The Study Of The Relationship Between Angiogenin And BFGF And Their Molecular Mechanisms

Angiogenin and basic fibroblast growth factor (bFGF) are two important growth factors during angiogenesis. They can regulate proliferation, differentiation and migration of the cell, thus play important roles during the process of tumor occurrence and development. However, the study of relationship between angiogenin and bFGF has not been reported. This study aims to investigate the relationship between angiogenin and bFGF and its molecular mechanism. Firstly, we successfully established the stable bFGF over-expressing and under-expressing transfectants with H7402 human hepatoma cells. We discovered that expression of angiogenin was decreased in the bFGF sense transfectants while increased in the bFGF antisense transfectants. In cell proliferation assay, we observed that the cell proliferation was increased in the bFGF sense transfectants and decreased in the bFGF antisense transfectants. These results showed that the endogenous bFGF may not only negatively regulate the angiogenin expression but also contribute to the overall cell proliferation in H7402 human hepatoma cells. Then we stably transfected angiogenin sense and antisense plasmids in HeLa human cervical cancer cells, and established the stable angiogenin over-expressing and under-expressing transfectants. We found that the angiogenin sense transfectants decreased the expression of bFGF and angiogenin antisense transfectants increased the expression of bFGF. The results further confirmed that angiopoietin negatively regulated the expression of bFGF.Our work confirmed that bFGF may negatively regulate the expression of angiogenin tumor cells and angiogenin negatively regulated the expression of bFGF in angiogenin-transfected cells. This is a new phenomenon in tumor angiogenesis factors research. To explore the molecular mechanism of relationship between angiogenin and bFGF not only helps further study of tumor angiogenesis but also contributes to anti-angiogenesis therapies. At present, there are only few clues about molecular regulation mechanism of angiogenin and bFGF. We make two pieces of hypothesis based on the existing reports: one is that angiogenin is a trans-acting factor involved in regulation of bFGFgene expression; the other is that there is a possible negative feedback regulation between angiogenin and bFGF. Firstly, luciferase reporter assay in 293T cells demonstrated that angiogenin inhibited bFGF promoter activity and participated in the expression regulation of bFGFgene. In the following experiments we localized the angiogenin-responsive region in the bFGF promotor using truncated promotor fragments, and the results showed that the angiogenin-responsive region is from -970bp to -834bp in the bFGF promotor. Secondly, we verified that a negative feedback existed in the relationship between angiogenin and bFGF. Stimulating the transfected 293T cells with exogenous bFGF, we found that the addition of exogenous bFGF compensated the inhibition of bFGF promoter activity by angiogenin. Therefore, we concluded that the negative feedback regulation between angiogenin and bFGF is through the inhibition of bFGF promotor activity and protein level, and further inhibited the expression of bFGFgene in negative feedback way. Data presented in this thesis contribute considerably to the understanding of the tumor angiogenesis research.