DNA 损伤诱导的 45α 基因过表达通过启动子低甲基化促进食管鳞状细胞癌。

Overexpression of DNA damage-induced 45 α gene contributes to esophageal squamous cell cancer by promoter hypomethylation.

机构信息

Department of Cardiothoracic Surgery, Second Xiangya Hospital of Central South University, Changsha, Hunan, PR China.

出版信息

J Exp Clin Cancer Res. 2012 Feb 8;31(1):11. doi: 10.1186/1756-9966-31-11.

BACKGROUND

Environmental factors-induced dysfunction of esophageal squamous epithelium, including genomic DNA impairment and apoptosis, play an important role in the pathogenesis of esophageal squamous cell cancer. DNA damage-induced 45α (GADD45α) has been found promoting DNA repair and removing methylation marker, Therefore, in this study we will investigate whether GADD45α expression is induced and its mechanism in esophageal squamous cell cancer.

METHODS

Two human esophageal squamous cell lines (ESCC), ECA109 and KYSE510 were cultured in RPMI-1640 medium supplemented with 10% fetal bovine serum (FBS). Lipofectamine 2000 was used to transfect cells. mRNA level of GADD45α was measured by reverse transcription-quantitive PCR (RT-qPCR), protein level of GADD45α was detected by western blot and Immunohistochemistry. Global DNA methylation of tissue sample was measured using the Methylamp Global DNA Methylation Quantification Ultra kit (Epigentek Group) and promoter methylation was measured by bisulfite sequencing.

RESULTS

GADD45a mRNA and protein levels were increased significantly in tumor tissue than that in adjacent normal tissue. Hypomethylation of global genomic DNA and GADD45α promoter were found in ESCC. The cell sensitivity to Cisplatin DDP was decreased significantly in Eca109 and Kyse510 cells, in which GADD45α expression was down-regulated by RNA interference (RNAi). In addition, silence of GADD45a expression in ESCC cells inhibited proliferation and promoted apoptosis.

CONCLUSION

Overexpression of GADD45α gene is due to DNA hypomethylation in ESCC. GADD45α may be a protective factor in DDP chemotherapy for esophageal squamous cell carcinoma.

背景

环境因素诱导的食管鳞状上皮功能障碍，包括基因组 DNA 损伤和细胞凋亡，在食管鳞状细胞癌的发病机制中起重要作用。已发现 DNA 损伤诱导的 45α（GADD45α）促进 DNA 修复并去除甲基化标记物，因此，在本研究中，我们将研究 GADD45α 是否在食管鳞状细胞癌中表达及其机制。

方法

培养人食管鳞状细胞系（ESCC）ECA109 和 KYSE510 细胞于添加 10%胎牛血清（FBS）的 RPMI-1640 培养基中。使用 Lipofectamine 2000 转染细胞。通过逆转录-定量 PCR（RT-qPCR）测量 GADD45α 的 mRNA 水平，通过 Western blot 和免疫组化检测 GADD45α 的蛋白水平。使用 Methylamp 全基因组 DNA 甲基化定量超试剂盒（Epigentek 集团）测量组织样品的全基因组 DNA 甲基化水平，并用亚硫酸氢盐测序法测量启动子甲基化。

结果

肿瘤组织中 GADD45a mRNA 和蛋白水平明显高于相邻正常组织。在 ESCC 中发现全基因组 DNA 和 GADD45α 启动子低甲基化。在 Eca109 和 Kyse510 细胞中，GADD45α 表达通过 RNA 干扰（RNAi）下调后，细胞对顺铂（DDP）的敏感性显著降低。此外，ESCC 细胞中 GADD45a 表达沉默抑制增殖并促进凋亡。