Role And Mechanism Of LSD1in The Androgen-Independent Phenotypic Transformation Of Prostate Cancer

Part I Construction and identification of recombinant lentiviral vectors over-expressing and interfering LSD1geneObjective To construct and identify recombinant lentiviral vectors over-expressing and interfering LSD1gene and to provide the basis for further experiments in vitro.Methods1. LSD1gene from plasmid LSD1was amplified via PCR technique and cloned into the expression plasmid of lentiviral vector, GV166, in order to produce the lentiviral expression vector, GV166-LSD1. PCR identification and sequencing assay were used for identifying the correct LSD1gene.293T cells were then transfected by GV166-LSD1. LSD1protein level in293T cells was assessed via Western Blot analysis. Recombinant lentiviruses carrying LSD1gene (LV-LSD1) were generated by293T cells following by co-transfection of GV166-LSD1and packaging plasmids pHelper1.0and pHelper2.0. The virus titer was examined. After LV-LSD1infected LNCaP cells, Real-time quantitative PCR and Western Blot analysis were used for measuring LSD1mRNA and protein expression.2. After LNCaP cells were infected by LV-LSD1, cell lines were selected by puromycin, and the successfully established cell lines were named as LNCaP-LSD1. LSD1mRNA and protein expression in LNCaP-LSD1cells were dectected by quantitative PCR and Western Blot analysis.3. According to siRNA sequence targeting LSD1gene identified by authoritative literatures, lentiviral interference plasmids vectors were constructed. The recombinant lentiviral plasmids were identified by PCR and sequencing. Then, recombinant lentivirus carrying LSD1-shRNA was produced by293T cells following by co-transfection of GV307and packaging plasmids pHelper1.0and pHelper2.0, and the virus titer was measured. After LNCaP-AI cells were infected by LV-LSD1-shRNA, LSD1mRNA and protein expression were dectected by Real-time quantitative PCR and Western Blot analysis.Results1. Recombinant lentiviruses carrying LSD1gene (LV-LSD1) were produced, and the titer was approximately2.0E+8TU/ml. After infected by LV-LSD1, LNCaP cells expressed more LSD1mRNA and protein.2. The monoclonal cell line LNCaP-LSD1stablely epressing LSD1gene was successfully established and expressed more LSD1mRNA and protein.3. The shRNA sequence targeting against LSD1was successfully packaged into the lentivirus, and the titer was approximately5.0E+7TU/ml. After infected by LV-LSD1-shRNA, LNCaP-AI cells expressed less LSD1mRNA and protein.Conclusions The recombinant lentiviral vector carrying LSD1gene (LV-LSD1) is constructed successfully. The over-expressing effects are produced in LNCaP-LSD1cells. The lentiviral vector of LSD1-shRNA is successfully constructed. The silencing effects appear in LNCaP infected by LV-LSD1-shRNA. The researchs will provide the basis for the further study on the role of LSD1in the androgen-independent phenotypic transformation of Prostate Cancer.Part II Role and Mechanism of LSD1in the Androgen-Independent Phenotypic Transformation of Prostate CancerObjective To investigate the role and mechanism of LSD1in the acquisition of androgen-independent phenotype by prostate cancer.Methods1. CCK-8assay was used to analysize the proliferation of LNCaP-LSD1cells and LNCaP cells treated with bicalutamide; CCK-8assay was used for detected the proliferation of LNCaP-AI cells and LNCaP cells treated with LSD1-siRNA, con-siRNA, pargyline, tranylcypromine alone or combination with R1881.2. The differences in the AR mRNA and protein of LNCaP-LSD1cells and LNCaP cells were measured by quantitative PCR and Western Blot analysis. ELISA was used to examine PSA secretion of LNCaP-LSD1cells and LNCaP cells treated with the different concentrations of bicalutamide. After treated with LSD1-siRNA, con-siRNA alone or combination with bicalutamide, LNCaP-AI cells was tested about PSA secretion by ELISA.3. The flow cytometry was used for analysizing the apoptosis of LNCaP-AI cells treated with etoposide alone or combination with LSD1-siRNA, con-siRNA. The differences in the p53, p21, Bax mRNA and protein of LNCaP-LSD1cells and LNCaP cells were assessed via quantitative PCR and Western Blot analysis. The level of p21, HDM2mRNA and protein was analysized by quantitative PCR and Western Blot in LNCaP-AI cells treated with LSD1-siRNA. Western Blot analysis was used to analysize the changes in Bax, PUMA, and survivin protein of LNCaP-AI cells treated with LSD1-siRNA or Con-siRNA.Results1. CCK-8assay showed that the growth of LNCaP cells was inhibited in the androgen-deprived medium or by Bicalutamide, but LNCaP-LSD1cells could sustainedly proliferate. The cell viability obviously decreased in LNCaP-AI cells and LNCaP cells treated with LSD1-siRNA, pargyline, or tranylcypromine. R1881-induced proliferation of LNCaP-AI cells and LNCaP cells was suppressed by LSD1-siRNA or tranylcypromine.2. RT-PCR and western blot indicated that the expression of AR mRNA and protein was the same in LNCaP-LSD1cells as in LNCaP cells. ELISA showed that the secreted PSA protein was apparently higher in LNCaP-LSD1cells than in LNCaP cells.10μmol/L Bicalutamide obviously reduced PSA in LNCaP cells, but did not influence PSA in LNCaP-LSD1cells and LNCaP-AI cells. The secreted PSA was suppressed by10μmol/L Bicalutamide in LNCaP-AI cells treated with LSD1-siRNA.3. FCM assay confirmed that the treatment with LSD1-siRNA increased LNCaP-AI cells apoptosis induced by etoposide. RT-PCR and western blot showed that LNCaP-LSD1cells expressed the same p53mRNA and protein as LNCaP cells, but less p21, Bax mRNA and protein. RT-PCR revealed that LSD1-siRNA increased the expression of p21and HDM2mRNA in LNCaP-AI cells. Western blot indicated that p21, HDM2, Bax, and PUMA protein were obviously up-regulated and survivin protein was down-regulated in LNCaP-AI cells treated with LSD1-siRNA.Conclusions LSD1probably plays a vital role in the AI-phenotypic transformation of prostate cancer. LSD1is over-expressed in the androgen-deprived condition, which could promote prostate cancer to obtain androgen-independent phenotype by activating AR-dependent signal pathway and suppressing p53-mediated apoptotic pathway.