Study the mechanism of SSAP Growth Effect in HEK293T cell and NIH3T3 cell

• Main Authors: Cheng-Wei Ou, 歐政瑋
• Other Authors: Chin-hsiang Chien
• Format: Others
• Language: zh-TW
• Published: 2005
• Online Access: http://ndltd.ncl.edu.tw/handle/80688465172558297331

碩士 === 國立陽明大學 === 生物化學研究所 === 93 === In recent years, it was found that sialidase may not only influence the glycoproteins and glycolipids on the cell surface but also influence the intra-cellular function.We found that transfected small sialidase gene, which expressed by Clostridium perfringens, to NIH3T3 cell and HEK293T cell could increase the cell proliferation rate of these two cell . ThereforeWe used small sialidase as a bait to perform yeast two-hybrid screening. Then,we found a novel protein which interacted with small silidase named small sialidase associated protein（SSAP）. After blast in NCBI GENEBANK ,we predicted that SSAP consists a SPRY domain with an unkonwn function domain localized in N-terminal and a ring finger domain localized in C-terminal. In order to investigate the function of SSAP, we used SSAP as a bait to perform yeast two-hybrid screening. Then,we found a protein containing two ubiquitin associated（UBA） domains which interacted with SSAP protein named Homo spaiens ubiquitin associated containing domain 1（HUAD1）.Accroding to the results of confocol microscopy observation, it’s suggested that both pEGFP-C1-SSAP and pEYFP-C1-HUAD 1 fusion protein localized in the cytosol but not in the nucleus of NIH3T3 cell. In MTT assay analysis, transient overexpression of SSAP or HUAD 1 would increase the proliferation rate of NIH3T3 cell and HEK293T cell .Moreover, cotransfected SSAP and HUAD 1 to NIH3T3 cell and HEK293T cell would increase the proliferation rate more remarkable. Besides that the results of FACS revealed that transient overexpression of SSAP or HUAD 1 would decrease the G1 phase percentage and increase the S phase percentage of cell cycle of NIH3T3 cell and HEK293T cell. Like the result of MTT assay, cotransfected SSAP and HUAD 1 to NIH3T3 cell and HEK293T would cause the cell cycle change more evident. Furthermore, we used western blot to check the expression of G1/S phaseCDK inhibitor p27 in NIH3T3 cell and HEK293T cell .The results suggested that cotransfect SSAP and HUAD 1 to NIH3T3 cell would decrease the expression of p27 but make no influence to HEK293T cell. Above all, the interaction between SSAP and HUAD 1 may play a role to increase cell proliferation rate in these two cell lines. Whether this kind of abilility could be a reason of cancerization would needs more deeply researches in the future.