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  • br Materials and methods br

    2020-10-15


    Materials and methods
    Acknowledgments This research was supported by funding from University of California Irvine to H.U.B.
    Introduction Oral squamous cell carcinoma (OSCC) is the fourth major cause of mortality among men in Taiwan and its incidence rates continue to rise annually. OSCC is closely associated with tobacco consumption and the chewing of betel nut (Areca catechu), which is widely available on the island. Two-thirds of OSCC patients in Taiwan seek medical treatment at an advanced stage. Despite some progress in OSCC treatments (surgery is still the primary treatment), the overall 5-year survival rate for OSCC patients is 50%, which has not significantly changed over the past 2 decades. It was estimated that 20–50% of patients without clinically evident cervical lymph node metastases do in fact have microscopic metastases, which lead to poor prognoses. It is, therefore, important to identify new diagnostic approaches and therapeutic targets for this deadly disease. Efforts to identify novel molecular predictors of behavior and therapeutic targets for OSCC have reported close relationships between several genetic and epigenetic alterations and the progressive development of OSCC.5, 6, 7, 8 Aberrant methylation of CpG islands in gene promoters is a common mechanism for suppressing gene expression in cancer cells. A process known as 9292 synthesis methylation can turn off the expressions of tumor-suppressor genes, just like a light switch, leading to oral cancer.9, 10, 11, 12, 13, 14 Silencing of cancer-associated genes by hypermethylation of CpG islands within the promoter and/or 5′-regions of many genes is a common feature of human cancer and is often associated with a transcriptional block and loss of the relevant protein, which is analogous to genetic loss-of-function mutations, such as point mutations and deletions.15, 16 Hypermethylation of the promoters of cancer-related genes is often associated with tumorigenesis and may also be involved in metastasis.17, 18, 19, 20, 21 Previous studies of OSCC reported the hypermethylation of several genes involved in DNA repair [O6-methylguanine-DNA-methyltranferase (MGMT)], tumor suppression (p16), detoxification of xenobiotics [glutathione S-transferase P (GSTP1)], and apoptosis-related gene [death-associated protein kinase (DAPK)].22, 23, 24 Although a consensus has emerged on hypermethylation, present studies disagree on methylation rates.24, 25, 26, 27, 28, 29, 30, 31, 32 Thus, two important questions are in order: to what degree does CpG island hypermethylation contribute to the genomic abnormalities in OSCC in Taiwan; and do hypermethylation events accumulate as cells progress toward metastasis? To detect the presence of neoplastic DNA methylation, we used a real-time quantitative methylation-specific polymerase chain reaction (MS-PCR) to analyze promoter hypermethylation rates of the p16, DAPK, MGMT, and GSTP1 genes in 64 OSCC tumors and corresponding adjacent normal mucosa. Twenty normal gingival mucosal samples obtained from healthy persons, who did not smoke or chew betel nut, served as a control group.
    Materials and methods
    Results
    Discussion Methylation is the main epigenetic modification in humans and changes in methylation patterns play important roles in tumorigenesis and metastasis. The MS-PCR technique enables precise mapping of methylation patterns in CpG islands of genomic DNA. This study found higher frequencies of overall hypermethylation in three genes (p16, MGMT, and DAPK) in primary tumors of oral cancer tissues (94%) than in adjacent tissues (70.3%). This result is similar to that of a study on Indian subjects, which found 87% of overall hypermethylation in the three genes of oral cancer tissues and 77% in adjacent tissues. Furthermore, our data showed that p16, DAPK, and MGMT hypermethylation rates in OSCC had CpG region frequencies of 67.2%, 45.3%, and 31.3% respectively, whereas p16, DAPK, and MGMT hypermethylation rates in adjacent tissues were 31.3%, 40.6%, and 25.0% respectively. The adjacent tissues represent clinically normal tissues with a high frequency of hypermethylation, which may indicate a molecular pathology, i.e., epigenetic aberrations and a relatively higher risk of progression toward malignant changes.