Microarray-Based Method for Quantificationally Detecting Methylation Changes of E-Cadherin Gene in Acute Leukemia.

Abstract

Aberrant DNA methylation of CpG sites is among the earliest and most frequent alterations in cancer. Several studies suggest that aberrant methylation of the CpG sites of the tumor suppressor gene is closely associated with carcinogenesis. So methylation detection is very important. The aim of this study was to describe a microarray-based method for quantificationally detecting changes of E-cadherin methylation in acute leukemia and to simply discuss the effect of microarray on detecting tumor methylation.This method used bisulfite-modified DNA as a template for PCR amplification, resulting in conversion of unmethylated cytosine, but not methylated cytosine, into thymine within CpG islands of interest. Five sets of oligonucleotide probes were designed to fabricate a DNA microarray to detect the methylation changes of E-cadherin gene CpG islands in acute leukemia. Each set contained a pair of methylated and unmethylated oligonucleotides for interrogating 3 or 4 CpG sites in close proximity. By TA cloning, PCR, sequencing, positive and negative DNA targets were obtained. A series of microarray hybridization were performed with mixtures of Cy3 labeled positive and negative DNA targets at different proportions. Next draw a standard curve by fluorescence intensity. Then leukemia samples DNA were abstracted and bisulfite-modified. Sample DNA targets were obtained by PCR amplification and were hybridized with the microarry. Finally the microarry was scanned with ScanArray Lite microarray analysis systems.Five linear relationships(R2=0.9660~0.9963) was established, which said the accuracy and reproducibility of the probes designed for microarray hybridization was good and could be used to eliminate background noise. The experimental results showed that the microarray assay could successfully detect five regions methylation changes of E-cadherin gene in every acute leukemia sample quantificationally. There were different degree of methylation in five acute leukemia samples and the hypermethylation region was the same. The test result was validated by gene sequencing.In tumors E-cadherin expression frequently is reduced, an event that contributes to tumor invasion and metastasis. The methylation of E-cadherin gene promoter is one important reason resulting in the silencing of expression. In normal peripheral blood mononuclear cells and bone marrow, E-cadherin is completely unmethylated. In our results, the methylation of E-cadherin is related to acute leukemia. And the same hypermethylation region may be the critical sites of methylation sites. As shown in this study, the use of a simple control system could test the accuracy and reproducibility of the probes designed for microarray hybridization. This control system can also be used to calibrate the levels of methylation changes detected in the investigated samples by microarray assay. With more and more methylated genes are found, microarray assay could be applied as a useful tool for mapping methylation changes in multiple CpG loci. It's more time-saving and more labor-saving than gene sequencing and can be readily used to high throughput analysis of DNA methylation. It will contribute significant information to our understanding of CpG island methylation in acute leukemia.