RESUMEN
Quantifying levels of DNA methylation in tumors is a useful approach for the identification of potential tumor suppressors and to find biomarkers that can be used as prognostic or therapeutic indicators. In the current study, we compared three methods commonly used for quantifying DNA methylation-bisulfite pyrosequencing, quantitative methylation-specific PCR (Q-MSP), and MethyLight-by focusing on the CpG island of the gene encoding the microRNA-34b and microRNA-34c (miR-34b/c); aberrant regulation of this miR is associated with various human malignancies, including gastric cancer. Standard curve analysis using control DNA samples demonstrated the highest quantitative accuracy in Q-MSP analysis. We also carried out methylation analysis using gastric mucosa specimens obtained from gastric cancer patients. We found a high correlation between methylation levels determined by Q-MSP and those determined by MethyLight (R(2)=0.952), whereas the results of bisulfite pyrosequencing and the other two methods were less well correlated (R(2)=0.864 and R(2)=0.804 for Q-MSP and MethyLight, respectively). This may reflect possible PCR bias in the pyrosequencing technique, which we show can be corrected for by applying a cubic approximate equation to the original data. Thus, although results obtained by the different DNA methylation analysis techniques are largely comparable, an appropriate correction may be necessary for stringent comparison.
Asunto(s)
Metilación de ADN , MicroARNs/genética , Reacción en Cadena de la Polimerasa/métodos , Análisis de Secuencia de ADN/métodos , Neoplasias Gástricas/genética , Anciano , Anciano de 80 o más Años , Islas de CpG/genética , Femenino , Predisposición Genética a la Enfermedad , Humanos , Masculino , Persona de Mediana Edad , Pronóstico , Regiones Promotoras Genéticas/genética , Neoplasias Gástricas/diagnóstico , Sulfitos/farmacologíaRESUMEN
Cancer cells secrete small membranous extracellular vesicles (EVs) into their microenvironment and circulation. Although their potential as cancer biomarkers has been promising, the identification and quantification of EVs in clinical samples remains challenging. Here we describe a sensitive and rapid analytical technique for profiling circulating EVs directly from blood samples of patients with colorectal cancer. EVs are captured by two types of antibodies and are detected by photosensitizer-beads, which enables us to detect cancer-derived EVs without a purification step. We also show that circulating EVs can be used for detection of colorectal cancer using the antigen CD147, which is embedded in cancer-linked EVs. This work describes a new liquid biopsy technique to sensitively detect disease-specific circulating EVs and provides perspectives in translational medicine from the standpoint of diagnosis and therapy.
Asunto(s)
Biomarcadores de Tumor/sangre , Neoplasias Colorrectales/sangre , Vesículas Secretoras/metabolismo , Basigina/metabolismo , Biomarcadores de Tumor/metabolismo , Estudios de Casos y Controles , Neoplasias Colorrectales/metabolismo , Ensayo de Inmunoadsorción Enzimática , Espacio Extracelular , Células HT29 , Humanos , Immunoblotting , Tetraspanina 28/metabolismo , Tetraspanina 29/metabolismo , Tetraspanina 30/metabolismoRESUMEN
DNA methylation is an epigenetic mark crucial in regulation of gene expression. Aberrant DNA methylation causes silencing of tumor suppressor genes and promotes chromosomal instability in human cancers. Most of previous studies for DNA methylation have focused on limited genomic regions, such as selected genes or promoter CpG islands (CGIs) containing recognition sites of methylation-sensitive restriction enzymes. Here, we describe a method for high-resolution analysis of DNA methylation using oligonucleotide tiling arrays. The input material is methylated DNA immunoprecipitated with anti-methylcytosine antibodies. We examined the ENCODE region ( approximately 1% of human genome) in three human colorectal cancer cell lines and identified over 700 candidate methylated sites (CMS), where 24 of 25 CMS selected randomly were subsequently verified by bisulfite sequencing. CMS were enriched in the 5' regulatory regions and the 3' regions of genes. We also compared DNA methylation patterns with histone H3 and H4 acetylation patterns in the HOXA cluster region. Our analysis revealed no acetylated histones in the hypermethylated region, demonstrating reciprocal relationship between DNA methylation and histone H3 and H4 acetylation. Our method recognizes DNA methylation with little bias by genomic location and, therefore, is useful for comprehensive high-resolution analysis of DNA methylation providing new findings in the epigenomics.
