Analysis of mechanism for VEGF expression in renal cell carcinoma

肾细胞癌中VEGF表达的机制分析

• 批准号: 08671852
• 负责人: MIZOKAMI Atsushi
• 金额: $ 1.47万
• 依托单位: Unuversity of Occupational and Environmental Health
• 依托单位国家: 日本
• 项目类别: Grant-in-Aid for Scientific Research (C)
• 财政年份: 1996
• 资助国家: 日本
• 起止时间: 1996 至 1997
• 项目状态: 已结题
• 来源: https://kaken.nii.ac.jp/grant/KAKENHI-PROJECT-08671852/
• 关键词: renal cell carcinoma; VEGF; promoter; TPA; promoter

I carried out northem blot analysis for VEGF mRNA in several renal cell carcinoma cell lines which I have. SN12C cells expressed VEGF mRNA most in four cell lines. Furthermore, 20 ng/ml TPA induced VEGF mRNA by 4 times of control. Actinomycin D treatment indicated that TPA regulated VEGF mRNA under the transcriptional level. Next, I amplified the 2.3 kb VEGF promoter region by PCR,subcloned into luciferase reporter plasmid. It was confirmed that this reporter has strong luciferase activity after transfected into SN12C cells, suggesting that this promoter has enough promoter activity for experiments. 20 ng/ml TPA induced this promoter activity by 5 times. Furthermore, TPA could induce promoter activity by 10 times even minimal VEGF promoter (59 bp promoter that included one GC box) was employed, indicating that TPA act on a basal transcriptional initiation complex and regulates VEGF mRNA expression. I also investigated important cis-acting elements that regulate VEGF expression. I constructed a serial deletion mutants of VEGF promoter region, transfected SN12C with these reporters, and compared luciferase activities. First, cis-acting elements were detected between 59 bp and 86 bp upstream of the transcription initiation site (TIS). Since this region included three GC boxes, these GC box was seemed to play an important role in basal transcription. Second, another cis-acting element was identified between 131 bp and 400 bp of TIS.Now I am subcloning this promoter region.

我对我所拥有的几个肾癌细胞系中的血管内皮生长因子基因进行了正常杂交分析。SN12C细胞在4种细胞系中表达最多的是VEGFmRNA。20 ng/mlTPA可诱导血管内皮生长因子的表达，是对照组的4倍。放线菌素D处理表明，TPA在转录水平上调节血管内皮细胞生长因子的表达。接下来，通过聚合酶链式反应扩增出2.3kb的血管内皮生长因子启动子区域，并将其亚克隆到荧光素酶报告载体中。证实该启动子在SN12C细胞中具有较强的荧光素酶活性，提示该启动子具有足够的启动子活性。20 ng/mlTPA可诱导5倍的启动子活性。此外，即使使用最小的VEGF启动子(59bp启动子，其中包括一个GC盒)，TPA也可以诱导启动子活性10倍，这表明TPA作用于一个基本的转录起始复合体，调节VEGFmRNA的表达。我还研究了调节血管内皮生长因子表达的重要顺式作用元件。构建了血管内皮生长因子启动子区的系列缺失突变体，并将其导入SN12C，比较其荧光素酶活性。首先，在转录起始点(TIS)上游59~86碱基之间检测到顺式作用元件。由于该区域包括三个GC盒，这些GC盒似乎在基础转录中起着重要作用。其次，在TIS的131~400碱基之间发现了另一个顺式作用元件，现将该启动子区域亚克隆。