宫颈癌抑癌基因PTEN启动子区甲基化状态的研究

发布时间：2018-05-20 02:03

本文选题：PTEN + 宫颈癌　；参考：《青岛大学》2014年硕士论文

【摘要】：PTEN(磷酸酶和张力蛋白同源物)是一种具有双蛋白质和脂质磷酸酶活性的肿瘤抑制基因。CpG岛甲基化是导致抑癌基因失活的一种机制,近年来许多学者从PTEN基因启动子区甲基化方向着手研究PTEN基因在肿瘤组织中下调的机制。然而关于宫颈癌组织中PTEN基因启动子区的甲基化状态存在争议,因此宫颈癌组织中PTEN基因启动子区甲基化状态及甲基化对PTEN基因表达的影响有待进一步研究。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以及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例宫颈正常组织作为研究对象,采用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,应用实时荧光定量PCR检测药物处理前后PTEN基因表达差异。结果 1.4种宫颈癌细胞系PTEN基因启动子区呈低甲基化状态,HPV阳性细胞系(HeLa, Caski)与HPV阴性细胞系(C-33A,HT-3) PTEN基因启动子甲基化率无显著性差异(CpGl:t=2.83, P=0.11; CpG2:t=1.00, P=0.42); 2.宫颈癌组织以及正常宫颈组织中PTEN基因启动子区均呈低甲基化状态,且两者间PTEN基因启动子甲基化水平无明显差异(CpGl:T=83.50,P=0.15; CpG2;T=34.5,P=0.720)； 3. Real-time qPCR显示,4种宫颈癌细胞系5-Aza-dC处理前后PTEN基因mRNA表达无明显差异(HeLa:t=-1.18, P=0.36; Caski:t=2.07,P=0.18;C-33A:t=-0.07, P=0.95; HT-3:t=-0.53, P=0.65)。结论 1.BSP联合TA克隆测序结果分析结果显示宫颈癌细胞系中HPV感染与PTIN基因启动子区甲基化程度无明显相关性； 2.BSP联合TA克隆测序结果比较及统计学分析提示宫颈癌组织中PTEN基因启动子区甲基化不是导致该基因表达下调的主要原因； 3.抑癌基因启动子高度甲基化被认为是除突变和缺失以外抑癌基因功能失活的关键机制,但仅应用MSP定性分析抑癌基因的甲基化水平具有一定的局限性,抑癌基因启动子区域甲基化研究中同时采用BSP联合TA克隆测序可更为客观的反映其甲基化状态。
[Abstract]:PTEN (phosphatase and tension protein congener) is a tumor suppressor gene with double protein and lipid phosphatase activity. CpG island methylation is a mechanism leading to the inactivation of tumor suppressor gene. In recent years, many researchers have started to study the down-regulation of PTEN gene in tumor tissues from the direction of promoter methylation of PTEN gene. However, the methylation status of PTEN promoter region in cervical cancer tissues is controversial. Therefore, the methylation status of PTEN promoter region and the effect of methylation on the expression of PTEN gene in cervical cancer tissues need to be further studied. Purpose Study on methylation status and expression of PTEN gene promoter in cervical cancer cell line and cervical tissue Method Four cervical cancer cell lines, HeLa, Caski, C-33A, HT-3, 18 cervical cancer tissues and 8 normal cervix tissues were selected to detect the methylation status of PTEN gene promoter by BSP combined with TA clone sequencing. Four cervical cancer cell lines cultured in vitro were treated with demethylated drug 5-aza-dC. The difference of PTEN gene expression before and after drug treatment was detected by real-time fluorescence quantitative PCR. Result 1.4 there was no significant difference in the methylation rate of PTEN promoter between the HPV-positive cell line (HeLa, Caski) and the HPV negative cell line (CpGl2: t1. 83, P0. 11, CpG2: t1. 00, P0. 42), and the HPV negative cell line (CpG2: t1. 00, P0. 42, P0. 42, CpG2: t1. 00, P0. 42), CpG2: t1. 00, P0. 42, CpG2: 1. 2. The promoter region of PTEN gene in cervical cancer and normal cervix tissues was hypomethylated, and there was no significant difference in methylation level of PTEN gene promoter between them. 3. Real-time qPCR showed that there was no significant difference in mRNA expression of PTEN gene in four cervical cancer cell lines before and after 5-Aza-dC treatment. The expression of PTEN gene was 0.36; Caski: t0. 07; C33: T0. 07, P0. 05; HT-3: t0. 53, p0. 65. Conclusion The results of 1.BSP combined with TA clone sequencing showed that there was no significant correlation between HPV infection and methylation degree of PTIN gene promoter in cervical cancer cell line. The results of 2.BSP combined with TA clone sequencing and statistical analysis showed that methylation of the promoter region of PTEN gene was not the main cause of down-regulation of PTEN gene expression in cervical cancer tissues. 3. Hypermethylation of tumor suppressor gene promoter is considered to be the key mechanism for the functional inactivation of tumor suppressor gene except mutation and deletion. However, only qualitative analysis of the methylation level of tumor suppressor gene by MSP has some limitations. In the study of methylation of tumor suppressor gene promoter region, BSP combined with TA clone sequencing can more objectively reflect the methylation status of tumor suppressor gene.
【学位授予单位】：青岛大学
【学位级别】：硕士
【学位授予年份】：2014
【分类号】：R737.33

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