RESUMEN
To investigate downstream molecular changes caused by mitogen-activated protein kinase (MEK) inhibitor treatment and further explore the impact of direct knockdown of early growth response-1 ( EGR1 ) in melanoma cell culture. RNA-sequencing (RNA-Seq) was performed to determine gene expression changes with MEK inhibitor treatment. Treatment with MEK inhibitor (trametinib) was then assessed in two cutaneous (MEL888, MEL624) and one conjunctival (YUARGE 13-3064) melanoma cell line. Direct knockdown of EGR1 was accomplished using lentiviral vectors containing shRNA. Cell viability was measured using PrestoBlueHS Cell Viability Reagent. Total RNA and protein were assessed by qPCR and SimpleWestern. RNA-Seq demonstrated a profound reduction in EGR1 with MEK inhibitor treatment, prompting further study of melanoma cell lines. Following trametinib treatment of melanoma cells, viability was reduced in both cutaneous (MEL888 26%, P â <â 0.01; MEL624 27%, P â <â 0.001) and conjunctival (YUARGE 13-3064 33%, P â <â 0.01) melanoma compared with DMSO control, with confirmed EGR1 knockdown to 0.04-, 0.01-, and 0.16-fold DMSO-treated levels (all P â <â 0.05) in MEL888, MEL624, and YUARGE 13-3064, respectively. Targeted EGR1 knockdown using shRNA reduced viability in both cutaneous (MEL624 78%, P â =â 0.05) and conjunctival melanoma (YUARGE-13-3064 67%, P â =â 0.02). RNA-Sequencing in MEK inhibitor-treated cells identified EGR1 as a candidate effector molecule of interest. In a malignant melanoma cell population, MEK inhibition reduced viability in both cutaneous and conjunctival melanoma with a profound downstream reduction in EGR1 expression. Targeted knockdown of EGR1 reduced both cutaneous and conjunctival melanoma cell viability independent of MEK inhibition, suggesting a key role for EGR1 in melanoma pathobiology.
Asunto(s)
Melanoma , Neoplasias Cutáneas , Humanos , Melanoma/tratamiento farmacológico , Melanoma/genética , Melanoma/metabolismo , Mitógenos , Dimetilsulfóxido , Neoplasias Cutáneas/tratamiento farmacológico , Neoplasias Cutáneas/genética , Neoplasias Cutáneas/patología , Quinasas MAP Reguladas por Señal Extracelular/metabolismo , Inhibidores de Proteínas Quinasas/farmacología , Quinasas de Proteína Quinasa Activadas por Mitógenos , ARN Interferente Pequeño , Línea Celular Tumoral , Proteínas Proto-Oncogénicas B-raf/genéticaRESUMEN
The laboratory rat (Rattus norvegicus) is an indispensable tool in experimental medicine and drug development, having made inestimable contributions to human health. We report here the genome sequence of the Brown Norway (BN) rat strain. The sequence represents a high-quality 'draft' covering over 90% of the genome. The BN rat sequence is the third complete mammalian genome to be deciphered, and three-way comparisons with the human and mouse genomes resolve details of mammalian evolution. This first comprehensive analysis includes genes and proteins and their relation to human disease, repeated sequences, comparative genome-wide studies of mammalian orthologous chromosomal regions and rearrangement breakpoints, reconstruction of ancestral karyotypes and the events leading to existing species, rates of variation, and lineage-specific and lineage-independent evolutionary events such as expansion of gene families, orthology relations and protein evolution.