The Study Of HEra Function On Cell Proliferation And Ethanol-induced Apoptosis

Homologues of Era, have been identified in all bacterium sequenced to date, and eukaryotic organisms, including mice and humans, form a new G-protein subfamily. In prokaryotic organism, Era is a GTPase essential for bacterial viability, it like appears to have an important role in the regulation of the bacterial cell-cycle and the processing and maturation of ribosome rRNA. In mammalian, the study of Era's function is focused in human Era, named hEra. The published data show that hEra is expressed in many tissues. Overexpression of mutated hEra or the reduction of Era results in the arrest of cell cycle. However, the exact fuction of Era is still not clear. Therefore, it is significance to further study the function of Era for underdstanding the physiological function of this new G-protein subfamily as well as the mechanism of cell proliferation and apoptosis.The hera gene, located at chromosome 17q11.2, was first cloned by Professor Sumin Chen of our lab in 1999. Thereafter, Professor Chen discovered that there existed two kind splices of hera: hEra and hEraII. The function of heraII is the main focus of our research group in the past years, but now hEra became the goal in this study. Published papers about hEra from internet of hEra and the previous results from our group about heraII tell us hEra have some roles related to cell proliferation and apoptosis. This study first emphasizes on the roles of hEra associated with cell proliferation, cell cycle and ethanol-induced apoptosis using a complete control mammalian expression system. Then HepG2, the model cell in ethanol-induced apoptosis study was used for hEra RNAi experiment by chemosynthesis siRNA duplexs. Finally, the expression pattern of hEra in hepatoma and pancreatic carcinoma were analyzed.The main results list as follows:1. hEra plays an important role in cell proliferation and ethanol-induced apoptosisThe system we used in the experiments contains vector pEGSH and receptor expression vector pERV3. After being induced by PonA, target gene can express. This inducible mammalian expression system can produce high expression level of target gene and turn on/off gene expression rapidly. It is a strong means to study gene function.Using this System, hEra was transfected into HEK293 cell lines in which pERV3 was stably transfected. After being induced by PonA, over-expressed wild type hEra could accelerate cell proliferation. The data of cell synchronization showed that over-expressed wild type hEra could accelerate cells into S phase, shorten the time of G1 phase (from 17.82 h to 14.13 h), decreased the number of G1 phase and increased that of S phase. Western blot assay discovered that over-expressed wild type hEra could prompt the protein expression of cyclin D. The RNAi of hEra in HepG2 cell by chemosynthesis siRNA obviously suppressed the cell growth. We also found over-expressed hEra could depressed ethanol-induced apoptosis, which molecular mechanism may be involved the c-jun and Bax signal transduction because over-expressed hEra could increase the c-jun at message RNA expression level and decrease the protein expression level of Bax.2. The G domain of hEra is important in cell proliferation and ethanol-induced apoptosis.To study the functions of G domains, 2 mutants were constructed: 1 point mutants hera S127N (the mutant points were located in Era G domain) and 1 truncated mutants hera1-343 (KH domain in C terminal were deleted). They were cloned into inducible expression vectors, and then these expression vectors were transfected into HEK293 cell lines in which pERV3 was stably transfected.After induced, hEra1-343 could accelerate cell proliferation, but hEra S127N doesn't. Besides, the similar phenomenon played in the ethanol-induced apoptosis: inducing the expression of hEra1-343 could depress this kind of apoptosis dramatically, but hEra S127N could not. These data illustrate that the G domain of hEra is important in cell proliferation and ethanol-induced apoptosis. To further study the role of hEra1-343 in ethanol-induced apoptosis, the message RNA expression level of AP-1 and c-myc and the protein expression level of Bax were detected. Results showed over-expressed hEra1-343 could increase the c-jun, decrease c-myc and Bax, which hints that hEra1-343 may be involved in some signal pathways and can play an important role dependently in cell.3. KH domain of hEra may be an“anchorage”to the whole protein.As the result 2, to study the functions of KH domains, we constructed 2 mutants: a point mutant hera S401N (the mutant points were located in Era C domain) and a truncated mutant hera?329 (KH domain in N terminal were deleted). They also be cloned into inducible expression vectors, and then these expression vectors were transfected into KV4 (HEK293 cell transfected pERV3 stably).After being induced, either of the two mutants could depress cell proliferation respectively. hEra ?329 can arrest cells in G2/M phase. Also, neither can alleviate the enthanol–induced apoptosis. Obviously, hEra ?329 and hEra S401N have an dominate negative effect on endogenous hEra.Based on the performance of wild type hEra and the mutants, we proposed a hypothesis: KH domain of hEra may be an anchorage and the G domain is the functional group. As mutation occurred in G domain, KH domain could efficantly located the protein on certain location where the normal physical activity of G domain is need, so the related cell procedure must be impaired, which may be further impact the cell proliferation. While mutation happened in C domain, KH domain cannot locate effectively, the whole G domain can play some roles as wild type hEra, example the performance of hEra1-343.Together, our results indicated that hEra is associated with cell proliferation and ethanol-induced apoptosis. KH domain of hEra may be an anchorage and the G domain is the functional group.