| BACKGROUND&OBJECTIVEProstate cancer is a major public health problem. According to reported estimates, prostate cancer is regarded as the second most common malignancy among men residing in the European Union and North America. In recent years, the morbidity rate of prostate cancer has been increasing steadily in China. For example, the annual morbidity rate of prostate cancer has increased by14%since1990. In contrast, the annual morbidity rate was quite stable in the1970s and1980s.Most cases of prostate cancer are responsive to androgen ablation therapy in the initial stages. However, many tumors eventually become androgen-refractory. Thus, these tumors become resistant to hormonal therapy with the passage of time. Eventually, metastatic phenotypes proliferate in patients suffering from prostate cancer.We have not been successful in devising an effective therapeutic approach to tackle cases of hormone-independent prostate cancer (HIPC). In fact, few biomarkers are capable of reasonably distinguishing aggressive and nonaggressive tumors after diagnosis. In other words, biomarkers with greater sensitivity and specificity can provide evidences for the diagnosis and prognosis of HIPC.Golgi phosphoprotein-3(GOLPH3) has several alternative names, such as GPP34, GMx33, MIDAS, and yeast Vps74p. It is a member of the trans-Golgi matrix family and binds to PtdIns(4)P-rich trans-Golgi membranes and MY018A. This indicates that a tensile force is required for efficient tubule and vesicle formation. Recently, several evidences suggest that GOLPH3is an oncogene, representing a first-in-class Golgi oncoprotein. GOLPH3, a novel oncogene, is commonly targeted for amplification in human cancer. Note that, an enhanced activation of mTOR signaling represents a molecular basis of GOLPH3’s oncogenic activity. However, research studies have seldom studied the correlation between GOLPH3expression and prognosis of Chinese patients with prostate cancer.In fact, very few studies have explored the transition from hormone-sensitive prostate cancer to HIPC.The role of GOLPH3gene in the development of malignant tumor and its mechanism research just getting started. This study focuses on stated the relationship between GOLPH3genes and the evolution of prostate cancer, and explore its potential signal network.METHODS1.Expression of GOLPH3in normal tissues and that related lesions The expression of GOLPH3was examined in specimens of28lung adenocarcinoma, gastric adenocarcinoma, colon adenocarcinoma, hepatocellular carcinoma, seminoma, endometrial adenocarcinoma and breast invasive ductal carcinoma,28paired nomal tissues by immunohistochemical EliVision method.The expression of GOLPH3was examined in specimens of139(36.48%) HDPC,102(26.77%) HIPC,21HIPC bone metastatic lesion,61(16.01%) high-grade prostatic intraepithelial neoplasia (HGPIN),20(5.25%) benign prostate hyperplasia (BPH),20(5.25%) normal prostate tissues by immunohistochemical EliVision method. 2.Effect of GOLPH3gene silencing on the biological characteristics of prostate cells2.1The expressions of GOLPH3gene in four prostate cell lines Dul45,PC-3,LNcap and BPH were detected by reverse transcriptase polymerase chain reaction (RT-PCR).2.2The expressions of GOLPH3protein in four prostate cell lines Dul45,PC-3,LNcap and BPH were detected by Western blot and immunofluorescense staining analysis.2.3We filtered effective interference fragments and transfected human prostate cancer cell lines, DU145/EGFP+with GOLPH3specific RNAi lentiviral vectors as well as controls. GOLPH3silenced cells were screened by blasticidin. Quantitative RT-PCR and Western blot analysis were used to detect the expressions of GOLPH3mRNA and protein, respectively. Flow cytometry, MTT assay,plate colony formation assay, transwell chamber and boydon chamber in vitro were used to assess the functional effects of GOLPH3silencing on tumor cell proliferation, invasion and metastasis.3. Exploring the possible molecular mechanism underlying GOLPH3-mediated multiple functions in prostate cancer using gene Gene expression microarray.The total of RNA of GOLPH3/EGFP+/GOLPH3-cells and the control were isolated and labeled by reverse transcription reaction for cDNA. Labeled cDNA were hybridized with cDNA microarray. After scanning and image processiong,the different gene expression profiling was investigated.RESULTSThe main results and findings are as follows:1.GOLPH3protein weakly positive expressed in normal tissues and parts of solid tumor tissues,such as colon adenocarcinoma. Intensely positive expression of GOLPH3was shown in lung adenocarcinoma, gastric adenocarcinoma, hepatocellular carcinoma, seminoma, endometrial adenocarcinoma and breast invasive ductal carcinoma.2.GOLPH3was expressed heterogeneously among the normal prostate tissue and related lesions. GOLPH3was weakly positive expressed in normal prostate(90%), BPH(95%),HGPIN(93.44%) and HDPC(85.61%), and the difference was not statistically (P=0.154). GOLPH3was intensely positive expressed in HIPC(81.37%) and bone metastatic lesion(95.24%). Compared with normal prostate, BPH, HGPIN and HDPC, the difference was nstatistically (P=0.234)3.RT-PCR, Western blot and immunofluorescense staining analysis show GOLPH3have different degrees of expression in transcription in PC-3,Du145,LNcap and BPH cell lines. The highest expression of GOLPH3was visualized in the Dul45and lower expressed in LNcap, PC-3and BPH.4.We constructed plasmids that expressed short hairpin RNAs that were targeted against GOLPH3under the control of the U6promoter by lentiviral vector. Three different sequences were originally selected for targeting the GOLPH3gene. We infected DU145/EGFP+cells with lentival vectors and examined GOLPH3mRNA and protein expression and found siGOLPH3-918was the most effective at blocking GOLPH3expression. Then, DU145/EGFP+cells were infected with pLenti6/si GOLPH3or pLenti6(control vector), and incubated with blasticidin to select blasticidin-resistant single clones. We found clone2exhibited a dramatic knock down of GOLPH3protein expression (Designated DU145/EGFP+/GOLPH3-). Western Blot and ICC analysis of the establishment of stable cells with knockdown of GOLPH3.5.DU145/EGFP+/GOLPH3-cells showed a significantly reduced proliferation compared with DU145/GFP+/mock cells as determined by in vitro MTT assay. In addition, DU145/EGFP+/GOLPH3-cells had a significant reduction in their ability to form colonies in plate as compared with DU145/GFP+/mock cells.The results of migration and invasion assays showed that the DU145/GFP+/mock cells posses a remarkable increasement in migration and invasiveness compared with DU145/EGFP+/GOLPH3-cells in vitro.6.Exploring the molecular mechanism underlying GOLPH3-mediated multiple functions in prostate cancer using gene microarray. Gene expression profiles of DU145/EGFP+/GOLPH3-and DU145/GFP+/mock cells were obtained and the microarray findings were confirmed by Real Time PCR. We obtained424of GOLPH3-related genes which165genes were down-regulated and259genes were up-regulated in DU145/EGFP+/GOLPH3-cells. The function of these424differentially expressed genes involved in cell cycle, regulation of cell cycle, cell motility, cell adhesion and interaction with the extracellular matrix. The pathway of these differentially expressed genes included TGF signalling pathway, Hedgehog pathway, MAPK signalling pathway and P53pathway.CONCLUSION1.Oncogene GOLPH3highly related with the evolution of prostate cancer.2.Oncogene GOLPH3plays an important role in promoting proliferation, metastasis and invasion of prostate cancer in vitro.3. TGF-β2, BMP7, CREB5, E2F2, ARRB2, TPK1, PLAT, SPP1, MAP2, CAV1and FLNA gene have close relationship with GOLPH3. GOLPH3may involve in many pathways to mediate evolution of prostate cancer, included TGF signalling pathway, Hedgehog pathway. We found GOLPH3may interacted with the extracellular matrix by AR-independent signaling pathway and regulated prostate cancer cell evolution. |
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