中文摘要：

背景与目的：抑癌基因Ras相关区域家族1A（Rasassociationdomainfamily1A，RASSFlA）启动子及第1外显子区cG位点高甲基化导致该基因沉默与多种恶性肿瘤的发生发展相关。本研究旨在探讨宫颈癌细胞系RASSFIA基因启动子及第1外显子区甲基化状态以及甲基化转移酶抑制剂5-氮杂-2’-脱氧胞苷（5-Aza-2’deoxycytidine，5-Azadc）作用对RASSFIA基因表达的影响。方法：采用5pmol／L（低浓度）和10pmol／L（高浓度）的5-Aza-dc作用于HeLa、Caski、HT-3以及C-33A等4种宫颈癌细胞系，分别采用甲基化特异PCR（methylation-specific PCR，MSP）和亚硫酸盐基因组测序法（bisulfite genome sequencing，BGS）检测5-Aza-dc处理前后RASSFlA基因启动子及第1外显子区甲基化状态，RT-PCR检测干预前后RASSFlA基因mRNA的转录表达。结果：HeLa和Caski两种HPV阳性细胞系RASSFlA基因启动子及第1外显子区均呈低甲基化状态，mRNA表达阳性。低浓度和高浓度5-Aza—dc作用后，mRNA表达未见明显改变（FHeLa=3．003，P=0．125；RaSki=0．045，P=0．956）。HT-3和C-33A两种HPV阴性宫颈癌细胞系RASSFlA基因启动子及第1外显子区则表现为高度甲基化状态，mRNA表达受到抑制。低浓度和高浓度5-Aza-dc作用后，HT-3和C-33A细胞系RASSFlA基因启动子及第1外显子区CG位点甲基化程度降低，检测到其mRNA表达，高浓度5-Aza-dc作用组表达水平明显高于低浓度组和细胞对照组（FHT-3=18．002，P=0．03；艮㈣=17．179，P=O．03），LSD-t检验显示差异有统计学意义（P〈O．05）。结论：HPV阳性和HPV阴性宫颈癌细胞系中RASSFIA基因启动子及第1外显子区甲基化状态不同；RASSFIA基因启动子及第1外显子区的高甲基化可抑制该基因表达；5-Aza-dc处理可使RASSFlA基因启动子及第1外显子区去甲基化，重新激活基因的表达，这种作用在一定范围内有剂量依赖性。

英文摘要：

Background and purpose： Loss or altered expression of Ras association domain family IA gene （RASSF1A） through DNA methylation has been associated with the pathogenesis of a variety of cancers, which suggests the tumor suppressor function of this gene. This study aimed to explore the effect of DNA methyltransferase inhibitor 5-Aza-2＇deoxycytidine （5-Aza-dc） on demethylation and expression of RASSF1A in cervical cancer cell lines. Methods： HPV positive cervical cancer cell lines HeLa and Caski, HPV negative cell lines HT-3 and C-33A were treated with two different concentration of 5-Aza-dc （5 ~tmol/L, 10 lxmol/L）. MSP （methylation-specific PCR） and Bisulfite genomic sequencing PCR （BGS） combined with TA clone were used to investigate methylation status of RASSF1A gene promoter and exon 1 before and after treatment of 5-Aza-dc. RASSFIA gene mRNA expressionwas detected by RT-PCR. Results： Two HPV positive cell lines showed hypomethylated RASSF1A promoters and expressed RASSF1A mRNA, and after treatment with 5-Aza-dc, the mRNA expression of RASSF1A did not change significantly （FHeLa=3.003, P=0.125; Fcaski=0.045, P=0.956）. Two HPV negative cervical cancer cell lines showed hypermethylation status of RASSF1A promoter and silenced RASSF1A. After treatment with 5-Aza-dc, demethylation occurred in the promoter region ofRASSF1A gene, which subsequently induced re-expression of this gene in HT-3 and C-33A. The F test （FHT-3= 18.002, P=0.03; Fc.33A= 17.179, P=0.03） and LSD-t test （P〈0.05） demonstrated that significant difference in the expression of RASSF1A was found upon two different concentrations drug treatment.Conclusion： The methylation status of promoter and exon 1 of RASSFIA gene in HPV positive and HPV negative cervical cancer cell lines are different. The promoter hypermethylation is correlated with RASSF1A gene expression in HPV negative cervical cancer cell line HT-3 and C-33A, and plays a key role in RASSF1A silencing. 5-Aza-dc may effectively reverse the methylation s