RESUMO
Aptamers for Ebola virus (EBOV) offer a powerful means for prevention and diagnostics. Unfortunately, few aptamers for EBOV have been discovered yet. Herein, assisted by magnetism-controlled selection chips to strictly manipulate selection conditions, a highly efficient aptamer selection platform for EBOV is proposed. With highly stringent selection conditions of rigorous washing, manipulation of minuscule amounts of magnetic beads, and real-time evaluation of the selection effectiveness, the selection performance of the platform was improved significantly. In only three rounds of selection, the high-performance aptamers for EBOV GP and NP proteins were obtained simultaneously, with dissociation constants ( Kd) in the nanomolar range. The aptamer was further applied to the detection of EBOV successfully, with a detection limit of 4.2 ng/mL. The whole detection process that consisted of sample mixing, separation, and signal acquisition was highly integrated and conducted in a magnetism-controlled detection chip, showing high biosafety and great potential for point-of-care detection. The method may open up new avenues for prevention and control of EBOV.
Assuntos
Aptâmeros de Nucleotídeos/metabolismo , Ebolavirus/isolamento & purificação , Magnetismo , Proteínas Virais/metabolismo , Aptâmeros de Nucleotídeos/química , Ebolavirus/metabolismo , Doença pelo Vírus Ebola/diagnóstico , Humanos , Limite de Detecção , Sistemas Automatizados de Assistência Junto ao Leito , Técnica de Seleção de Aptâmeros , Proteínas Virais/análiseRESUMO
Our previous studies show that adenosine-induced apoptosis is involved in endoplasmic reticulum stress in HepG2 cells. In this study, we have investigated whether knockdown of GRP78 by short hairpin RNA (shRNA) increases the cytotoxic effects of adenosine in HepG2 cells. The adenovirus vector-delivered shRNA targeting GRP78 (Ad-shGRP78) was constructed and transfected into HepG2 cells. RT-PCR assay was used to determine RNA interference efficiency. Effects of knockdown of GRP78 on adenosine-induced cell viabilities, cell-cycle distribution and apoptosis, as well as relative protein expressions were determined by flow cytometry and/or Western blot analysis. The intracellular Ca2+ concentration was detected by laser scanning confocal microscope. Mitochondrial membrane potential (ΔΨm) was measured by a fluorospectrophotometer. The results revealed that GRP78 mRNA was significantly downregulated by Ad-shGRP78 transfection. Knockdown of GRP78 enhanced HepG2 cell sensitivity to adenosine by modulating G0/G1 arrest and stimulating Bax, Bak, m-calpain, caspase-4 and CHOP protein levels. Knockdown of GRP78 worsened cytosolic Ca2+ overload and ΔΨm loss. Knockdown of caspase-4 by shRNA decreased caspase-3 mRNA expression and cell apoptosis. These findings indicate that GRP 78 plays a protective role in ER stress-induced apoptosis and show that the combination of chemotherapy drug and RNA interference adenoviruses provides a new treatment strategy against malignant tumors.
Assuntos
Adenosina/toxicidade , Antineoplásicos/farmacologia , Apoptose/efeitos dos fármacos , Proteínas de Choque Térmico/metabolismo , RNA Interferente Pequeno/metabolismo , Adenoviridae/genética , Antineoplásicos/metabolismo , Proteínas Reguladoras de Apoptose/metabolismo , Carcinoma Hepatocelular/metabolismo , Carcinoma Hepatocelular/patologia , Caspases Iniciadoras/metabolismo , Regulação para Baixo/efeitos dos fármacos , Chaperona BiP do Retículo Endoplasmático , Pontos de Checagem da Fase G1 do Ciclo Celular/efeitos dos fármacos , Vetores Genéticos/metabolismo , Proteínas de Choque Térmico/antagonistas & inibidores , Proteínas de Choque Térmico/genética , Células Hep G2 , Humanos , Neoplasias Hepáticas/metabolismo , Neoplasias Hepáticas/patologia , Potencial da Membrana Mitocondrial/efeitos dos fármacos , Interferência de RNA , RNA Mensageiro/metabolismo , Transdução de Sinais/efeitos dos fármacos , Fator de Transcrição CHOP/metabolismoRESUMO
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.
Assuntos
Carcinoma Hepatocelular/patologia , Estresse do Retículo Endoplasmático/fisiologia , Neoplasias Hepáticas/patologia , RNA Longo não Codificante/metabolismo , Proteína Supressora de Tumor p53/metabolismo , Animais , Apoptose/fisiologia , Western Blotting , Chaperona BiP do Retículo Endoplasmático , Citometria de Fluxo , Células Hep G2 , Xenoenxertos , Humanos , Imuno-Histoquímica , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Nus , Reação em Cadeia da Polimerase Via Transcriptase ReversaRESUMO
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.