宫颈癌细胞系及宫颈组织PTEN基因启动子区甲基化状态的检测与分析

Detection and analysis of methylation status at the PTEN promotor in cervical cancer cell lines and cervical tissues

目的:探讨宫颈癌细胞系及宫颈组织中PTEN基因启动子甲基化状态对其表达的影响。方法:选取体外培养的4种宫颈癌细胞系HeLa、Caski、C-33A、HT-3,以及18例宫颈癌组织和8例宫颈正常组织为研究对象。应用重亚硫酸盐测序PCR(bisulfite genomic sequencing PCR,BSP)联合TA克隆测序检测PTEN基因启动子甲基化状态;采用去甲基化药物5-氮杂-2'-脱氧胞苷(5-Aza-dC)处理体外培养的4种宫颈癌细胞系,RT-PCR法检测处理前后PTEN基因mRNA转录表达的差异,并与本课题组前期5-Aza-dC作用前后4种细胞系表达谱芯片PTEN基因的差异进行比较分析。结果:4种宫颈癌细胞系、宫颈癌组织及正常宫颈组织中PTEN基因启动子区均呈低甲基化状态;且宫颈癌组织与正常宫颈组织PTEN基因启动子甲基化水平无显著差异(T=34.5,P=0.720)。表达谱芯片中4种细胞系PTEN基因差异倍数均>0.5且<2。RT-PCR显示,5-Aza-dC处理前后4种细胞系中PTEN mRNA表达无显著差异(HeLa:t=0.384,P=0.738;Caski:t=0.073,P=0.949;C-33A:t=0.097,P=0.931;HT-3:t=0.073,P=0.542)。结论:宫颈癌组织及宫颈癌细胞系中未发现PTEN基因启动子区CpG岛的高甲基化,PTEN基因表达的差异与该基因启动子区甲基化无显著相关性。

Objective: To investigate methylation status at PTEN gene promoter and influence maybe caused by which on PTEN expression in cervical cancer cell lines and cervical tissues. Methods: Methylation status of PTEN gene promoter were investigated in four kinds of cervical cancer cell lines HeLa,Caski,C-33A,HT-3,18 cases of cervical cancer and 8 cases of normal cervical tissue throw bisulfite genomic sequencing PCR( BSP) combined with TA clone for sequencing. All cervical cancer cell lines were treated with 5-Aza-2'-deoxycytidine( 5-AzadC). Reverse transcription PCR( RT-PCR) were used to compare the changes of PTEN gene expression on before and after the treatment of 5-Aza-dC in cervical cell lines. And differences of PTEN gene expression were compared between the foregoing results of RT-PCR and previous Agilent Human Whole Genome Expression Profiling( 4 × 44K) chips detected by our group. Results: The CpG island of PTEN gene promoter showed hypomethylation in both 4 kinds of cancer cell lines and cervical tissues,what's more,methylation level had no significant differences of cervical cancer tissue and normal ones( T = 34. 5,P = 0. 720). Previous chip resultsshows PTEN gene fold difference was > 0. 5 and < 2,and RT-PCR results showed no significant difference between before and after the treatment of 5-Aza-dC in 4 kinds of cervical cell lines( HeLa: t =0. 384,P =0. 738; Caski: t =0. 073,P =0. 949; C-33A: t =0. 097,P =0. 931; HT-3: t = 0. 073,P = 0. 542),which was consistent with chip results. Conclusions: PTEN promoter hypermethylation is a rare event in cervical tissues and cervical cancer cell lines. On the other hand,expression differences of PTEN gene between cervical cancer tissues and normal ones have no significant correlation with promoter hypermethylation.