RESUMO
Nuclear RNA interference (RNAi) is mediated by the canonical RNAi machinery and can lead to transcriptional silencing, transcriptional activation, or modulation of alternative splicing patterns. These effects transpire through changes in histone and DNA modifications via RNAi-mediated recruitment of chromatin-modifying enzymes. To prove that nuclear RNAi occurs and modulates transcription in human cells, we used live-cell imaging to detect and track nuclear RNAi transcriptional repression in single living human cells. While employing reporter genes constructed with inducible promoters and cognate-inducible short hairpin RNA (shRNA) targeted against the reporter coding region, we have characterized the dynamics of the nuclear RNAi process in living human cells. We show that the silencing effect is mediated through the nascent mRNA, followed by activity of histone methylating enzymes, but not through DNA methylation.
Assuntos
Proteínas de Fluorescência Verde/genética , Imagem Molecular/métodos , Interferência de RNA , Núcleo Celular/genética , Metilação de DNA/efeitos dos fármacos , Epigênese Genética , Regulação da Expressão Gênica , Proteínas de Fluorescência Verde/análise , Proteínas de Fluorescência Verde/metabolismo , Células HEK293 , Histona Metiltransferases , Histona-Lisina N-Metiltransferase/antagonistas & inibidores , Histona-Lisina N-Metiltransferase/genética , Histona-Lisina N-Metiltransferase/metabolismo , Humanos , Hibridização in Situ Fluorescente/métodos , Microscopia de Fluorescência/métodos , Piperazinas/farmacologia , Regiões Promotoras Genéticas , Quinazolinas/farmacologia , Precursores de RNA/genética , RNA Interferente Pequeno , Sítio de Iniciação de TranscriçãoRESUMO
Inactivation of tumor suppressor and metastasis suppressor genes via epigenetic silencing is a frequent event in human cancers. Recent work has shown new mechanisms of epigenetic silencing, based on the occurrence of long noncoding promoter-spanning antisense and/or sense RNAs (lncRNAs), which constitute part of chromatin silencing complexes. Using reverse transcription polymerase chain reaction (RT-PCR), we have started to scan "triple negative" and Her2-overexpressing breast cancer cell lines for directional/bidirectional transcription through promoters of tumor suppressor and metastasis suppressor genes known to be epigenetically silenced in vivo. Surprisingly, we found that RT-PCR-amplified products were obtained at high frequency in the absence of exogenous primers. These amplified products resulted from RT priming via transcripts originating from promoter or upstream spanning regions. Consequently, this priming overruled directionality determination and led to false detection-identification of such lncRNAs. We show that this prevalent "no primer" artifact can be eliminated by treating the RNA preparations with periodate, performing RT reactions at highly elevated temperatures, or a combination of both. These experimental improvements enabled determination of the presence and directionality of individual promoter-spanning long noncoding RNAs with certainty. Examples for the BRMS1 metastasis suppressor gene, as well as RAR-ß2 and CST6 human tumor suppressor genes, in breast carcinoma cell lines are presented.
Assuntos
Artefatos , RNA não Traduzido/genética , Reação em Cadeia da Polimerase Via Transcriptase Reversa/métodos , Linhagem Celular Tumoral , Cistatina M/genética , Primers do DNA/genética , Humanos , Metástase Neoplásica , Proteínas de Neoplasias/genética , Regiões Promotoras Genéticas/genética , RNA Antissenso/genética , Receptores do Ácido Retinoico/genética , Proteínas RepressorasRESUMO
Glioblastoma multiforme (GBM) is a highly aggressive malignant brain tumor. Despite some recent improvement in the treatment of this malignancy, life expectancy of GBM patients remains extremely low. Therefore, continuous efforts to develop new treatment modalities are mandatory. A novel approach to cancer treatment is the use of targeted treatments, alone and in combination with other therapies. In this study, we evaluated the effects of novel combinations of conventional anti-cancer treatments (temozolomide or irradiation) with the targeted drug, imatinib, or with psychotropic drugs, belonging to the selective serotonin reuptake inhibitors (SSRIs) and phenothiazine subclasses, as well as combination of imatinib with psychotropic agents, on a human U87 glioblastoma cell line. The combination of temozolomide with imatinib or the psychotropic drugs resulted in an additive anti-proliferative effect, while the combination of irradiation and the psychotropic agents resulted in a less than additive effect on cell proliferation. A marked synergistic anti-proliferative effect of imatinib combined with the psychotropic drugs fluoxetine, sertraline or perphenazine was demonstrated. None of the single or combined treatments led to a reduction in the expression of phosphorylated MAP kinase. However, a marked synergistic reduction in the expression of the key regulatory molecule, pAKT, was detected, following the combined treatment of the cells with the imatinib/psychotropics combination. This down-regulation of pAKT may mediate the synergistic anti-proliferative interaction of imatinib with the psychotropic agents. Although the concentrations of the psychotropic agents used in this and other in vitro studies were beyond the clinically relevant blood levels in humans, recent studies have demonstrated anti-proliferative effects in vivo, using sertraline in a human colon cancer model. Thus, it seems that further in vivo studies combining imatinib with psychotropic agents, especially fluoxetine and sertraline, are warranted.