Study On Methylation Status And Expression Of Rassfia Gene Promoter And Exon1in Cervical Carcinoma

High-risk human papillomaviruses (HR-HPVs) were the important etiology factor of cervical cancer. HPV viral oncoproteins E6and E7can bind to p53and retinoblastoma gene products and degrade them, respectively. Epigenetic modification including DNA methylation at cytosines can inactivate numerous tumor suppressor genes (TSG) and is now recognized as a major contributor to the development of cancer. It is an important mechanism that silence of tumor suppressor gene induced by methylation of its promoter. But how and where HPVs interact with host gene is not well understood.Background and purpose:The Ras associated domain family gene1A (RASSF1A) is one of tumor suppressor genes (TSGs) thought to inactivate because of hypermathylation in many major cancers. We sought to explore methylation status in cervical cancer and investigate the changes of methylation state and expression of RASSF1A gene in cervical cancer cell lines.Methods:Methylation-specific Polymerase Chain Reaction (MSP) and Bisulfite genome sequencing (BGS) in combination with TA clone were used to investigate methylation of RASSF1A in four cervical cancer cell lines (HeLa; CaSki; HT-3and C-33A) and a series of48primary cervical cancer tissues (45HPV-positive and3HPV-negative) and15cervical normal tissues. DNA-demethylating agent5-Aza-2-deoxycytidine (5-Aza-dc) was used to demethylate the RASSF1A promoter and the exon1in four cell lines. Real time-PCR was used to detect RASSF1A gene mRNA expression.Results:①The methylation status of RASSFIA gene promoter and exon1by MSP were U type in two HPV positive cervical carcinoma cell lines (HeLa and CaSki) and M type in two HPV negative cervical carcinoma cell lines.After treatment with5-Aza-dc, the methylation status change to M+U type in two HPV negative cell lines, whereas no change was found in two HPV positive cell lines.②For cervical tumors and cell lines combined, methylation rate of RASSFIA gene was9.6%(5/52),but was absent in all normal cervical tissues,there was no statistical significance(Fisher’s exact test; PΓ=0.2691); For cervical tumors and cell lines combined, Fisher’s exact test (P=0.00027) indicates a reverse correlation between hypermathylation of promoter of RASSFIA and the presence of HPV.③Detailed methylation analysis of each individual site in the RASSF1A CpG island by bisulfite genome sequencing (BGS). BGS was conducted in all cell lines and5samples of cervical cancer and1samples of normal control. Bisulfite genome sequencing analysis confirmed the MSP results. The area of the CpG-rich region around the transcription initiation site of RASSFIA gene between the nucleotides-271and434, which included64CpG sites. Only scattered methylated CpG sites were detected in HeLa and Caski and densely methylated CpG sites in the RASSF1A promoter were found in C-33A and HT-3cell lines, after treatment with5-Aza-dc, demethylation occurred in the promoter region of RASSF1A gene.④There was no significant difference about RASSFIA expression between cervical cancer tissues and normal cervical tissues(t=0.686, P=0.069), and there was no difference between HPV positive and HPV negative cervial cancer samples(t=0.085, P=0.756).⑤F test (FHT-3=85.598, P=0.00; FC-33A=12.476, P=0.00) and LSD-t test (P＜0.05) demonstrate that significant difference in the expression of RASSFIA is found upon two different concentration drug treatment. As a control, no change in the expression of RASSFIA was observed in Caski cell line (FCaski=1.08, P=0.398).Conclusion:①Reverse correlation between hypermathylation of promoter of RASSFIA and the presence of HPV in cervial cancer tissues and cell lines.②The promoter hypermethylation is correlated with RASSFIA gene expression in HPV negative cervical cancer cell line HT-3and C-33A, and plays a key role in RASSFIA silencing.5-Aza-dc may effectively reverse the methylation status of RASSFIA gene promoter in cervical cancer HT-3and C-33A cells and reactivated gene expression silenced by aberrant hypermethylation.③Hypermethylation of RASSFIA occurs only in a subset of cervical cancers. We found no methylation RASSFIA promoter in HPV-positive squamous cell carcinomas and normal tissues.④RASSFIA gene may not involved in the development of cervical cancer through its promoter and exon methylation, it is not entirely clear the pathways and mechanisms which RASSFIA act in the process and this need further study.