An Comparative Investigation On Effects Of P52SHC1 And P52SHC3 On The Cell Cycle Of Neurons, Nsc And PC12 Cells

Adult neuron has been out of cell cycle and has no ability to divide and proliferate any more. It was reported that the homolog, SHC1 and SHC3, had totally different distribution in nervous system: the former existed in neural stem/progenitor cells which were found to be largely devoid of SHC3 while the later expressed only in post-mitotic neurons where SHC1 was not found. This phenomenon provides us a base to study the molecular mechanism that neurons are absent from cell cycle. In order to explain the mechanism, effects of p52SHC1 and p52SHC3 on the cell cycle of dividing cells (NSC) and non-dividing cells (neurons) need to be studied. So in this paper, we study the influence of RNAi silencing of SHC3 mRNA and SHC1 mRNA or overexpression of SHC1 and SHC3 on the cell cycles of neurons and NSC, respectively, discuss the effects of overexpressed SHC1, SHC3 and their domains on PC12 cell cycle, investigate the molecular mechanism that the CH1+SH2 domain of SHC3 promotes the proliferation of PC12 cells.This paper mostly includes the following three parts:Partâ… : The effects of SHC1 and SHC3 on the cell cycle of neurons and NSC. To investigate the effects of SHC1 and SHC3 on the cell cycle of neurons, primary neurons were isolated from rat cortex and then enriched to more than 99% purity which testified by SHC3 immuno-staining. Then, lentivirus mediated RNAi was used to silence the mRNA of SHC3 and adenovirus was used to express SHC1 in neurons. After the silencing of SHC3 mRNA, part of neurons re-enter to the cell cycle accompany by upregulation of Cyclin D1, Cyclin E, Cyclin A, CDK2 and phospholated CDK2 (p-CDK2). When SHC1 was expressed, the cell cycle of neurons was not changed, but the levels of Cyclin A, CDK2 and p-CDK2 were elevated. In order to study the effect of SHC1 and SHC3 on the cell cycle of NSC, NSC was isolated from rat cortex and then identified. Then, lentivirus mediated RNAi was used to silencing the mRNA of SHC1 and adenovirus was used to express SHC3 in NSC. After the silencing of SHC1 mRNA, the cell cycle of NSC was blocked and the expression of Cyclin E and Cyclin A was downregulated. When SHC3 was expressed, the cell cycle of NSC was also blocked and expression of Cyclin D1, Cyclin E and Cyclin A was downregulated.Partâ…¡: The effects of overexpressed SHC1, SHC3 and their domains on PC12 cell cycle. In this part, the ORFs of p52SHC1 and p52 SHC3 were successfully cloned from mouse, and then eukaryotic expression vectors of SHC1, SHC3 and their domains (including SHC1, SHC1-PTB, SHC1-PTB+CH1, SHC1-SH2, SHC1-CH1+SH2, SHC3, SHC3-PTB, SHC3-PTB+CH1, SHC3-SH2 and SHC3-CH1+SH2) were constructed. The PC12 cell cycle and expression levels of cell cycle related proteins (Cyclin A, Cyclin D1, Cyclin E and CDK2) were detected by flow cytometry and Western Blot, respectively, after PC12 cells were transfected with these vectors. The fact that existence of CH1 domain affected the functions of PTB domain and SH2 domain of SHC1 and SHC3 was found in the 36h and 60h transfected PC12 cells through flow cytometry analysis. So the CH1 domain was considered when the functions of PTB domain and SH2 domain were investigated. The results of Western Blot and flow cytometry showed that SHC1 promoted the expression of CDK2 and decreased the number of G0/G1 phase cells, while SHC3 lowered the expression of Cylin A and cyclin E and arrested the cell cycle to G0/G1 phase; the PTB domain of SHC1 up regulated the amount of Cyclin D1 and down regulated the amount of Cyclin A, but had little effect on cell cycle of PC12 cells, while PTB domain of SHC3 down regulated the amount of Cyclin A, Cyclin D1 and Cyclin E and increased the number of G0/G1 phase cells; both the SH2 domains of SHC1 and SHC3 enhanced the expression of Cyclin D1 and Cyclin E and caused decrease of G0/G1 phase cells, but SH2 domain of SHC3 also promoted the expression of CDK2, so the decrease of G0/G1 phase cells triggering by over expression of SH2 domain of SHC3 was greater than that of SH2 domain of SHC1. Comparing the functions of different domains in SHC1 and SHC3, it can be infered that to different molecules, the PTB domain and SH2 domain of SHC3 seems more functional to the cell cycle than those of SHC1 and to the same molecule, the functional difference of PTB domain and SH2 domain of SHC1 seems little, but the function of PTB domain of SHC3 is stronger than that of SH2 domain.Partâ…¢: Melecular mechanism that SH2 domain of SHC3 influences the PC12 cell cycle . The binding energy of CH1 domain in different ways with GRB2 was computed and the result showed that the binding energy of normal pattern(from N-terminal to C-terminal) was higher than that of adverse pattern(from C-terminal to N-terminal). Because cell proliferation can be caused by GRB2 related signal pathways, so the higher binding energy, the more proliferated cells. Firstly, we amplified the SH2 domain and CH1 domain of SHC3 by PCR, inverted the location of SH2 domain and CH1 domain by changing CH1+SH2 into SH2+CH1, and constructed the eukaryotic expression vector of SHC3-SH2+CH1. Secondly, CH1+SH2 and SH2+CH1 expression vectors were transfected into PC12 cells. At last, Co-IP was used to detected the binding differences between CH1+SH2 and SH2+CH1 with endogenous GRB2 and flow cyometry and Western Blot were used to detected the changes of cell cycle and expression levels of different cell cycle related proteins after transfected for 36h. The results suggested that CH1+SH2 had higher affinity with GRB2 than that of SH2+CH1 and CH1+SH2 promoted the amounts of cyclin D1, cyclin E and CDK2 and reduced the number of G0/G1 phase cells which indicated that CH1+SH2 enhanced the proliferation of PC12 cells, but SH2+CH1 down regulated the expression of cyclin A, cyclin D1 and cyclin E and increased the number of G0/G1 phase cells which meant the cell proliferation was inhibited.The study of effects of SHC1 and SHC3 on the cell cycle of neurons and NSC shows that SHC3 plays an important role in mainteining the quiescent condition of neurons and may provide potential target for curing the central nervous system degenerative diseases.It can be concluded the PTB domain of SHC3, plays a main role in the function of SHC3 and CH1 domain of SHC3 has direction in transmitting the cell signals to the downstream signal molecules which infers that different binding ways of CH1 domain to the downstream molecules may cause different cytological effect.