ABSTRACT
INTRODUCTION: The recent DNA methylation studies on cancers have revealed the necessity of profiling an entire human genome and not to restrict the profiling to specific regions of the human genome. It has been suggested that genome-wide DNA methylation analysis enables us to identify the genes that are regulated by DNA methylation in carcinogenesis. METHODS: So, we performed whole-genome DNA methylation analysis for human lung squamous cell carcinoma (SCC), which is strongly related with smoking. We also performed microarrays using 21 pairs of normal lung tissues and tumors from patients with SCC. By combining these data, 30 hypermethylated and down-regulated genes, and 22 hypomethylated and up-regulated genes were selected. The gene expression level and DNA methylation pattern were confirmed by semiquantitative reverse-transcriptase polymerase chain reaction and pyrosequencing, respectively. RESULTS: By these validations, we selected five hypermethylated and down-regulated genes and one hypomethylated and up-regulated gene. Moreover, these six genes were proven to be actually regulated by DNA methylation by confirming the recovery of their DNA methylation pattern and gene expression level using a demethylating agent. The DNA methylation pattern of the CYTL1 promoter region was significantly different between early and advanced stages of SCC. CONCLUSION: In conclusion, by combining the whole-genome DNA methylation pattern and the gene expression profile, we identified the six genes (CCDC37, CYTL1, CDO1, SLIT2, LMO3, and SERPINB5) that are regulated by DNA methylation, and we suggest their value as target molecules for further study of SCC.
Subject(s)
Carcinoma, Squamous Cell/genetics , DNA Methylation , Gene Expression Regulation, Neoplastic , Gene Expression , Lung Neoplasms/genetics , Adaptor Proteins, Signal Transducing/genetics , Adult , Aged , Aged, 80 and over , Blood Proteins/genetics , Carcinoma, Squamous Cell/pathology , Cysteine Dioxygenase/genetics , Cytokines/genetics , Down-Regulation , Gene Expression Profiling , Genome-Wide Association Study , Humans , Intercellular Signaling Peptides and Proteins/genetics , LIM Domain Proteins/genetics , Lung Neoplasms/pathology , Male , Microarray Analysis , Middle Aged , Neoplasm Staging , Nerve Tissue Proteins/genetics , Serpins/genetics , Up-RegulationABSTRACT
Tissue microdissection is appropriate for separating pure cells from heterogeneous tissues. Recently, we have focused on whole genome DNA methylation patterns of lung squamous cell carcinoma (SCC) and needed to obtain the appropriate counterpart cells of lung SCC. However, in some regions of human tissues, such as in bronchial epithelium, it is difficult to apply tissue microdissection as a means to isolate pure cells from a heterogenous mixture of cells. Accordingly, we developed the pop brush method to retrieve sufficient amounts of pure bronchial epithelium from gross lung specimens, and this method enables us to study epigenetic variations of lung SCC.
Subject(s)
Bronchi/pathology , Cell Separation/methods , Epithelial Cells/pathology , Specimen Handling/methods , Cell Line, Tumor , Humans , Microdissection , Reproducibility of ResultsABSTRACT
To elucidate the proteins related to the development and progress of esophageal squamous cell carcinoma (ESCC), we performed gene expression profile analysis of 14 ESCC tissues. We identified 182 genes that were commonly upregulated (p < 10(-5)) and 54 genes that were downregulated in ESCC tissues (p < 10(-6)). In order to validate the gene expression profiles, we did semiquantitative RT-PCR analysis and found 11 genes upregulated in greater than 70% of 19 tissue samples. We further examined the protein expression level and the distribution patterns of four genes: aurora kinase B (AURKB), periostin (POSTN), heat shock protein 47 (HSP47), and matrix metalloprotease 1 (MMP1). Ultimately, three genes (AURKB, HSP47, POSTN) were verified to be increased at both the mRNA and protein levels. Among them, AURKB and HSP47 were found to increase in the early development stage of ESCC and POSTN might be considered to function in the cell-cell interactions between cancer cell and adjacent stromal cells. Accordingly, these studies may provide valuable information for the identification of the genes that are necessary to study further their functions. Moreover, these genes might be used as potential diagnostic or therapeutic target molecules for ESCC patients.
