ABSTRACT
<p><b>OBJECTIVE</b>To detect the expression of the nucleostemin (NS) gene in prostate cancer PC-3, LNCaP and DU145 cells, and to study the effect of the NS gene on the proliferation of PC-3 cells after its silencing.</p><p><b>METHODS</b>The protein and mRNA expressions of NS in PC-3, LNCaP and DU145 cells were respectively detected by immunohistochemical staining and RT-PCR. An NS-specific short-hairpin RNA (shRNA) expression plasmid was used to transfect the PC-3 cells (NS-shRNA-PC-3), followed by observation of the changes of the NS gene and the proliferation and apoptosis of the cells.</p><p><b>RESULTS</b>The NS gene was highly expressed in the three types of cells. After the transfection, the NS expression and the proliferation of the NS-siRNA-PC-3 cells were remarkably reduced, while the percentage of the GO/G1 cells and the early apoptosis of the PC-3 cells obviously increased. A marked decrease was observed in the neoplasm forming ability of the NS-siRNA-PC-3 cells in the nude mice.</p><p><b>CONCLUSION</b>NS is highly expressed in prostate cancer cells. The proliferation of PC-3 cells is remarkably reduced and the early apoptosis of PC-3 cells increased after silencing the NS gene by NS-specific shRNA.</p>
Subject(s)
Animals , Humans , Male , Mice , Apoptosis , Carrier Proteins , Genetics , Cell Line, Tumor , Cell Proliferation , GTP-Binding Proteins , Gene Silencing , Mice, Nude , Neoplasm Proteins , Genetics , Nuclear Proteins , Genetics , Prostatic Neoplasms , RNA, Small Interfering , Reverse Transcriptase Polymerase Chain Reaction , TransfectionABSTRACT
<p><b>OBJECTIVE</b>To screen the genes and possible signal transduction pathways involved in the mechanism of nucleostemin (NS) in the proliferation of prostate cancer.</p><p><b>METHODS</b>Oligonucleotide DNA microarray was used to screen the genome changes after knocking-down expression of NS in PC-3 cells and quantitative real-time PCR was used to further confirm the important differentially expressed genes.</p><p><b>RESULTS</b>219 differentially expressed genes were found and theses genes were involved in cell cycle, cell proliferation, signal transduction, cell apoptosis and cell differentiation, etc. INK4 family genes (p15, p16, p18) were up-regulated and cyclin D1, HDAC1 were down-regulated, the main action points were CDK4/6-cyclin D and pRb-E2F1 complexes.</p><p><b>CONCLUSION</b>NS may promote the progression of prostate cancer by inhibiting the expression of p15, p16, and p18 in PC-3 cells. NS is an important G(1)/S checkpoint regulator and its regulatory activity has been certified at gene level.</p>
Subject(s)
Humans , Male , Apoptosis , Cell Cycle , Cell Line, Tumor , Cell Proliferation , Cyclin D1 , Metabolism , Cyclin-Dependent Kinase Inhibitor Proteins , Metabolism , GTP-Binding Proteins , Genetics , Gene Expression Profiling , Gene Expression Regulation, Neoplastic , Gene Knockdown Techniques , Histone Deacetylase 1 , Metabolism , Nuclear Proteins , Genetics , Oligonucleotide Array Sequence Analysis , Prostatic Neoplasms , Genetics , Metabolism , Pathology , RNA Interference , Signal TransductionABSTRACT
<p><b>OBJECTIVE</b>To explore the expression of the nucleostemin (NS) gene in prostate cancer (PCa) tissues and its clinical significance.</p><p><b>METHODS</b>We detected the NS expression in PCa, benign prostatic hyperplasia (BPH) and high grade prostatic intraepithelial neoplasia (HGPIN) tissues by RT-PCR and immunohistochemistry, and analyzed the correlation between the expression of the NS protein and the clinical variables of PCa.</p><p><b>RESULTS</b>The NS mRNA level was markedly higher in the PCa than in the BPH tissues. The rates of strongly positive, positive and weakly positive expressions of the NS protein were 48.8%, 36.6% and 12.2% in PCa, 4.0%, 32.0% and 56.0% in BPH, and 5.0%, 25.0% and 60.0% in HGPIN, respectively. The expression level of the NS protein was significantly higher in PCa than in BPH and HGPIN (P < 0.05). The expression of the NS gene was negatively correlated with the degree of cell differentiation in the PCa tissues, the worse the differentiation, the higher the NS expression level.</p><p><b>CONCLUSION</b>The NS gene is highly expressed in PCa tissues and may have an important role in the adverse differentiation and malignant proliferation of prostate cancer.</p>
Subject(s)
Aged , Humans , Male , Middle Aged , Carrier Proteins , Genetics , GTP-Binding Proteins , Immunohistochemistry , Neoplasm Staging , Nuclear Proteins , Genetics , Prostatic Neoplasms , Genetics , Metabolism , Pathology , RNA, Messenger , Genetics , Metabolism , Reverse Transcriptase Polymerase Chain ReactionABSTRACT
<p><b>BACKGROUND</b>Nucleostemin is essential for the proliferation and survival of stem and cancer cells, but it is unknown whether this newly identified molecule is involved in prostate cancer pathogenesis.</p><p><b>METHODS</b>Total RNA and protein were extracted from prostate cancer tissues and PC-3, LNCap and DU145 cell lines. The nucleostemin mRNA and protein expression were measured by RT-PCR and Western blot. Immunohistochemistry was also used to detect the nucleostemin protein expression in prostate cancer tissues and PC-3 cells. A nucleostemin specific, short hairpin RNA, expression plasmid was used to transfect PC-3 cells. The changes of nucleostemin gene were detected and the proliferative capacity of the cells was determined.</p><p><b>RESULTS</b>Nucleostemin was highly expressed in prostate cancer tissues and cell lines. Nucleostemin expression level in the silencer group PC-3 cells remarkably reduced. The proliferation rate of silencer group PC-3 cells decreased and the percentage of G1 stage cells increased. The neoplasm forming capacity in nude mice of the silencer group PC-3 cells decreased significantly.</p><p><b>CONCLUSIONS</b>Nucleostemin is highly expressed in prostate cancer tissues and cell lines. The proliferative capacity of PC-3 cells is remarkably reduced after silencing nucleostemin gene expression.</p>
Subject(s)
Animals , Humans , Male , Mice , Apoptosis , Carrier Proteins , Genetics , Physiology , Cell Cycle , Cell Line, Tumor , Cell Proliferation , GTP-Binding Proteins , Nuclear Proteins , Genetics , Physiology , Prostatic Neoplasms , Genetics , Pathology , RNA, MessengerABSTRACT
<p><b>OBJECTIVE</b>To construct a recombinant human CD59 gene containing intercellular adhesion molecule-2 promoter for high level endothelial-specific expression in xenotransplantation.</p><p><b>METHODS</b>ICAM-2 promotor fragment and CD59-intron 1 fragment were produced by PCR from the human blood genome, and then clone these fragments into a pcDNA3-CD59 eukaryotic expression vector which was followed by digestion with the specific restricted endonuclease (for example: EcoRI, Hind III). The ICAM-2 promoter and CD59-intron 1 fragments were identified by PCR, and sequencing. The recombinant was then transfected into pig aorta endothelial cells with Lipofection, and the expression was measured by flow cytometer.</p><p><b>RESULTS</b>Products of the sequences measured were in accord with the frames of the gene bank. The expression of the protein of this recombinant was positive.</p><p><b>CONCLUSION</b>The CD59 recombinant gene is constructed successfully, providing a basis for transgenic research.</p>