The Effect Of HIV-1 Nef On Prostate Cancer Cells Proliferation And Its Molecular Mechanism

Objective: To investigate the effect of HIV-1 negtive factor (Nef) on prostate cancer cells proliferation and its potential molecular mechanism.Methods: (1) A pair of PCR primers for Nef gene with EcoR I and SalⅠrestriction enzyme cut sites was designed according to the sequence registered in GenBank. Then the genome of plasmid PCINL was taken as template and Nef gene was amplified using PCR. Subsequently, amplified gene fragments were digested with the two enzymes mentioned above and then cloned into pCI-neo vector to create recombinant eukaryotic expression plasmid designated as pNef, which was introduced with Flag sequence to facilitate the detection of protein. After identification with enzyme digestion and nucleotide sequences analysis, recombinant pNef DNA was transient transfected into prostate cancer cells. The expression of pNef protein in prostate cancer cells was detected by western blot (WB). (2) Investigate the effect of Nef on prostate cancer cells proliferation and apoptosis by MTT assay and flow cytometroy (FCM), respectively. (3) Detect the expression level of Androgen Receptor (AR) and the activity of Prostate Special Antibody (PSA) promoter in the prostate cancer cells transient transfected Nef through WB and Luciferase Reporter Assay, respectively. (4) Exploit the effect of Nef on the activity of PTEN/PI3K/Akt and NF-κB signallings in prostate cancer cells using WB and ELISA. Results: (1) Nucleotide sequences analysis indicated that the isolated and cloned pNef sequence length was 651 bp. The isolated Nef sequence was 100% homology with Nef gene previously registered in GenBank. The specific bands were both detected at the expected place by Western blot. (2) The results of MTT assay and FCM indicated that Nef slightiy inhibited prostate cancer cells proliferation and induced apoptosis in vitro. (3) The consequences of WB and Luciferase Reporter Assay showed that Nef down-regulated AR expression level and PSA promoter activity. (4) Further WB and ELISA demonstrated that Nef partially blocked the activity of PTEN/PI3K/Akt and NF-κB signallings in prostate cancer cells.Conclusion: (1) Nef gene could be correctly expressed in prostate cancer cells. (2) Nef could slightly inhibited prostate cancer cells proliferation, meanwhile, induced apoptosis. (3) Mechanistic studies show that ectopic expression of Nef could down-reuglate AR expression level and PSA promoter activity. Furthermore, overexpression Nef in prostate cancer cells could not only partially block PTEN/PI3K/Akt signaling, but also inhibit NF-κB signallings.