A Study Of Regulation And Function For Several Angiogenesis And Lymphangiogenesis Related Genes

Genes are the genetic basis of life. Understanding gene function and its regulation is the key to understanding of the human physiological processes and human diseases. This dissertation was focused on the molecular regulation and new function of several angiogenesis or lymphangiogenesis related genes. The dissertation includes three major sections.Section1:MicroRNA-503targets FGF2and VEGFA and inhibits tumor angiogenesis and growth.FGF2and VEGFA are the two of the most potent angiogenic factors and regulated by hypoxia through transcription factor HIF-la. This study has found that miR-503can simultaneously down-regulate FGF2and VEGFA by a posttranscriptional mechanism. Bioinformatic analysis identified potential binding sites for miR-503at3’-UTRs of both FGF2and VEGFA. Luciferase assays using FGF2and VEGFA3’-UTR reporters showed that down-regulation of FGF2and VEGFA by miR-503was directly through the targeted miR-503binding sites. Transfection of miR-503mimics significantly down-regulated the expression of both FGF2and VEGFA at both mRNA and protein levels. Also, this study identified that the expression of miR-503is markly repressed in HCC cells and primary tumors due to a potential epigenetical mechanism. Overexpression of miR-503reduced tumor angiogenesis in vitro in a marigel based angiogenesis assay and in vivo in a hepatoma xenograft mouse model. The study also revealed that miR-503expression was down-regulated by hypoxia through HIF-1α, which provides a novel molecular mechanism for induction of FGF2and VEGFA expression under hypoxia by a microRNA-mediated mechanism. These results identify a miRNA that targets both FGF2and VEGFA in cancers, demonstrate the anti-angiogenesis role of miR-503in tumorigenesis, and provide a novel mechanism for hypoxia induced FGF2and VEGFA through HIF-la mediated inhibition of miR-503.Section2:Transcriptional activation of the Proxl gene by HIF-la and HIF-2a in response to hypoxia.Prox1encodes a homeobox transcription factor critical to organ development, but its regulation is poorly understood. Here, this study showed that Proxl expression is induced by hypoxia, and controlled by a hypoxia-response element (HRE) at the Proxl promoter/regulatory region and HIF-la/HIF-2a. EMS A and ChIP assays demonstrated the direct interaction of the HRE with HIF-la and HIF-2a. Overexpression of HIF-la and HIF-2a increased activation of the Proxl promoter, whereas knockdown of HIF-la and HIF-2a inhibited the activation. These data reveal a novel molecular mechanism for regulation of Proxl expression in response to hypoxia and provide new insights into Proxl-controlled processed such as lymphangiogenesis.Section3:AGGF1acts as a tumor suppressor through interaction with p53.AGGF1was previously cloned and identified as the first disease gene for Klippel-Trenaunay syndrome (KTS), which is a congenital vascular disease associated with capillary and venous malformations or varicose veins. However, the function and molecular regulation of the AGGF1gene is largely unknown. This study was focused on the function and molecular mechanism of AGGF1in tumor cells. The study showed that AGGF1acts as a tumor suppressor in cancers. Overexpression AGGF1in cancer cells by transfecting a mammalian expression plasimid for human AGGF1induces cell apoptosis, inhibits cell proliferation, and induces cell cycle arrest, as well as promotes DNA repair and inhibits cancer cell colony formation. Knockdown of AGGF1by siRNAs in cancer cells leads to a dramatically increase of cell proliferation, a promotion of cell cycle progression, and resistance to apoptosis, as well as reduction of DNA repair capacity and increasing of cancer cell colony formation. Also, a number of amino acids alterations (gene mutation) for AGGF1in primary tumor tissues have been reported. Thus, we functionally characterized AGGF1as a tumor suppressor gene.Mechanically, the present study found that AGGF1is located in nuclear in cancer cells, and interacts with p53through its FHA domain, which stabilizes p53by preventing its degradation and increases its expression at protein level. Also, the interaction between AGGF1and p53leads to a dramatical induction of phosphorylation and acetylation of p53 at multiple-sites, and activation of p53function. Thus, the p53signal cascade is induced and a large series of downstream genes are activated.