Department of Biochemistry, College of Natural Sciences, Chungnam National University, Daejeon, Republic of Korea.
Mol Carcinog. 2010 Mar;49(3):259-70. doi: 10.1002/mc.20596.
To clarify the regulatory mechanism of GW112 gene expression, 5'-flanking region of the human GW112 gene was isolated and characterized in the present study. 5'-RACE analysis showed a single transcription start site, which is located 142 nucleotides upstream of the translation initiation site. Transient transfection studies with serial deletion constructs and close examination of the sequences identified a putative NF kappaB binding sequence between -442 and -430, which could be responsible for efficient expression of the GW112 gene. Indeed, GW112 gene was found to be regulated by NF kappaB signals including overexpressed p65 and I kappaB alpha, IKK inhibitor, and proteasome inhibitor. Binding of NF kappaB to its putative site was confirmed by EMSA and ChIP assays. These results suggest that NF kappaB is an essential regulatory factor for GW112 transcription. Based on this finding, we next confirmed that inhibition of GW112 expression could induce apoptosis in the presence of cytotoxic agent in gastric cancer cells. Furthermore, knocking-down or overexpression of GW112 gene in gastric cancer cells demonstrated that GW112 has an antiapoptotic property against the cytotoxic agents-induced apoptosis. Taken together, these results suggest that GW112 could be an important mediator in NF kappaB-dependent tumorigenesis of digestive tract tissues.
为了阐明 GW112 基因表达的调控机制，本研究分离并鉴定了人 GW112 基因的 5'侧翼区。5'-RACE 分析显示单一转录起始位点，位于翻译起始位点上游 142 个核苷酸处。通过连续缺失构建体的瞬时转染研究，并仔细检查序列，鉴定出一个推定的 NF-κB 结合序列，位于-442 至-430 之间，这可能是 GW112 基因有效表达的原因。事实上，发现 GW112 基因受 NF-κB 信号的调控，包括过表达的 p65 和 IκBα、IKK 抑制剂和蛋白酶体抑制剂。NF-κB 与其推定位点的结合通过 EMSA 和 ChIP 测定得到证实。这些结果表明 NF-κB 是 GW112 转录的必需调节因子。基于这一发现，我们接下来证实了在胃癌细胞存在细胞毒性药物的情况下，抑制 GW112 表达可诱导细胞凋亡。此外，在胃癌细胞中敲低或过表达 GW112 基因表明，GW112 对细胞毒性药物诱导的细胞凋亡具有抗凋亡作用。综上所述，这些结果表明 GW112 可能是 NF-κB 依赖性消化道组织肿瘤发生的重要介质。
