肝癌中NFAT2基因启动子区CpG岛甲基化状态的研究

发布时间：2018-01-04 18:21

本文关键词：肝癌中NFAT2基因启动子区CpG岛甲基化状态的研究　出处：《江苏大学》2017年硕士论文　论文类型：学位论文

【摘要】：目的肝细胞性肝癌(Hepatocellular carcinoma,HCC)的高患病率与低生存率严重危害着大众健康。在探寻肝癌的有效早期诊断和分子靶向治疗方面,对其致癌分子机制的了解不可或缺。有研究表明活化T细胞核因子(nuclear factor of activated T cells,NFAT)家族与肿瘤的发生与生长有关,其中活化T细胞核因子2(NFAT2)基因在肝癌组织与癌旁组织中表达差异最大,较癌旁组织,在癌组织中表达下降。本文即是从表观遗传学角度,探究肝细胞性肝癌(HCC)中NFAT2基因表达下降的机制,探索该基因启动子区CpG岛甲基化的状态。并进一步验证甲基化对NFAT2基因表达的影响。方法通过实时荧光定量聚合酶链反应(quantificational real-time PCR,qRT-PCR)检测组织标本NFAT2 mRNA的表达,通过蛋白质免疫印迹(Western Blot,WB)检测组织标本蛋白的表达,验证癌组织与癌旁组织中NFAT2的表达改变情况。通过重硫酸盐测序法(bisulfite sequencing PCR,BSP)研究肝癌组织与癌旁组织及不同肝细胞系NFAT2启动子的甲基化状态。通过甲基化酶抑制剂(5-氮杂-2’-脱氧胞苷,5-aza-dC)的细胞加药试验进一步探索NFAT2启动子区的甲基化状态及其对NFAT2表达的影响。结果肝癌组织中NFAT2 mRNA和蛋白水平低于癌旁组织(P0.05)。癌组织中NFAT2基因启动子CpG岛甲基化百分比高于癌旁组织(33.0%±13.9%VS.21.6%±8.3%)(P=0.003)。人肝癌细胞系(HuH7、HepG2、Hep3B)中NFAT2基因启动子CpG岛甲基化频率高于人正常肝细胞系(L02)(34.8%±7.3%,40.4%±10.3%and 37.0%±10.1%VS.16.2%±6.9%)(P0.01)。Spearman相关分析显示NFAT2 mRNA水平与其启动子甲基化程度呈负相关(r=-0.661,P=0.027)。添加甲基化酶抑制剂(5-aza-dC)的PLC细胞(实验组)NFAT2 mRNA和蛋白的表达高于添加二甲基亚枫(dimethyl sulfoxide,DMSO)的PLC细胞(对照组)(P0.05)。结论肝癌中NFAT2基因启动子区CpG岛处于高甲基化状态,并且该甲基化可能参与NFAT2表达降低的调控。
[Abstract]:Objective Hepatocellular carcinoma was used in hepatocellular carcinoma (HCC). The high prevalence rate and low survival rate of HCC seriously endanger public health. In order to explore the effective early diagnosis and molecular targeted therapy of liver cancer. It is necessary to understand the molecular mechanism of its carcinogenesis. Some studies have shown that nuclear factor of activated T cells can be activated. The expression of activated T cell nuclear factor 2 (NF-2NFAT2) gene in HCC and paracancerous tissues was higher than that in paracancerous tissues. From the perspective of epigenetics, we explore the mechanism of the decrease of NFAT2 gene expression in hepatocellular carcinoma (HCC). To explore the status of CpG island methylation in the promoter region of the gene, and to further verify the effect of methylation on the expression of NFAT2 gene. Quantificational real-time PCR. The expression of NFAT2 mRNA was detected by qRT-PCR, and the expression of protein was detected by Western blot. To verify the change of NFAT2 expression in cancer tissues and adjacent tissues, bisulfite sequencing PCR was detected by bisulfate sequencing. The methylation status of NFAT2 promoter in hepatocellular carcinoma tissues, paracancerous tissues and different liver cell lines was studied by means of 5-aza-2-deoxycytidine (deoxycytidine), a methylase inhibitor. 5-aza-dc). The methylation status of NFAT2 promoter and its effect on the expression of NFAT2 in HCC were further explored by cell addition assay. Results the levels of NFAT2 mRNA and protein in HCC tissues were lower than those in paracancerous tissues (P < 0.05). The percentage of CpG island methylation of NFAT2 promoter in cancer tissue was higher than that in paracancerous tissue (33.0% 卤13.9VS.21.6% 卤8.3%). Human hepatoma cell line HuH7. The methylation frequency of CpG island of NFAT2 gene promoter was higher than that of human normal liver cell line L02H3B (34.8% 卤7.3%). 40.4% 卤10.3 and 37.0% 卤10.1 vs 16.2% 卤6.9%. Spearman correlation analysis showed that the level of NFAT2 mRNA was negatively correlated with its promoter methylation. The expression of NFAT2 mRNA and protein in PLC cells supplemented with methylase inhibitor 5 aza-dC was higher than that in the experimental group (P < 0. 027). Dimethyl sulfoxide. Conclusion the CpG island in the promoter region of NFAT2 gene in HCC is hypermethylated. And this methylation may be involved in the regulation of NFAT2 expression reduction.
【学位授予单位】：江苏大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R735.7

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