Cervical Cells HPV16 Virus DNA Methylation Correlation With Cervical Lesions

Background and Objectives:Cervial cancer is one of the most common gynecologic malignancy all over the world. Human papillomavirus(HPV) infection has a direct relationship with cervical cancer. Epigenetic events play an important role in the devolopment of cancer. We can find abnormal methylation patterns in almost all types of cancer and methylation involves the whole genome. This study is designed to analyze the methylation patterns of the11CpG dinucleotides within LCR enhancer and E6promoter. Supplied with clinical data, we try to define correlations between HPV16DNA methylation and pathology. The possible relation between specific methylation and cervical cancer is also discussed. This study will make contribution to explore further the mechanism of HPV16’s oncogenictity and find candidate biomarker for the progression of cervical neoplasia.Material and Methods:All clinical TCT samples were obtained from patients examined during regular gynecological diagnosis in PUMCH from Nov,2009to Jun,2010. We totally collected324TCT samples,244samples HPV infected. By multiple primers’ PCR,146samples were confirmed as single HPV type infected and98samples were mixed HPV types infected. Among them51samples were single HPV16infected. After the step of DNA extraction, we chose30samples in all to complete the research(26samples from single HPV16infected group and4samples from mixed HPV types infected group).Taking the clinical information into account, we divided them into four groups:6samples from asmptomatic patients,7samples from patients with LSIL,10samples from patients with HSIL,7samples from patients with squamous carcinomas. Moreover, SiHa cells were supplied from the cell center of CAMS. All carcinoma samples and part of HSIL samples were histologically confirmed by the pathological department of PUMCH.All DNA preparations were done with QIAamp DNA Mini Kit. Bisulfite modifications were done with EZ DNA Methylation-Gold Kit. The modified DNA was amplified in the form of two amplicons:LCR enhancer and E6promoter. The presence of PCR products was verified by agarose gel electrophoresis, and confirmed amplicons were cloned with pEASY-T1Cloning Vector. Five independent E.coli colonies derived from each sample were randomly selected for sequencing.Descriptive and tabular statistics were used to explore data. Graphical representations of CpG methylation frequency were estimated from the data. Additionally, the software SPSS13.0for windows was also used for data analysis. Fisher’s exact test was used to test the hypotheses that the methylation frequency of SCC group was greater than the methylation frequency of specimens from other groups. Student’s t Test was used to test the hypotheses that the observed methylation frequency at each site in each diagnostic outcome category was statistically significantly greater than zero.Results:1、We evaluated1013CpG sites in this study. Altogether, we found45methylated CpGs, i.e.,4.4%of all CpGs were methylated. Among all11CpG sites, no methylation was found at the positions7533,7551,7680and7860, about2%of the CpGs at the positions7674,7692were methylated, about8%of the CpGs at the positions31,37,43,52,58were methylated.2、Most methylations were contributed from SCC group. For SCC group, about8%of the CpGs at the positions7674were methylated, more than20%of the CpGs at the positions31,37,43,52,58were methylated.The observed methylation frequency at these sites in SCC category was statistically significantly greater than zero (Student’s t Test, P<0.001). And the methylation frequency of SCC group was significantly greater than the methylation frequency of HSIL group (Fisher’s exact test,P<0.01)3、Unfortunately, some of our samples did not contain sufficient DNA to complete this study. Though there is a nearly complete lack of methylation in LSIL/NORMAL groups, we did not find statistical significant difference between the methylation frequency of LSIL/NORMAL groups and other groups for statistical reasons.4、We sequenced5independent copies of SiHa cells. Methylated CpGs were found in the LCR Enhancer amplicon.Conclusions:1、Very few methylation was found in the LCR Enhancer amplicon. About8%of the CpGs in the E6promoter amplicon were methylated.2、Most methylations were contributed from SCC group. The methylation frequency of SCC group was significantly greater than the methylation frequency of HSIL group. 3、There is a nearly complete lack of methylation in LSIL/NORMAL groups.4、DNA methylation patterns in the HPV16genomes of the SiHa cell line is heterogeneous.