:Proteomic Analysis For PTEN Knock Out Mouse Model Identified The Target Gene Chop And The Establishment Of The C-myc Inducible Cell Line

Objective:Proof of principle of the use of quantitative proteomics (Super-SILAC) to determine protein level changes in PTEN knock out prostate cancer mouse model.Methods:PTEN knockout mice-derived cell line was labeled with Lysine and arginine bearing non-radioactive isotopes (Stable Isotope Labeling with Amino acids in Cell culture-SILAC) for9generation until>95%of incorporation. Protein extract from the labeled cells were mixed in1:1ratio with prostate lobes from either PTEN-/-or PTEN+/+mice. Protein purified, digested and resulting peptides analyzed by MS/MS. H refers to heavy and represents the labeled cell line, and L refers to Light and represents the tissues (KO or WT). Individual H/L ration was calculated fro each case and the compared between KO and WT.Results:2000proteins were identified in each experiment. Although only one animal from each group (KO and WT) was used a preliminary gene ontology classification was performed and results suggest that PTEN-/-prostate show an increase in the expression of proteins related with cell adhesion (Collagen, laminin, integrins...) and protein transport (RAB proteins...) while down regulate proteins relates with cell cycle and chromosome organization (MAPK7and Septin6or Histone2, respectively).Conclusion:Quantitative proteomic comparison from genetically modified animal models can be performed using stably labeled cell lines as internal control.Part two:The Changes Caused by PTEN Deletion in Prostate Cancer CellsObjective:Combined with the data from the Proeomic analysis,Loss of PTEN function, results in reduction of a seris of the gene of unfolded protein response. Chop gene incharge of the apoptosis function in UPR. To study the relationship between the Chop and PTEN in prostate cancer cell line.Methods:Firstly we used siRNA PTEN knock down the PTEN gene in the22Rv1cell line. Then we used realtime PCR to test the mRNA of the Chop. MTT and Flowcytometer were used to test cell viability and apoptosis. WesternBlot was used to test the protein level of the target gene.Results:In the prostate cancer cell line22Rv1, The deletion of PTEN gene increase cell viability and reduce the apoptosis,aslo downregulated the Chop gene.Conclusion:The deletion of PTEN gene may induce the apoptosis and increase cell viability in Prostate cancer cells through mechanism of up-regulation of p-Akt, aslo downregulated the Chop gene.Part three:Role of Chop in the regulation of growth and apoptosis in human prostate cancer cellObjective:Chop, also known as gadd153, is a highly stress-inducible gene that is robustly expressed following disruption of homeostasis in the endoplasmic reticulum (ER)(so-called ER stress). Although all reported types of ER stress induce expression of Chop, its role in the stress response has remained largely undefined. Since we have proved deletion of PTEN can down-regulated Chop gene, the fuction of Chop in prostate cancer cells has never been clearly demonstrated.Methods:Firstly we used siRNA PTEN knock down the PTEN gene in the22Rvl cell line. Then we used realtime PCR to test the mRNA of the Chop.We employed22Rv1in which Chop is knocked down or overexpressed. MTT and Flowcytometer were used to test cell viability and apoptosis. WesternBlot was used to test the protein level of the target gene.Results:In the prostate cancer cell line, overexpression of chop lead the cell to apoptosis and reduced the cell viability. Investigation of mechanisms contributing to this effect revealed that elevated chop expression results in the down regulation of p-Akt expression.Conclusion:Chop gene may induce the apoptosis in Prostate cancer cells through mechanism of down-regulation of p-Akt..Part four:The establishment of the c-myc inducible cell lineObjective:To establish an stable cell line which can inducibly overexpress c-myc gene.Methods:The gene of interest is cloned into the multiple cloning site of the inducible expression vector, and the resulting construct cotransfected with the regulatory plasmid, pcDNA6/TR. into mammalian cells. After transfection, cells are treated with tetracycline to derepress the hybrid CMV/TetO2promoter in the inducible expression vector and induce transcription of your gene of interest.Results:Establish an cell line which can inducibly overexpress c-myc geneConclusion:Recent studies in human tissues have indicated that MYC appears to be activated at the earliest phases of prostate cancer in prostatic intraepithelial neoplasia.To identify the role of c-myc in the progression of prostate cancer,we Establish an cell line which can inducibly overexpress c-myc gene for further research.