Methylation status of the long control region of HPV 16 in clinical cervical specimens

Abstract

DNA methylation is one of the regulatory pathways that modulate human papillomavirus (HPV) gene expression. To obtain detailed methylation information on crucial areas of the long control region (LCR) of HPV 16 and to clarify the significance of methylation in clinical cervical lesions, 80 clinical samples were examined to determine the methylation status of the HPV 16 promoter and enhancer core using bisulfite modification and pyrosequencing. Seventy samples [26 of cervical carcinoma (CC), 13 of cervical intraepithelia neoplasia (CIN) III, 17 of CIN I-II and 14 of asymptomatic HPV 16 infection] were successfully examined. Analysis of the general methylation status of HPV 16 LCR in the 70 clinical specimens revealed 43 (61.4%) with methylation in the promoter and/ or enhancer core of HPV 16. The proportion of methylated samples was highest in CC specimens (84.6%), followed by asymptomatic infection (71.4%) and CIN III (46.2%), while the proportion of methylated samples was lowest in CIN I-II specimens (29.4%). The methylation status of eight CpGs in HPV 16 LCR was determined in detail. In general, the methylation of CpGs was more common in the promoter than in the enhancer core region. The methylation frequencies of the eight CpGs ranged from 14.6±7.2 to 33.7±23.0% in individual methylated CpG cases. The methylation pattern of all eight CpGs methylated in the promoter and enhancer core was more common in CC, and the pattern of scattered methylated CpGs was relatively more prevalent in asymptomatic infections. Our study demonstrates that DNA methylation is a common phenomenon in HPV 16 LCR clinical specimens, and may function as a host defense mechanism. While hypomethylation is probably associated with the initiation of neoplasia, hyper-methylation in cervical cancer may be a reflection of the host defense mechanism. In the regulation of transcription, methylation is of more importance in the HPV 16 promoter than in the enhancer core.

Methylation of Human Papillomavirus Type 16 CpG Sites at E2-Binding Site 1 (E2BS1), E2BS2, and the Sp1-Binding Site in Cervical Cancer Samples as Determined by High-Resolution Melting Analysis–PCR

“…Some studies reported decreased E2BS methylation during lesion progression (30)(31)(32) or a trend of a lower risk of incident precancerous and cancerous lesions for patients with high E2BS methylation (33,34). On the contrary, methylation of the five CpGs located in the E2BS1 and E2BS2 and Sp1-binding site (Sp1BS) has frequently been associated with cervical cancer (28,29,(35)(36)(37)(38), and a gradual increase in methylation during lesion progression toward cancer has been reported in some studies (29,36,37,39). Nevertheless, most of these studies did not include the full spectrum of cervical disease progression from normal to cancer.…”

Increased methylation of human papillomavirus type 16 DNA is associated with the severity of cervical lesions in infected females from northeast China

“…The methylation proportions of cervical swab DNA samples at the 5 CpG sites (nt 31, 37, 43, 52 and 58) may aid evaluation of the incidence of cervical cancer. Among the DNA samples studied in the present study, an increased number of methylated CpG sites were identified in the HPV 16 promoter region, compared with the enhancer core, suggesting that the methylation of the HPV 16 promoter may be of increased importance, compared with that of the enhancer (21). CpG site 7,859, which is located in the origin of replication, exhibited no methylation in the DNA samples from patients with HSIL, hypomethylation in DNA samples from patients with LSIL (2.70%) and hypermethylation in DNA samples from patients with CC (24.32%).…”

“…A study examining the methylation patterns of 19 HPV 16 CpG site dinucleotides within the L1 gene and the long control region observed the frequency of methylation to be highest in CC and lowest in CINs (low and high grade) (37). In another study, the methylation status of 8 CpG sites in the HPV 16 promoter region and enhancer core were examined in clinical DNA samples using pyrosequencing, demonstrating that the proportion of methylated DNA samples was highest in CC, followed by asymptomatic infection and CIN3, and was lowest in CIN1/2 (21). The discrepancy of the methylation status between these results may be due to the methods utilized for methylation evaluation and the classification of cervical disease grade and differences between ethnic groups.…”

“…DNA methylation is an epigenetic mechanism that is able to inhibit gene expression directly by interfering with transcription factor binding, or indirectly by recruiting histone deacetylases through methyl-DNA-binding proteins (20,21). Previous studies have demonstrated that DNA methylation is also an important method of regulating the expression of the HPV 16 gene by blocking certain transcription factor binding sites, and is associated with the presence of cervical lesions (22,23).…”

“…The present study observed that the proportion of methylation in DNA samples at HPV 16 3'-L1 and the LCR increased as cervical lesions progressed. The increased methylation proportion in CC samples appears paradoxical, as the DNA methylation mechanism generally leads to transcriptional suppression (21). However, certain HPV genomes exist in the episomal, unmethylated form in cancer cells, which maintains the transformation of viral genomes (38).…”