The Study Of REGγ's Role In Oncogenic Signal Pathways And Its Certain Modification

Proteasome degradation pathway is regulated by a precise mechanism of degrading specific proteins. It plays a critical role in many cellular pathways, which need to be actived by the proteasome activator. Proteasome Activator REGγcan significantly enhance the catalytic activity of the proteasome. It is demonstrated that REGy can degrade its target proteins-p21 and SRC-3 in an ubiquitin and ATP-independent proteolytic manner, which broke through the previous concept of that REGy/20S proteasome only can facilitate the hydrolysis of small peptides1. Besides, REGy may be involved in cell proliferation, anti-apoptosis and tumorigenesis. It is significant to research the biological mechanism of REGy.First, we have predicted the relation between the key genes in cancer signal pathways and REGy by summary and analysis of numerous public database by bioinformatics means. Then, we verify the predict by some molecular bio logy experiments such as RT-PCR technology. It is a relatively new study method for screening genes which are interested. And all of this will provide a novel perspective and research direction for the prevention and treatment of cancer.There are mainly four parts of researching the function of REGy in cancer signal pathways and the mechanismof REGy's modifications:1. The effect of REGy's phosphorylation on its functionWe transfect REGy wide type or REGy mutants which is phosphorylation site deletion(S156A,S170A,S170D,S174A,S174D,S248A,S248D,S170/174A,S170/174D,S248/252A和S248/252D) into the MEF REGy-/-cell line, then explore their interaction with p21 separately by immunofluresence analysis. The experiments results demonstrate that compared with REGy wide type, there is a significant reduction in the capacity of REGγS248/252 A to degrade p21.2. The effect of REGy's SUMOylation on its functionWe co-trans feet p21 and REGγwide type or REGy(6KR) mutants which is SUMOylation deletion into the 293T cell line, then explore their interaction between REGγand p21 by immunoprecipita- -tion analysis. The experiments results demonstrate that compared with REGy wide type, there is a significant reduction in the capacity of REGy 6KR (SUMOylation- -deficient) to degrade p21.3. The binding sites of p21 and REGyWe co-transfect REGy and p21 wide type or p21 mutants(D156-161,R155A,156A,L157A,I158A,F159A, S160AandK161A) into the293T cell line, then explore their interaction between REGy and p21 by immunoprecipitation analysis. The experiments results demonstrate that compared with p21 wide type, there is a significant reduction in the capacity of p21 F159A to bind with REGy.4. The REGy's function in cancer signal pathwaysWe detect the relation between the key genes in cancer signal pathways and REGy by RealTime-PCR technology in ARO, A549, HCT116 and HepG2 four kinds of cancer cell lines of the trend predict from bio informatics analysis. The results demonstrate that most of the relation from RealTime-PCR experiments have the consisitent trend of bioinformatics predict.In summary, there are a series of research of REGy's modifications and its function on the interaction between p21 and REGγ, besides, we also study the relation between the genes referred to cancer signal pathways. Accordingly, we get some conclusion, and which lay a foundation for further explore of REGy's function and regulation.