Abstract
Double C2-like domain β (DOC2B) gene encodes for a calcium-binding protein, which is involved in neurotransmitter release, sorting, and exocytosis. We have identified the promoter region of the DOC2B gene as hypermethylated in pre-malignant, malignant cervical tissues, and cervical cancer cell lines by methylation-sensitive dimethyl sulfoxide-polymerase chain reaction and bisulfite genome sequencing; whereas, it was unmethylated in normal cervical tissues (p < 0.05). The promoter hypermethylation was inversely associated with mRNA expression in SiHa, CaSki, and HeLa cells and treatment with demethylating agent 5-aza-2-deoxycytidine restored DOC2B expression. The region -630 to +25 bp of the DOC2B gene showed robust promoter activity by a luciferase reporter assay and was inhibited by in vitro artificial methylation with Sss1 methylase prior to transient transfections. Overexpression of the DOC2B gene in SiHa cells when compared with controls showed significantly reduced colony formation, cell proliferation, induced cell cycle arrest, and repressed cell migration and invasion (p < 0.05). Ectopic expression of DOC2B resulted in anoikis-mediated cell death and repressed tumor growth in a nude mice xenograft model (p < 0.05). DOC2B expressing cells showed a significant increase in intracellular calcium level (p < 0.05), impaired AKT1 and ERK1/2 signaling, and induced actin cytoskeleton remodeling. Our results show that promoter hypermethylation and silencing of the DOC2B gene is an early and frequent event during cervical carcinogenesis and whose reduced expression due to DNA promoter methylation may lead to selective cervical tumor growth.
Highlights
DOC2B promoter hypermethylation is an early and frequent event in cervical cancer
The copy number variation (CNV) analysis was performed by searching in genomic variant database
A total of 10 fragments showed the characteristics of CpG islands and 6 fragments were found within the specific genes; whereas, the remaining fragments were found outside the gene coordinates
Summary
DOC2B promoter hypermethylation is an early and frequent event in cervical cancer. Results: DOC2B hypermethylation induces transcriptional repression, reactivated by demethylation; ectopic expression increases Ca2ϩ flux and inhibits key characteristics of tumorigenesis including proliferation, motility, and invasion. Significance: DNA methylation regulates DOC2B gene expression in cervical cancer. Our results show that promoter hypermethylation and silencing of the DOC2B gene is an early and frequent event during cervical carcinogenesis and whose reduced expression due to DNA promoter methylation may lead to selective cervical tumor growth. DOC2B is suggested to be involved in Ca2ϩ-dependent intracellular vesicle trafficking, ion and phospholipids binding, neurotransmitter release, and transporter activity (5, 6) It interacts with syntaxin binding protein 4 and MunC18c (7) leading to facilitation of exocytosis. We have demonstrated for the first time that DOC2B gene promoter hypermethylation as an early and frequent event in cervical cancer leads to down-regulation of its expression and subsequently to altered function in cervical cancer. Our data suggests DOC2B may act as a negative growth regulator due to its impact on several tumor-associated functions in cervical cancer
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