Analysis of mechanism for VEGF expression in renal cell carcinoma

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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.