Analysis of mechanism for VEGF expression in renal cell carcinoma

我对自己拥有的几种肾细胞癌细胞系进行了VEGF mRNA的northem blot分析。4种细胞系中SN12C细胞表达VEGF mRNA最多。20 ng/ml TPA对VEGF mRNA的诱导作用是对照组的4倍。放线菌素D处理表明，TPA在转录水平下调节VEGF mRNA。接下来，我通过PCR扩增了2.3 kb的VEGF启动子区域，并将其亚克隆到荧光素酶报告质粒中。证实该报告子转染SN12C细胞后具有较强的荧光素酶活性，提示该启动子具有足够的实验启动子活性。20 ng/ml TPA对该启动子活性的诱导作用为5倍。此外，即使使用最小的VEGF启动子（包含一个GC盒的59 bp启动子），TPA也能诱导10倍的启动子活性，表明TPA作用于基础转录起始复合物，调节VEGF mRNA的表达。我还研究了调节VEGF表达的重要顺式作用元件。我构建了VEGF启动子区序列缺失突变体，用这些报告基因转染SN12C，比较荧光素酶活性。首先，在转录起始位点（transcription initiation site， TIS）上游59 ~ 86 bp处检测到顺式作用元件。由于该区域包含三个GC盒，这些GC盒似乎在基础转录中起重要作用。其次，在TIS的131 ~ 400 bp之间鉴定出另一个顺式作用元件。现在我正在亚克隆这个启动子区域。

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.