMPK/mTOR/ULK1 signaling pathway played a protective role in the apoptotic procession. Inhibition of autophagy may effectively enhance the anticancer effect of Ado in human hepatoblastoma HepG2 cells.
Asunto(s)
Adenosina/farmacología , Apoptosis/efectos de los fármacos , Autofagia/efectos de los fármacos , Hepatoblastoma/tratamiento farmacológico , Neoplasias Hepáticas/tratamiento farmacológico , Proteínas Quinasas Activadas por AMP/genética , Proteínas Quinasas Activadas por AMP/metabolismo , Adenosina/uso terapéutico , Homólogo de la Proteína 1 Relacionada con la Autofagia/metabolismo , Supervivencia Celular/efectos de los fármacos , Cromonas/farmacología , Relación Dosis-Respuesta a Droga , Chaperón BiP del Retículo Endoplásmico , Estrés del Retículo Endoplásmico/efectos de los fármacos , Puntos de Control de la Fase G1 del Ciclo Celular/efectos de los fármacos , Técnicas de Silenciamiento del Gen , Células Hep G2 , Hepatoblastoma/patología , Humanos , Péptidos y Proteínas de Señalización Intracelular/metabolismo , Neoplasias Hepáticas/patología , Morfolinas/farmacología , ARN Interferente Pequeño/metabolismo , Transducción de Señal/efectos de los fármacos , Transducción de Señal/genética , Sirolimus/farmacología , Serina-Treonina Quinasas TOR/metabolismoRESUMEN
Long non-coding RNAs (lncRNAs) play important roles in diverse biological processes. Although downregulation of lncRNA maternally expressed gene 3 (MEG3) has been identified in several types of cancers, little is known concerning its biological role and regulatory mechanism in hepatoma. Our previous studies demonstrated that MEG3 induces apoptosis in a p53-dependent manner. The aim of the present study was to determine whether endoplasmic reticulum (ER) stress is involved in MEG3induced apoptosis. Recombinant lentiviral vectors containing MEG3 (LvMEG3) were constructed and transfected into HepG2 cells. A 3(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay, RTPCR, flow cytometry, western blot analysis, immunofluorescence and immunohistochemistry were applied. Transfected HepG2 cells were also transplanted into nude mice, and the tumor growth curves were determined. The results showed that the recombinant lentivirus of MEG3 was transfected successfully into the HepG2 cells and the expression level of MEG3 was significantly increased. Ectopic expression of MEG3 inhibited HepG2 cell proliferation in vitro and in vivo, and also induced apoptosis. Ectopic expression of MEG3 increased ER stressrelated proteins 78kDa glucoseregulated protein (GRP78), inositolrequiring enzyme 1 (IRE1), RNAdependent protein kinaselike ER kinase (PERK), activating transcription factor 6 (ATF6), C/EBP homologous protein (CHOP), caspase3, as well as p53 and NFκB expression accompanied by NFκB translocation from the cytoplasm to the nucleus. Furthermore, inhibition of NFκB with Bay117082 decreased p53 expression in the MEG3transfected cells. These results indicate that MEG3 inhibits cell proliferation and induces apoptosis, partially via the activation of the ER stress and p53 pathway, in which NFκB signaling is required for p53 activation in ER stress.
Asunto(s)
Carcinoma Hepatocelular/patología , Estrés del Retículo Endoplásmico/fisiología , Neoplasias Hepáticas/patología , ARN Largo no Codificante/metabolismo , Proteína p53 Supresora de Tumor/metabolismo , Animales , Apoptosis/fisiología , Western Blotting , Chaperón BiP del Retículo Endoplásmico , Citometría de Flujo , Células Hep G2 , Xenoinjertos , Humanos , Inmunohistoquímica , Ratones , Ratones Endogámicos BALB C , Ratones Desnudos , Reacción en Cadena de la Polimerasa de Transcriptasa InversaRESUMEN
Long non-coding RNA MEG3 is suggested to function as a tumor suppressor. However, the activation mechanism of MEG3 is still not well understood and data are not available on its role under adenosine-induced apoptosis. In this study, HepG2 cells were treated with adenosine or 5-AzacdR. Methylation status of MEG3 promoter was detected by methylation specific PCR (MSP) and MEG3 expression was determined by qRT-PCR. PcDNA3.1-MEG3 recombinant plasmid was constructed and transfected to hepatoma HepG2 and Huh7 cells. Cell growth, morphological changes, cell-cycle distribution and apoptosis were analyzed by MTT assay, fluorescence microscopy and flow cytometry. The mRNA and protein expression levels were detected by qRT-PCR and western blot analysis. MEG3 binding proteins were screened by the improved MS2 biotin tagged RNA affinity purification method. The co-expression network of MEG3 was generated by GO analysis and ILF3 was identified as MEG3 binding protein by RNA pulldown and western blot analysis. Both adenosine and 5-Aza-CdR increased MEG3 mRNA expression and the CpG island of MEG3 gene in HepG2 cells was typical hypermethylation. Ectopic expression of MEG3 inhibited hepatoma cell growth in a time-dependent manner, resulted in cell cycle arrest and induced apoptosis. Ectopic expression of MEG3 increased p53, caspase-3 mRNA and protein levels, decreased MDM2 and cyclin D1 mRNA and protein levels, as well as ILF3 protein expression in HepG2 cells. These findings are the first to identify that adenosine increases MEG3 expression by inhibition of DNA methylation and its antitumor effects is involved in MEG3 activation. ILF3 may participate in the anticancer regulation of MEG3 by interacting with MEG3.