RESUMO
Tumor progression is driven by genetic mutations, but little is known about the environmental conditions that select for these mutations. Studying the transcriptomes of paired colorectal cancer cell lines that differed only in the mutational status of their KRAS or BRAF genes, we found that GLUT1, encoding glucose transporter-1, was one of three genes consistently up-regulated in cells with KRAS or BRAF mutations. The mutant cells exhibited enhanced glucose uptake and glycolysis and survived in low-glucose conditions, phenotypes that all required GLUT1 expression. In contrast, when cells with wild-type KRAS alleles were subjected to a low-glucose environment, very few cells survived. Most surviving cells expressed high levels of GLUT1, and 4% of these survivors had acquired KRAS mutations not present in their parents. The glycolysis inhibitor 3-bromopyruvate preferentially suppressed the growth of cells with KRAS or BRAF mutations. Together, these data suggest that glucose deprivation can drive the acquisition of KRAS pathway mutations in human tumors.
Assuntos
Neoplasias Colorretais/genética , Genes ras , Glucose/metabolismo , Mutação , Proteínas Proto-Oncogênicas B-raf/genética , Animais , Linhagem Celular Tumoral , Proliferação de Células , Neoplasias Colorretais/metabolismo , Regulação Neoplásica da Expressão Gênica , Marcação de Genes , Transportador de Glucose Tipo 1/genética , Transportador de Glucose Tipo 1/metabolismo , Glicólise/efeitos dos fármacos , Humanos , Ácido Láctico/metabolismo , Camundongos , Camundongos Nus , Transplante de Neoplasias , Análise de Sequência com Séries de Oligonucleotídeos , Piruvatos/farmacologia , Transplante HeterólogoRESUMO
MicroRNAs (miRNAs) are a class of small noncoding RNAs that have important regulatory roles in multicellular organisms. The public miRNA database contains 321 human miRNA sequences, 234 of which have been experimentally verified. To explore the possibility that additional miRNAs are present in the human genome, we have developed an experimental approach called miRNA serial analysis of gene expression (miRAGE) and used it to perform the largest experimental analysis of human miRNAs to date. Sequence analysis of 273,966 small RNA tags from human colorectal cells allowed us to identify 200 known mature miRNAs, 133 novel miRNA candidates, and 112 previously uncharacterized miRNA* forms. To aid in the evaluation of candidate miRNAs, we disrupted the Dicer locus in three human colorectal cancer cell lines and examined known and novel miRNAs in these cells. These studies suggest that the human genome contains many more miRNAs than currently identified and provide an approach for the large-scale experimental cloning of novel human miRNAs in human tissues.