RESUMEN
Transfer-RNA-derived small RNAs (tsRNAs; also called tRNA-derived fragments) are an abundant class of small non-coding RNAs whose biological roles are not well understood. Here we show that inhibition of a specific tsRNA, LeuCAG3'tsRNA, induces apoptosis in rapidly dividing cells in vitro and in a patient-derived orthotopic hepatocellular carcinoma model in mice. This tsRNA binds at least two ribosomal protein mRNAs (RPS28 and RPS15) to enhance their translation. A decrease in translation of RPS28 mRNA blocks pre-18S ribosomal RNA processing, resulting in a reduction in the number of 40S ribosomal subunits. These data establish a post-transcriptional mechanism that can fine-tune gene expression during different physiological states and provide a potential new target for treating cancer.
Asunto(s)
ARN Pequeño no Traducido/genética , ARN de Transferencia de Leucina/genética , Proteínas Ribosómicas/biosíntesis , Ribosomas/genética , Ribosomas/metabolismo , Animales , Apoptosis/efectos de los fármacos , Apoptosis/genética , Emparejamiento Base , Secuencia de Bases , Carcinoma Hepatocelular/genética , Carcinoma Hepatocelular/patología , Carcinoma Hepatocelular/terapia , Proliferación Celular/efectos de los fármacos , Proliferación Celular/genética , Supervivencia Celular/efectos de los fármacos , Supervivencia Celular/genética , Femenino , Células HCT116 , Células HEK293 , Células HeLa , Humanos , Neoplasias Hepáticas/genética , Neoplasias Hepáticas/patología , Neoplasias Hepáticas/terapia , Masculino , Ratones , Oligonucleótidos Antisentido/genética , Oligonucleótidos Antisentido/farmacología , Oligonucleótidos Antisentido/uso terapéutico , Biosíntesis de Proteínas/efectos de los fármacos , Biosíntesis de Proteínas/genética , ARN Mensajero/química , ARN Mensajero/genética , ARN Mensajero/metabolismo , ARN Ribosómico 18S/genética , ARN Ribosómico 18S/metabolismo , ARN Pequeño no Traducido/antagonistas & inhibidores , ARN de Transferencia de Leucina/antagonistas & inhibidores , Proteínas Ribosómicas/genética , Subunidades Ribosómicas Pequeñas de Eucariotas/metabolismo , Ribosomas/efectos de los fármacos , Especificidad por Sustrato/genética , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
Herpes zoster (HZ) remains a significant health burden with millions of cases in North America and Europe annually. HZ is frequently followed by long-term pain or post-herpetic neuralgia (PHN). Although effective vaccines for HZ are available, currently used nucleotide analogues often have limited effectiveness against HZ and especially PHN, so there remains a need for additional antiviral therapies for HZ. We recently identified a population of small non-coding RNA (sncRNA) encoded by Varicella Zoster Virus (VZV) and showed that single locked-nucleic acid antagonists (LNAA) to some sncRNA can modulate VZV replication in cell culture. In this work, we explored the antiviral effects of combinations of LNAA oligonucleotides targeting VZVsncRNA. Combinations of LNAA targeting three VZVsncRNA encoded in and near a critical viral regulatory gene were additive, achieving 96 % reduction in virus growth in a cell line. VZV growth was also inhibited by more than 90 % in primary human skin fibroblast cultures by individual and combinations of LNAA to VZVsncRNA. The inhibition by VZVsncRNA was specific and not a consequence of innate immune responses since LNAA to a different VZVsncRNA enhanced VZV growth. Targeted VZVsncRNA lack homologous sequences in the human transcriptome suggesting that LNAA to them would have reduced cytotoxicity if used as therapeutics. These results support further development of oligonucleotides targeting VZVsncRNA as a novel treatment for HZ.
Asunto(s)
Antivirales/farmacología , Herpesvirus Humano 3/efectos de los fármacos , Ácidos Nucleicos/farmacología , ARN Pequeño no Traducido/genética , Fibroblastos , Herpes Zóster/tratamiento farmacológico , Herpesvirus Humano 3/fisiología , Humanos , Oligonucleótidos/síntesis química , ARN Pequeño no Traducido/antagonistas & inhibidores , Replicación Viral/efectos de los fármacosRESUMEN
Most herpesviruses use both host and viral small non-coding RNAs (sncRNA), especially microRNA, to modulate infection. Bioinformatic analyses of NGS data obtained from Varicella Zoster virus (VZV)-infected cells predicted 24 VZVsncRNA, seven of which were confirmed to be expressed in infected fibroblasts and neurons using stem-loop quantitative reverse transcription PCR (SL-PCR). We here assayed for the expression of all 24 of the bioinformatically predicted VZVsncRNA in cells productively infected by VZV using SL-PCR. 23 of the 24 predicted sequences were detected in VZV-infected ARPE19 cells and 19 of the 24 sequences in infected human neurons generated by two methods from embryonic stem cells. We also show that blocking one of two newly-tested VZV-encoded sncRNA using locked nucleotide antagonists significantly increased viral replication. These findings suggest that further study of VZV encoded sncRNA could elucidate an additional level of regulation of the life cycle of this pathogenic human herpesvirus.
Asunto(s)
Células Epiteliales/virología , Herpesvirus Humano 3/genética , Neuronas/virología , ARN Pequeño no Traducido/genética , ARN Viral/genética , Células Cultivadas , Biología Computacional , Perfilación de la Expresión Génica , Herpesvirus Humano 3/fisiología , Humanos , ARN Pequeño no Traducido/antagonistas & inhibidores , ARN Viral/antagonistas & inhibidores , Replicación Viral/genéticaRESUMEN
Viral infection initiates an array of changes in host gene expression. Many viruses dampen host protein expression and attempt to evade the host anti-viral defense machinery. Host gene expression is suppressed at several stages of host messenger RNA (mRNA) formation including selective degradation of translationally competent messenger RNAs. Besides mRNAs, host cells also express a variety of noncoding RNAs, including small RNAs, that may also be subject to inhibition upon viral infection. In this review we focused on different ways viruses antagonize coding and noncoding RNAs in the host cell to its advantage.