The Effect of HIV-1 Nef on Prostate Cancer Cells Proliferation and Its Molecular Mechanism
|School||Nanjing Medical University|
|Course||Department of Urology|
|Keywords||HIV-1 Nef Prostate cancer cells Apoptosis Signaling pathway|
Objective: To investigate the human immunodeficiency virus type 1 (human immunodeficiency virus type 1, HIV-1) encoding the viral replication factor (Negative factor, Nef) negative regulation of prostate cancer (prostate cancer) cell growth and reproduction of the impact of its molecular mechanism . Method: (1) registered under GenBank the NEF gene nucleotide sequence PCR primers were designed in the EcoR I and Sal I digestion sites were introduced at their 5 'ends; to contain the Nef gene pCINL plasmid as a template, the Nef gene was amplified dual enzyme then subcloned into the eukaryotic expression vector pCI-neo. In order to facilitate protein detection, the introduction of Flag sequence at the 5 'end of the downstream primer construct recombinant eukaryotic expression plasmid. Recombinant plasmid by restriction enzyme digestion, nucleic acid sequencing and analysis after transient transfection of prostate cancer, detect the Nef gene expression from the protein level by immunoblotting (Western blot, WB). (2) the use of MTT assay and flow cytometry (flow cytometroy, FCM) detection Nef on prostate cancer cell proliferation and apoptosis. (3) WB and luciferase report experimental (Luciferase Reporter Assay) Determination of expression level of the Nef of prostate cancer in the androgen receptor (Androgen Receptor, AR) and a prostate-specific antibody (Prostate Special Antibody, PSA) promoter activity. (4) with the WB and ELISA experiments preliminary study of the role of Nef of PTEN/PI3K/Akt and NF-κB signaling pathway in prostate cancer cells. Results: (1) successfully constructed a recombinant eukaryotic expression plasmid containing Nef gene, nucleic acid sequence analysis showed that the Nef gene contained 651 bp, cloned the Nef gene sequences registered in GenBank 100% homologous to the introduction of the Flag sequence completely correct; RT-PCR and WB detected specific bands in Nef expected position. (2) MTT experiments and flow cytometry mild Nef in vitro can inhibit prostate cell proliferation and induced apoptosis. (3) WB and luciferase report experiments in vitro, Nef can down the level of expression of AR in prostate cancer cells as well as the activity of the PSA promoter specificity. (4) WB and ELISA showed that Nef can at least partially blocked in PTEN/PI3K/Akt and the activity of the NF-κB signaling pathway in prostate cancer cells. Discussion: (1) HIV-1 Nef gene in prostate cancer cells in the correct expression. (2) Nef protein is at least slightly inhibited prostate cancer cell proliferation and induced apoptosis. (3) Nef protein expression levels of AR and PSA promoter activity downregulation, Nef potential mechanism for inhibiting the proliferation of prostate cancer cells. (4) Nef protein may by partially blocking the activity of the the PTEN/PI3K/Akt and NF-κB signaling pathway, which induced apoptosis.