Abstract：
OBJECTIVE: 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.
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.
CONCLUSIONS: 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.
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.
CONCLUSIONS: 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.
摘要：
目的：HPVCpG位点甲基化在宫颈癌发生及预后中的作用机制尚不清楚。我们研究了E2和E6的CpG位点去甲基化的影响，这是HPV16整合的必需基因，对宫颈癌细胞的表达，一体化,和扩散。
方法:体外用不同浓度的去甲基化化合物5-氮杂-dc(0、5、10、20μmol/l)处理HPV16阳性(Caski)细胞。干预之后,通过TBS检测HPV16E2和E6的甲基化状态，实时荧光定量PCR和Westernblot检测E2和E6mRNA和蛋白的表达水平,CCK8检测细胞增殖活性,FCM检测细胞周期和凋亡。GraphPadPrism版本8.4.2和R版本4.2.3用于相关数据分析。
结果：随着5-aza-dc干预浓度的增加，HPV16E2和E6CpG位点的甲基化水平逐渐降低。随着E2和E6甲基化率的降低，E2表达增加，E2/E6比值增加，E6表达降低，Caski细胞的生长抑制率增加。E2和E6的表达分别与其甲基化程度呈负相关和正相关。E2/E6mRNA与蛋白的比值与E2和E6的甲基化程度呈负相关。
结论：去甲基化可作为影响HPV表达和持续感染的前瞻性治疗方法。为病毒感染的临床治疗提供了新的理论依据。
方法:体外用不同浓度的去甲基化化合物5-氮杂-dc(0、5、10、20μmol/l)处理HPV16阳性(Caski)细胞。干预之后,通过TBS检测HPV16E2和E6的甲基化状态，实时荧光定量PCR和Westernblot检测E2和E6mRNA和蛋白的表达水平,CCK8检测细胞增殖活性,FCM检测细胞周期和凋亡。GraphPadPrism版本8.4.2和R版本4.2.3用于相关数据分析。
结果：随着5-aza-dc干预浓度的增加，HPV16E2和E6CpG位点的甲基化水平逐渐降低。随着E2和E6甲基化率的降低，E2表达增加，E2/E6比值增加，E6表达降低，Caski细胞的生长抑制率增加。E2和E6的表达分别与其甲基化程度呈负相关和正相关。E2/E6mRNA与蛋白的比值与E2和E6的甲基化程度呈负相关。
结论：去甲基化可作为影响HPV表达和持续感染的前瞻性治疗方法。为病毒感染的临床治疗提供了新的理论依据。
