Transcriptional Downregulation Of GDDR Facilitates The Expression Of VEGF Under Hypoxia In Gastric Cancer Cell

Object:To difine the underlying mechanism by which HIFs down-regulat GDDR at the mRNA level in hypoxic conditions and provide a potential correction with down-regulateion of GDDR and development and progression of cancer. Then to investigate the contribution of GDDR in the progression of regulation of VEGF and tumor angiogenesis under the low oxygen tensions.Methods:To assess the direct role of transcription factors HIF-1 in regulating GDDR we performed transient co-transfection of its expression plasmids and the reporter gene construct. Comparison of GDDR mRNA expression in the hypoxic cells and control cells by using quantitive Real time PCR. To analyze the function of GDDR in the expression of VEGF at the mRNA and protein level, we culture the GDDR-deficient SGC-7901 cells and the cells transfected stably with GDDR under low oxygen tensions; while siRNA(small interference RNA)studies for GDDR in GDDR-sufficient GES-1 cells reveal effects on VEGF in hypoxic circumstances.Expression of VEGF mRNA studied by quantitive Real time PCR,VEGF protien was studied by ELISA.Results:The main findings of our studies as follow:1. The promoter region of the GDDR was amplified from human genomi cDNA.we successfully constructed a GDDR luciferase reporter victor.2. The GDDR promoter is responsive to HIF-1αby using the Dual-Luciferase Reporter Assay system.The quantitive Real time PCR results showed that the mRNA level decreased by the transfection HIF-1αunder hypoxic conditions.3. The mRNA and protien level of VEGF was induced by HIF-αin low oxygen cricumstance. An alternative model of oxygen sensing involving production of VEGF in hypoxia has been proposed4. In the hypoxic cell model system,the mRNA and protien level of VEGF in GDDR stably transfection SGC-7901 cells was less efficient in upregulating in the GDDR-deficient SGC-7901 cells,but it was higher than the GDDR stably transfection SGC-7901 cells non-stimulated by low oxygen tensions.5. GDDR-siRNA is an efficient tool for gene silencing. Inhibition of GDDR with siRNA was performed resulting in a significant upregulation of VEGF in GDDR-sufficient GES-1 cells. Conclusion:GDDR is down-regulated at the mRNA level, through concerted action of HIF-1αactivated by low oxygen tensions.VEGF is less efficient upregulating own to the expressiong of GDDR restored in SGC-7901cells under hypoxic circulations.GDDR is a tumor suppressor gene.