Effects of CpG sites methylation modification of HPV16 integration essential gene on the proliferation of cervical cancer cells

Purpose The mechanism of methylation of HPV CpG sites in the occurrence and prognosis of cervical carcinogenesis remains unclear. We investigated the effects of demethylation of the CpG sites of E2 and E6, essential genes of HPV16 integration, on cervical cancer cell expression, integration, and proliferation. Materials and Methods HPV16-positive (Caski) cells were treated with different concentrations of the demethylation compound 5-aza-dc (0, 5, 10, 20 μmol/l) in vitro. After the intervention, the methylation statuses of HPV16 E2 and E6 were detected by TBS, the expression levels of E2 and E6 mRNA and protein were detected by real-time PCR and western blot, cell proliferation activity was detected by CCK8, and cell cycle and apoptosis were determined by FCM. GraphPad Prism version 8.4.2 and R version 4.2.3 were used for relevant data analyses. Results The methylation levels of HPV16 E2 and E6 CpG sites decreased gradually with increasing 5-aza-dc intervention concentrations. With decreasing E2 and E6 methylation rates, E2 expression increased, the E2/E6 ratio increased, E6 expression decreased, and the growth inhibition rate of Caski cells increased. E2 and E6 expression were negatively and positively correlated with their degrees of methylation respectively, while the E2/E6 mRNA to protein ratio was negatively correlated with the methylation degrees of E2 and E6. Conclusion Demethylation can be used as a prospective treatment to affect HPV expression and persistent infection, providing a new theoretical basis for the clinical treatment of viral infections (AU)

Effects of CpG sites methylation modification of HPV16 integration essential gene on the proliferation of cervical cancer cells.

PURPOSE: The mechanism of methylation of HPV CpG sites in the occurrence and prognosis of cervical carcinogenesis remains unclear. We investigated the effects of demethylation of the CpG sites of E2 and E6, essential genes of HPV16 integration, on cervical cancer cell expression, integration, and proliferation. MATERIALS AND METHODS: HPV16-positive (Caski) cells were treated with different concentrations of the demethylation compound 5-aza-dc (0, 5, 10, 20 µmol/l) in vitro. After the intervention, the methylation statuses of HPV16 E2 and E6 were detected by TBS, the expression levels of E2 and E6 mRNA and protein were detected by real-time PCR and western blot, cell proliferation activity was detected by CCK8, and cell cycle and apoptosis were determined by FCM. GraphPad Prism version 8.4.2 and R version 4.2.3 were used for relevant data analyses. RESULTS: The methylation levels of HPV16 E2 and E6 CpG sites decreased gradually with increasing 5-aza-dc intervention concentrations. With decreasing E2 and E6 methylation rates, E2 expression increased, the E2/E6 ratio increased, E6 expression decreased, and the growth inhibition rate of Caski cells increased. E2 and E6 expression were negatively and positively correlated with their degrees of methylation respectively, while the E2/E6 mRNA to protein ratio was negatively correlated with the methylation degrees of E2 and E6. CONCLUSION: Demethylation can be used as a prospective treatment to affect HPV expression and persistent infection, providing a new theoretical basis for the clinical treatment of viral infections.