ABSTRACT
In addition to conventional cytology, liquid-based cytology (LBC) is also used for immunocytochemistry and gene analysis. However, an appropriate method to obtain high quality DNA for next-generation sequencing (NGS) using LBC specimens remains controversial. We determined the optimal conditions for fixation with an alcohol-based fixative for LBC and DNA extraction using cultured cancer cell lines and clinical specimens. The extracted DNA was processed for NGS after the DNA quality was confirmed based on the DNA concentration and degree of degradation. The optimal conditions for cultured cells to obtain high quality DNA were to fix the cells at a density of 6 × 103 or 2 × 104 cells/mL and to use the magnetic bead-based DNA extraction method. Even after storing the fixed cells for 90 days, DNA extracted using the above and other extraction kits, including membrane-based methods, did not undergo degradation. Furthermore, 5-year-old residual LBC samples demonstrated high DNA quality that was suitable for NGS. Furthermore, a cancer genome panel analysis was successfully performed with DNA extracted from cultured cells fixed at 6 × 103 cells/mL for 90 days, and with DNA from residual LBC samples even after 1 year of storage. Residual LBC samples may be a useful source of DNA for clinical NGS to promote genome-based cancer medicine.
Subject(s)
Cytodiagnosis/methods , Genes, Neoplasm/genetics , Genetic Testing/methods , Neoplasms/diagnosis , Specimen Handling/methods , Cell Line, Tumor , DNA/genetics , High-Throughput Nucleotide Sequencing/methods , Humans , MCF-7 Cells , Neoplasms/geneticsABSTRACT
BACKGROUND AND PURPOSE: High de novo expression of MUC5AC (a gastric-type secreted mucin) is observed in many types of pancreatobiliary neoplasms, including precursor lesions. In this study, we show that the DNA methylation pattern is intimately correlated with MUC5AC expression in ten cancer cell lines (breast, lung, pancreas, and colon). METHODS: The CpG methylation status of the MUC5AC promoter from -3855 to +321 was mapped using MassARRAY analysis, which utilizes base-specific cleavage of nucleic acids. ChIP assays and micro-RNA (miRNA) microarray expression profiling were also carried out in both MUC5AC-positive cells and in those with no or low MUC5AC expression. RESULTS: In the distal region from -3718 to -3670 of the promoter, MUC5AC-negative cancer cells (e.g., MDA-MB-453) were highly methylated, whereas MUC5AC-positive cells (e.g., MCF-7) had low methylation levels. The modification status of histone H3 lysine 9 (H3-K9) was also closely related to MUC5AC expression. Expression levels of miRNAs in the cancer cells were not correlated with MUC5AC expression. CONCLUSION: Our results indicate that MUC5AC is regulated by CpG methylation and histone H3-K9 modification of the MUC5AC promoter distal region, but not by miRNAs. An understanding of the epigenetic regulation of MUC5AC may be of importance for the diagnosis of carcinogenic risk in the pancreatobiliary system.
Subject(s)
Biliary Tract Neoplasms/genetics , Gene Expression Regulation, Neoplastic , Mucin 5AC/genetics , Pancreatic Neoplasms/genetics , Promoter Regions, Genetic , RNA, Neoplasm/genetics , Biliary Tract Neoplasms/metabolism , Biliary Tract Neoplasms/pathology , Cell Line, Tumor , DNA Methylation , Genetic Predisposition to Disease , Humans , Immunohistochemistry , Mucin 5AC/biosynthesis , Pancreatic Neoplasms/metabolism , Pancreatic Neoplasms/pathology , Reverse Transcriptase Polymerase Chain Reaction , Time FactorsABSTRACT
MUC1 is a transmembrane mucin that is highly expressed in various cancers and correlates with malignant potential. Important cancer-related genes such as p16 and E-cadherin are controlled epigenetically; however, MUC1 has been overlooked in epigenetics. Herein, we provide the first report that MUC1 gene expression is regulated by DNA methylation and histone H3 lysine 9 (H3-K9) modification of the MUC1 promoter. The recently developed MassARRAY assay was performed to investigate the DNA methylation status of 184 CpG sites from -2,753 to +263. Near the transcriptional start site, the DNA methylation level of MUC1-negative cancer cell lines (e.g., MDA-MB-453) was high, whereas that of MUC1-positive cell lines (e.g., MCF-7) was low. Histone H3-K9 modification status was also closely related to MUC1 gene expression. Furthermore, MUC1 mRNA expression in MUC1-negative cells was restored by treatment with the DNA methylation inhibitor 5-aza-2'-deoxycytidine. Our results indicate that DNA methylation and histone H3-K9 modification in the 5' flanking region play a critical role in MUC1 gene expression, and this study defines MUC1 as a new member of the class of epigenetically controlled genes. An understanding of the epigenetic changes of MUC1 may be of importance for diagnosis of carcinogenic risk and prediction of outcome for cancer patients.
