title: HPV16 oncogenes E6 or/and E7 may influence the methylation status of RASSFIA gene promoter region in cervical cancer cell line HT-3.
abstract: Both human papillomavirus (HPV) infection and the aberrant Ras associated domain family gene 1A (RASSF1A) promoter methylation status participate in the pathogenesis of cervical cancer. Some studies suggest that E6, and E7 are involved in the pathogenetic mechanisms of RASSF1A. We mainly explored a possible involvement of HPV16 oncogenes E6 or/and E7 in RASSF1A promoter methylation status and possible roles of RASSF1A gene methylation in cervical cancer. Bisulfite genomic sequencing (BGS) PCR combined with TA clone, methylation-specific PCR (MSP) were used to analyze methylation status of the RASSF1A gene promoter in HPV16/18-positive and HPV-negative cervical cancer cell lines; ectopically expressed HPV16 E6, E7 and E6/E7 cervical cancer cell lines; normal cervical and cervical cancer tissues. The mRNA and protein expression of RASSF1A was detected by RT-PCR and western blotting. Re-expression and downregulated promoter methylation status were detected in the ectopically expressed HPV16 E6 and E7 cervical cancer cell line HT-3. The methylation status and expression of RASSF1A could be downregulated or reactivated by 5-Aza-dc in HT-3 and C33A cells. Additionally, statistics showed significant hypermethylation of RASSF1A in cervical cancer samples compared to that in normal cervical samples (P<0.05). The false negative rate (FNR) was 6.25% by HC2 method, when reconfirmed by HPV detection combining the MY09/11, GP5+/6+ and SPF1/2 methods. The ectopic expression of HPV16 E6 and/or E7 may be involved in aberrant methylation and expression of the RASSF1A gene. RASSF1A gene expression could be regulated by its promoter methylation status. Additionally, the false negativity of the HPV detection may contribute to the uncertain relationship between HPV infection and aberrant RASSF1A promoter methylation.
authors.affiliation: Department of Obstetrics and Gynecology, Peking University People's Hospital, Beijing 100044, P.R. China.
authors.forename: Fufen
authors.initials: F
authors.lastname: Yin
authors.affiliation: Department of Obstetrics and Gynecology, Affiliated Hospital of Qingdao University, Qingdao, Shandong 266000, P.R. China.
authors.forename: Ning
authors.initials: N
authors.lastname: Wang
authors.affiliation: Department of Obstetrics and Gynecology, People's Hospital of Huangdao District, Qingdao, Shandong 266000, P.R. China.
authors.forename: Shanshan
authors.initials: S
authors.lastname: Wang
authors.affiliation: Department of Obstetrics and Gynecology, Affiliated Hospital of Qingdao University, Qingdao, Shandong 266000, P.R. China.
authors.forename: Fengsheng
authors.initials: F
authors.lastname: Yu
authors.affiliation: Department of Obstetrics and Gynecology, Affiliated Hospital of Qingdao University, Qingdao, Shandong 266000, P.R. China.
authors.forename: Xin
authors.initials: X
authors.lastname: Sun
authors.affiliation: Department of Obstetrics and Gynecology, Affiliated Hospital of Qingdao University, Qingdao, Shandong 266000, P.R. China.
authors.forename: Xiao
authors.initials: X
authors.lastname: Yu
authors.affiliation: Department of Medical Microbiology, Qingdao University Medical College, Qingdao, Shandong 266000, P.R. China.
authors.forename: Bing
authors.initials: B
authors.lastname: Luo
authors.affiliation: Department of Pathology, Magee-Womens Hospital, University of Pittsburgh Medical Center, Pittsburgh, PA 15213-3180, USA.
authors.forename: Chengquan
authors.initials: C
authors.lastname: Zhao
authors.affiliation: Department of Obstetrics and Gynecology, Affiliated Hospital of Qingdao University, Qingdao, Shandong 266000, P.R. China.
authors.forename: Yankui
authors.initials: Y
authors.lastname: Wang
mesh_terms: D045744:Cell Line, Tumor; D019175:DNA Methylation; D015536:Down-Regulation; D044127:Epigenesis, Genetic; D005188:False Negative Reactions; D005260:Female; D015972:Gene Expression Regulation, Neoplastic; D052162:Human papillomavirus 16 / Q000235:genetics* / Q000378:metabolism / Q000472:pathogenicity; D006801:Humans; D009856:Oncogene Proteins, Viral / Q000235:genetics / Q000378:metabolism*; D050725:Papillomavirus E7 Proteins / Q000235:genetics*; D030361:Papillomavirus Infections / Q000235:genetics* / Q000378:metabolism / Q000473:pathology; D011401:Promoter Regions, Genetic; D012097:Repressor Proteins / Q000235:genetics / Q000378:metabolism*; D017422:Sequence Analysis, DNA; D025521:Tumor Suppressor Proteins / Q000235:genetics*; D002583:Uterine Cervical Neoplasms / Q000235:genetics / Q000378:metabolism / Q000473:pathology / Q000821:virology*
chemical_list: C068651:E6 protein, Human papillomavirus type 16; D009856:Oncogene Proteins, Viral; D050725:Papillomavirus E7 Proteins; C411762:RASSF1 protein, human; D012097:Repressor Proteins; D025521:Tumor Suppressor Proteins; C059731:oncogene protein E7, Human papillomavirus type 16