Effect And Mechanisms Of Human P100 Protein On HeLa Cell Cycle

Objectives:Cell cycle is an ordered set of events, culminating in cell growth and division into two daughter cells. The related proteins orderly expression and cooperation are the preconditions for the cell cycle to run normally. Cell cycle regulation mechanisms mainly include several factors, such as cyclins, cyclin-dependent kinases(CDKs) and CDK inhibitors(CKIs), which form a complex regulatory network. Alteration of any part of these regulation mechanisms might result in uncontrolled cell proliferation even cancer. p100 protein is a multi-function protein which contains four repeated SN (Staphylococcal Nuclease) domains and one Tudor-SN (TSN) domain. As a dual regulator of gene transcription and pre-mRNA splicing, the SN domains of p100 protein promote the transcription activity mediated by STAT6, and function in signal transduction pathway mediated by cytokines. Furthermore, the TSN domain of p100 protein interacts with snRNPs (small nuclear ribonucleoproteins) complexes, accelerate spliceosome assembly. Since both cell cycle and transcription are the basic processes in organism, it is naturally for us to assume that p100 protein probably participate the transcription regulation of some cell cycle related proteins. The objective of this project is to figure out the impact of p100 protein on HeLa cell cycle and its molecular mechanism.Methods:The report is composed of three parts. In partâ… , promoter chip (ChIP-GLAS) and gene expression profile microarray were applied on HeLa-pSG5-p100-flag to search for the potential target gene regulated by p100 protein. In partâ…¡, a stable transfectant was constructed, which suppressed the p100 protein expression continuously and stably. After small hairpin RNA (shRNA) targeting human p100 gene was designed, recombinant vector pGenesil-shRNA-hp100 was constructed and transfected into Hela cells by lipofectamine. The transfected HeLa cells were screened by G418 to establish stable transfectant. In partâ…¢, we underline the impact and mechanisms of p100 protein on cell cycle. Three tests were included in this part. Firstly, the association between p100 protein and HeLa cell cycle was detected by flow cytometry assay. The cells were synchronized by thymidine double block assay. The cell cycle distribution was detected by flow cytometry after PI staining. Secondly, the impact of p100 protein on cell proliferation ability in HeLa cells was measured by MTS assay. Finally, two proteins-Chkl and CDKN2C (p18) were selected to investigate the relation between p100 protein and cell cycle. Setting P-actin and GAPDH as internal control, Western blotting and Real-time PCR were applied to measure the protein level and mRNA level of CDKN2C and Chkl respectively.Results:Partâ… :The results of ChIP-GLAS show that several promoters of cell cycle related genes have high binding affinity with p100 protein, including CDK4, CDKN2A, ChK1, E2F1 and so on. Partâ…¡:A recombinant expression plasmid which could express siRNA was constructed and transfected into HeLa cells. A stable transfeactant HeLa-p100 I which stably suppressed the p100 protein expression was established by G418 screening. The suppression efficiency of transient and stable transfectant were 47.59% and 62.86% respectively measured by Western blotting. Partâ…¢:This part includes 3 tests. The results of test 1 indicate that when p100 protein level altered, either increase or decrease, will have impact on HeLa cell cycle distribution-prolong G1 phase and shorten G2 and S phase. The results from test 2 demonstrate that p100 protein overexpression promotes cell proliferation. The results from test 3 show us:â‘ p100 protein down-regulates CDKN2C (p18) protein expression:transiently or stably suppression of p100 protein expression will significantly elevate both the protein level and the mRNA level of CDKN2C (p18).â‘¡p100 protein up-regulates Chkl protein expression.Conclusions:1. p100 protein is associated with HeLa cell cycle.2. p100 protein promotes HeLa cell proliferation.3. p100 protein down-regulates CDKN2C protein level, but up-regulate Chkl protein level in HeLa cells..