Analysis Of The Status Of RASSF2A Gene Promoter Methylation In Cervical Cancer And The Clinical Significance Of The RASSF2A Gene Promoter Methylation

BackgroundCervical cancer is one of the most common human malignancies in terms of incidence and mortality rates. Human papillomavirus infection is the most significant risk factor in the etiology, and more than90%of cervical squamous carcinomas and50%of adenocarcinomas contains HPV DNA. The presence of HPV in nearly all cervical cancer implies the highest worldwide attributable fraction so far reported for a specific reason of any major human malignancy. However, HPV infection is necessary but insufficient to cause cervical cancer. Epidemiological studies have suggested that malignant transformation is a rare consequence of infection, indicating other unidentified genetic alterations are also involved. Although incidence and mortality are decreasing owing to the implementation of routine screening in early stage, cervical cancer still remains the second most common malignancy in women worldwide. The overall survival rate was still no significant improvement and the5-year survival rate of cervical cancer in stage Ⅰ is beyond80%, while stage II is50%-60%, stage Ⅲ is25%-40%and stage Ⅳ is5%-15%. The exact mechanism of cervical cancer development has not been fully elucidated, and the treatment effect of it is needed to be improved. Therefore, looking for the molecular markers related to development and progression of cervical cancer and target molecules is the problem that we need to solve urgently.For the past few years, with the development of molecular biology research, the importance of epigenetic change in the carcinogenesis has been realized. Such mechanisms may include DNA methylation, histone modification, genomic imprinting, maternal effects and RNA editing. DNA methylation plays an important role in tumor generation and development. Aberrant methylaion of CpG islands at the promoter region of a gene is an epigenetic change that induces the transcriptional silencing of tumor suppressor genes.RAS proteins plays a key role in controlling the activity of several essential signaling pathways, such as Rho GTPases, and MAPK, which regulate cell proliferation, morphology and apoptosis. Alteration of RAS signaling can result in tumorigenesis. During the past few years, a group of proteins with RAS binding domains were designated as negative effectors. They were designated as the RAS association family which currently includes10members. So far, nine members of RASSF gene family have been shown to be inactivated or downregulated in human neoplasms. RASSF2A as a tumor suppressor gene is frequently inactivated following methylation of its promoter in colorectal, gastric, pancreatic ductal and nasopharyngeal cancers. Still, the precise role of RASSF2A in cervical cancer remains unclear.In this study, we investigated these epigenetic alterations in cervical cancer, focusing on DNA methylation of RASSF2A. We analyzed the frequency of methylation and explore whether the methylation status of RASSF2A was associated with the loss of RASSF2A expression in cervical cancer. We further evaluated the correlation between RASSF2A methylation in cervical cancer and patients’clinicopathological characteristics. The cervical cancer cell lines also were treated with the methyltransferse inhibitor5-aza-2’-deoxycytidine (5-aza-dC) to investigate the promoter methylation status of the RASSF2A gene in carcinogenesis of cervical cancer. Part One Analysis of the status of RASSF2A gene promoter methylation in cervical cancer and its correlation with pathological featuresObjectiveTo investigate the expression and promoter hypermethylation status of RASSF2A gene in cervical cancer. To explore the correlation between the expression silence of RASSF2A gene and promoter hypermethylation.MethodsRT-PCR was used to examine the expression of RASSF2A in46cervical cancer tissue specimens,38cervical intraepithelial neoplasis (CIN) tissue specimens and25normal controls. The promoter methylation of RASSF2A was evaluated with MSP method.Results1.25of25(100%) normal cervical tissues were positive for RASSF2A mRNA, and27of38(71%) were detected in CIN tissues. Among46cervical cancer samples analyzed, only16of46(35%) were positive for RASSF2A. The positive rate of RASSF2A expression in cervical cancer was significantly lower than that in CIN (x2=10.96, P<0.01). The positive rate of RASSF2A expression in CIN was significantly lower than that in normal cervical samples (x2=8.77, P<0.01), and The positive rate of RASSF2A expression in CINII-III was significantly lower than that in CINI (x2=9.031, P<0.01)2.Twenty-five cases was detected RASSF2A promoter hypermethylation among46cases (56.5%) of cervical cancer tissues. But only seven cases were found methylation status in38(18.4%) CIN and no methylation status in normal cervical cases, which was distinctly different from the promoter hypermethylation status of cervical cancer (x2=12.67,22.30, P<0.01). The positive rate of RASSF2A methylation in CIN was significantly higher than that in normal cervical samples(x2=5.181, P<0.05), and The positive rate of RASSF2A methylation in CINI was significantly lower than that in CINII-Ⅲ (x2=5.01, P<0.05)3.The positive for RASSF2A expression in cervical cancer cases without methylation (57.1%) was significantly higher than that of cervical cancer tissues with promoter hypermethylation(16%)(x2=8.52, p<0.01)4.In our study, the positive lymph node metastasis exhibited an increased promoter methylation frequency for RASSF2A (x2=5.828, p<0.05). There were no significant associations between the methylation frequencies for RASSF2A gene to age, clinical stage, histological grade and types of pathology.ConclusionRASSF2A was absent or slight expression in cervical cancer tissues. It indicated RASSF2A play a role as suppressor gene in cervical cancer. RASSF2A gene promoter methylation was a frequent epigenetic event in cervical cancer and had a significant correlation with oncogenesis and lymph node metastasis. Its methylation may be used as a potential epigenetic biomarker for diagnosis and prognosis in cervical cancer. Part Two Analysis of the status of RASSF2A gene promoter methylation in cervical cancer cell linesObjectiveTo detect promoter hypermethylation and expression of RASSF2A in cervical cancer cells (HeLa, CaSki, SiHa and C33A) with MSP after5-aza-dC treatment.MethodsRT-PCR was used to examine the expression of RASSF2A in cervical cancer cells before and after5-aza-dC treatment. The promoter methylation of RASSF2A was detected with MSP methods, then we detect the changes of biological behavior of cervical cancer cells with MTT and flow cytometry.Results1.Almost complete RASSF2A hypermethylation were found in HeLa and SiHa cells. Partial methylation detected in CaSki and C33A cells.2.After5-aza-dC treatment, the relative expression of RASSF2A mRNA and protein increased significantly in cervical cancer cells.3.The HeLa cells were treated with5-aza-dC to demethylation, the cell proliferation capacity was decreased, and the apoptosis rate was increased.ConclusionThe high methylation status of the RASSF2A gene promoter in cells suggests that RASSF2A gene promoter hypermethylation status may play an important role in the molecular mechanisms of the occurrence and development in cervical cancer. It also indicates that the RASSF2A gene promoter methylation may be associated with low expression of RASSF2A. RASSF2A might be a potential therapeutic target for the treatment of cervical cancer.