The Influence Of Sonic Hedgehog Signaling Pathway On The VEGF Expression In Tumor Cells

Background and PurposeIt was proved that VEGF (vascular endothelial growth factor) which is one of TAFs (tumor-angiogenesis factors), is essential for tumor angiogenesis and play a key role in promoting tumor growth and metastasis. Because of that, it had become a hot research topic of tumor biotherapy.Sonic hedgehog (SHH) signaling pathway, which is important in human embryonic development for angiogenesis, does not activate in healthy adults. However, it has been reported that when the SHH signaling pathway was blocked, the expression of VEGF was lower and angiogenesis would be reduced in the animal models of pathologically angiogenic remodelling in eyes. It has been reported that SHH signaling pathway was activated in a wide variety of tumor, and there was some relationship between SHH signaling pathway and VEGF, but the detailed mechanism is still unrevealed. So, we intend to investigate whether VEGF is a target gene of this signaling pathway, and blocking this signaling pathway is down-regulation of the expression of VEGF in the tumor cells.Methods1. The special primers of targeted VEGF gene were designed. The aimed fragment of VEGF gene was amplified with PCR, then inserted into PMD19-T vector to obtain stander vector of VEGF gene. A stander curve was established with this vector.2. The SHH signaling pathway was blocked with cyclopamine which was special blocking agent of this pathway. The expressions of VEGF mRNA and protein were detected by fluorescence quantitative PCR (FQ-PCR) and Western blot.3. The pGenesill-Gli1 vector was transfected into tumor cell Hep3B, which was a recombinant plasmid of RNA interference specific to Gli1, a nuclear transcription factor in SHH signaling pathway. Then FQ-PCR and Western blot were performed to measure the expressions of VEGF mRNA and protein respectively.4. The influence of down-regulation of VEGF with combined cyclopamine and siRNA was investigated; expressions of VEGF were tested just like above-mentioned.Results1. The stander vector for FQ-PCR was proved to be constructed successfully by DNA sequencing. FQ-PCR showed that amplified products were special. The relationship of threshold cycles and plasmid copy number was approximately linearity, when the copy number was between 102~107. The method of FQ-PCR for detecting VEGF gene was established.2. Compared with control group, the levels of VEGF expression were decreased in both cyclopamine group and siRNA group. The inhibition of VEGF with combination of siRNA and cyclopamine was more significantly.ConclusionsThe established FQ-PCR method can provide a rapid and accurate assay for the VEGF mRNA expression. SHH signaling pathway could regulate the expressions of VEGF mRNA and protein, and VEGF gene is a target gene of this signaling pathway, and it is under the regulation of nuclear transcription factor Gli1. The combination of siRNA and low molecular inhibitor such as cyclopamine could down-regulate VEGF expression, and that could be a new treatment pathway of hepatocellular carcinoma.