RESUMEN
Epigenetic regulation of gene expression is well known mechanism that regulates cellular senescence of cancer cells. Here we show that inhibition of DNA methyltransferases (DNMTs) with 5-azacytidine (5-AzaC) or with specific small interfering RNA (siRNA) against DNMT1 and 3b induced the cellular senescence of human umbilical cord blood-derived multipotent stem cells (hUCB-MSCs) and increased p16(INK4A) and p21(CIP1/WAF1) expression. DNMT inhibition changed histone marks into the active forms and decreased the methylation of CpG islands in the p16(INK4A) and p21(CIP1/WAF1) promoter regions. Enrichment of EZH2, the key factor that methylates histone H3 lysine 9 and 27 residues, was decreased on the p16(INK4A) and p21(CIP1/WAF1) promoter regions. We found that DNMT inhibition decreased expression levels of Polycomb-group (PcG) proteins and increased expression of microRNAs (miRNAs), which target PcG proteins. Decreased CpG island methylation and increased levels of active histone marks at genomic regions encoding miRNAs were observed after 5-AzaC treatment. Taken together, DNMTs have a critical role in regulating the cellular senescence of hUCB-MSCs through controlling not only the DNA methylation status but also active/inactive histone marks at genomic regions of PcG-targeting miRNAs and p16(INK4A) and p21(CIP1/WAF1) promoter regions.
Asunto(s)
Senescencia Celular/genética , ADN (Citosina-5-)-Metiltransferasas/metabolismo , Proteínas de Unión al ADN/metabolismo , MicroARNs/genética , Proteínas Nucleares/metabolismo , Proteínas Proto-Oncogénicas/metabolismo , Proteínas Represoras/metabolismo , Factores de Transcripción/metabolismo , Azacitidina/farmacología , Western Blotting , Ciclo Celular/efectos de los fármacos , Ciclo Celular/genética , Proliferación Celular/efectos de los fármacos , Células Cultivadas , Senescencia Celular/efectos de los fármacos , Inmunoprecipitación de Cromatina , Islas de CpG/genética , ADN (Citosina-5-)-Metiltransferasa 1 , ADN (Citosina-5-)-Metiltransferasas/antagonistas & inhibidores , Proteínas de Unión al ADN/genética , Proteína Potenciadora del Homólogo Zeste 2 , Inhibidores Enzimáticos/farmacología , Citometría de Flujo , Humanos , Inmunohistoquímica , Células Madre Multipotentes/citología , Células Madre Multipotentes/metabolismo , Proteínas Nucleares/genética , Complejo Represivo Polycomb 1 , Complejo Represivo Polycomb 2 , Proteínas Proto-Oncogénicas/genética , Proteínas Represoras/genética , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Factores de Transcripción/genética , ADN Metiltransferasa 3BRESUMEN
The OCT4A gene, a POU homeodomain transcription factor, has been shown to be expressed in embryonic stem cells (ESC) as well as hUCB-MSCs. In this study, the roles played by OCT4A in hUCB-MSCs were determined by stably inhibiting OCT4A with lenti-viral vector-based small hairpin RNA (shRNA). A decreased rate of cell proliferation was observed in OCT4-inhibited hUCB-MSCs. Down-regulation of CCNA2 expression in OCT4-inhibited hUCB-MSCs was confirmed by RT-PCR and real-time RT-PCR analysis in three genetically independent hUCB-MSC clones. Adipogenic differentiation was also suppressed in OCT4-inhibited hUCB-MSCs. The up-regulation of DTX1 and down-regulation of HDAC1, 2, and 4 expressions may be related to this differentiation deformity. The expression of other transcription factors, including SOX2, REX1 and c-MYC, was also affected by OCT4 inhibition in hUCB-MSCs. In conclusion, these finding suggest that OCT4A performs functionally conserved roles in hUCB-MSCs, making its expression biologically important for ex vivo culture of hUCB-MSCs.