RESUMO
The tumour suppressor gene (Rb1) is necessary for the maintenance of telomere integrity in osteoblastic cells. We now show that the compaction of telomeric chromatin and the appropriate histone modifications of telomeric DNA are both dependent upon Rb1-mediated transcription of the telomere-derived long non-coding RNA TERRA. Expression of TERRA was reduced in Rb1 haploinsufficient cells, and further decreased by shRNA-mediated reduction of residual Rb1 expression. Restoration of Rb1 levels through lentiviral transduction was sufficient to reestablish both transcription of TERRA and condensation of telomeric chromatin. The human chromosome 15q TERRA promoter contains predicted retinoblastoma control elements, and was able to confer Rb1-dependent transcription upon a promoterless reporter gene. Chromatin immunoprecipitation revealed preferential binding of phosphorylated over non-phosphorylated Rb1 at the TERRA promoter. As Rb1-deficient cells show increased genomic instability we suggest that this novel non-canonical action of Rb1 may contribute to the tumour suppressive actions of Rb1.
Assuntos
Heterocromatina/ultraestrutura , Histonas/genética , Osteoblastos/metabolismo , RNA Longo não Codificante/genética , Proteína do Retinoblastoma/genética , Telômero/metabolismo , Animais , Osso e Ossos/citologia , Osso e Ossos/metabolismo , Cromossomos Humanos Par 15 , Regulação da Expressão Gênica , Vetores Genéticos/química , Vetores Genéticos/metabolismo , Instabilidade Genômica , Heterocromatina/metabolismo , Histonas/metabolismo , Humanos , Lentivirus/genética , Lentivirus/metabolismo , Camundongos , Camundongos Transgênicos , Osteoblastos/citologia , Fosforilação , Cultura Primária de Células , Regiões Promotoras Genéticas , RNA Longo não Codificante/metabolismo , RNA Interferente Pequeno/genética , RNA Interferente Pequeno/metabolismo , Proteína do Retinoblastoma/antagonistas & inibidores , Proteína do Retinoblastoma/metabolismo , Telômero/ultraestrutura , Transcrição GênicaRESUMO
BACKGROUND: MiR-221/-222 are frequently overexpressed in breast cancer and are associated with increased malignancy. The specific modification of microRNAs (miRNAs) expression could be a promising strategy in breast cancer therapy, leading to the suppression of tumourigenic processes in tumour cells. METHODS: MiR-221/-222 expressions were analysed in 86 breast cancer tissues by quantitative RT-PCR and tested for correlation with immunohistochemistry data and clinical follow-up. In vitro assays were conducted using human breast cancer cell lines with lentiviral overexpression of miR-221/-222. RESULTS: In tumour tissues, miR-221/-222 were associated with the occurrence of distant metastases. In particular, high levels of miR-221 were revealed to have a high prognostic impact for the identification of significantly different groups with advanced tumours. MiR-221/-222 overexpression strongly increased cell proliferation and invasion in vitro. Following miR-221/-222 overexpression an increased uPAR expression and cell invasion were observed. CONCLUSION: This study demonstrates a significant role for highly expressed miR-221/-222 in advanced breast cancers allowing for the identification of significantly different prognostic groups, particularly for HER2-positive and lymph-node-positive breast cancers. Considering that miR-221/-222 are strongly involved in cell invasion, these miRNAs may be promising markers for breast cancer prognosis and therapy.
Assuntos
Neoplasias da Mama/patologia , MicroRNAs/fisiologia , Idoso , Biomarcadores Tumorais/genética , Neoplasias da Mama/genética , Diagnóstico Diferencial , Progressão da Doença , Feminino , Regulação Neoplásica da Expressão Gênica/fisiologia , Células HEK293 , Humanos , Invasividade Neoplásica , Metástase Neoplásica , Estadiamento de Neoplasias , Prognóstico , Células Tumorais CultivadasRESUMO
Cyclin D1 overexpression is the hallmark of mantle cell lymphoma (MCL). However, the importance of cyclin D1 in the maintenance and progression of the disease remains to be defined. The aim of this study was to elucidate the role of cyclin D1 overexpression using an efficient cyclin D1-shRNA and a lentiviral system in well-characterized MCL cell lines. Surprisingly, the knockdown of cyclin D1 led to a moderate retardation in growth, without induction of apoptosis. The cyclin D1-shRNA-transduced MCL cells showed a 15% shift from S phase to G(1) phase of the cell cycle, a weak induction of p27(Kip1), decreased Rb (Ser807/811) phosphorylation, and a consistent upregulation of cyclin D2 mRNA and protein expression. However, double knockdown of cyclins D1 and D2 did not intensify the effects observed with cyclin D1 knockdown alone. These data suggest that the moderate effects of cyclin D1 downregulation on survival and proliferation are likely the result of compensatory cyclin-independent mechanisms governing proliferation or alternatively, secondary genetic events that make cyclin D1 dispensable. These findings have important implications for MCL therapy, as strategies targeting only cyclin D1 function might be hampered by compensatory regulatory mechanisms, resulting in a low probability of treatment response.
