The Studies On The Expression Of Novel Golgi Proteins In Prostate Cancer And The Mechanism Of GOLPH3Promotes Prostate Cancer Metastasis

Part Ⅰ. The expression and significance of novel Golgi proteins in prostate cancer cells and tissuesObjective:The aim of this study was to investigate the characteristic expression and clinical pathological significance of GOLPH3/GOLPH2, novel Golgi proteins, in prostate cancer cell lines and tissues.Methods:(1) We investigated the GOLPH3mRNA and protein levels in the four most commonly used prostate cancer cell lines by quantitative real-time polymerase chain reaction and Western blot analysis, respectively.(2) Furthermore, we investigated the localization of GOLPH3/GOLPH2in prostate cancer cell DU145by immunofluorescence.(3) We performed a comprehensive GOLPH3/GOLPH2expression analysis in prostate using a tissue microarray (including50different cases of prostate adenocarcinoma along with20cases of benign prostatic hyperplasia and10cases of normal prostate tissues) and analyzed differential expression of GOLPH3/GOLPH2in prostate tissues.Results:(1) Novel Golgi proteins GOLPH3/GOLPH2were significantly expressed in prostate cancer cell lines, i.e., PC-3, DU145, LNCaP, and22RV1, among those cells, the expression of GOLPH3/GOLPH2were much higher in androgen-independent cell lines (PC-3and DU145) than those detected in androgen-dependent cell lines (LNCaP and22RV1).(2) The immunofluorescence results demonstrated that GOLPH3/GOLPH2were located at the trans-Golgi and cis-Golgi in prostate cancer cell DU145, respectively.(3) Tissue microarray immunohistochemistry demonstrated that GOLPH3/GOLPH2proteins were differently expressed in prostate cancer, benign prostatic hyperplasia, and normal prostate tissues. Positive expression of GOLPH3in prostate cancer, benign prostatic hyperplasia, and normal prostate was64%(32/50),30%(6/20), and20%(2/10), respectively, and that of GOLPH2in prostate cancer, benign prostatic hyperplasia, and normal prostate was92%(46/50),50%(10/20), and40%(4/10), respectively, and there were significant differences (P<0.05). However there were no significant differences between GOLPH3/GOLPH2overexpression and pathological variables of prostate cancer including pathological stage and Gleason grade (P>0.05).Conclusions:(1) Novel Golgi proteins GOLPH3/GOLPH2are significantly expressed in prostate cancer cell lines and prostate cancer tissues.(2) GOLPH3/GOLPH2aberrant expression in prostate cancer cells may be related to hormone-sensitive.(3) GOLPH3/GOLPH2aberrant expression may be an important characteristic of prostate cancer. Analysis of the characteristic expression of GOLPH3/GOLPH2can be useful for the diagnosis of prostate cancer.Part Ⅱ. The effect of GOLPH3RNAi silencing on the biological behavior of prostate cancer cell PC-3Objective:The aim of this study was to evaluate the effect of GOLPH3RNAi silencing on the ability of proliferation, invasion, and migration of prostate cancer cell PC-3, further to investigate roles of GOLPH3in the initiation and metastasis of prostate cancer. Methods:(1) Seven pairs shRNA primers were designed and synthesised, and the pUEGH vector was digested with restrictive endonuclease EcoRI and BamHI, then the linearized pUEGH linked with shRNA. The resulting vector was transformed into GeneHogs. Restrictive endonuclease digestion analysis was used to verify the location and integrity of the inserted fragment. Lentiviral expression plasmid and lentiviral packaging mix co-transfected into293T cells, then the transfections were packed into the lentiviral vector pUEGH-GOLPH3shRNA.(2) The expression of GOLPH3mRNA and protein levels in prostate cancer cells transfected with lentiviral was investigated by qPCR and Western blot assay, respectively, then the effect of RNAi interference was to evaluate.(3) The effect of proliferation and growth of PC-3cells transfected with GOLPH3RNAi was detectd by the MTT assay. The ability of invasion and migration of PC-3cells transfected with GOLPH3shRNA was detected using a transwell system.Results:(1) Lentiviral vector pUEGH-GOLPH3shRNA was successfully constructed confirmed by PCR and enzymatic digestion.(2) After lentiviral vector of GOLPH3shRNA and negative control lentiviral vector transfected into PC-3cells, the qPCR and Western blot analysis demonstrated that the expression of GOLPH3mRNA levels and protein levels in PC-3cells transfected with lentiviral vector of GOLPH3shRNA was decreased79.40%and75.35%compared with the negative lentiviral vector transfected cells (P<0.05). GOLPH3shRNA inhibition rate of GOLPH3at the mRNA and protein levels was very satisfactory.(3) Cell proliferation assay showed that lentiviral vector of GOLPH3shRNA group had no significant effect on the proliferation of PC-3cells, compared with the negative control lentiviral vector group (P>0.05). Invasion and migration assays showed that the number of the transfected-GOLPH3shRNA PC-3cells passed through the membrane was significantly decreased than the negative control group (P<0.05). After GOLPH3gene silencing, the ability of PC-3cells invasion and migration was significantly inhibited.Conclusions:(1) These results show that the stable GOLPH3gene silencing-transfection of prostate cancer cells has been successfully constructed.(2) GOLPH3gene silencing by lentivirus-mediated RNA interference effectively inhibits the expression of GOLPH3in prostate cancer cell PC-3.(3) After GOLPH3gene silencing, prostate cancer cells proliferation and cell growth are not significantly inhibited, however, prostate cancer cells invasion and migration are significantly decreased. These findings show that GOLPH3play an important role in the process of prostate cancer metastasis, and downregulation of the GOLPH3by RNAi technology is expected to become an effective new approach for the treatment of prostate cancer.Part Ⅲ. Exploring molecular mechanisms of GOLPH3RNAisuppresses invasion and migration of prostate cancer cell PC-3 Objective:To explore molecular mechanisms of the shRNA-mediated GOLPH3silencing suppresses invasion and migration of prostate cancer cell PC-3.Methods:(1) The expression of metastasis-related genes and factors, i.e., IGF1, Furin, MMP1, MMP2, MMP3, MMP9, MMP10, MMP14, TIMP2, VIM, MSN, E-Cadherin, CD44, MYH9, VEGF-A, and ACTR2in the stable GOLPH3RNAi-transfection of prostate cancer cells and its control stable cell lines was investigated at mRNA levels by qPCR.(2) The expression of positive metastasis-related genes and factors confirmed at mRNA levels in the stable GOLPH3RNAi-transfection of prostate cancer cells and its control stable cell lines was further investigated at protein levels by the Western blot assay.Results:(1) The qPCR results demonstrated that the expression of IGF1, MMP2, MMP9, MSN, VIM, E-Cadherin, and TIMP2in PC-3cells transfected with GOLPH3RNAi at mRNA levels was decreased, of which IGF1, MMP2, MMP9, and VIM mRNA expression was decreased significantly (P<0.05). While the expression of Furin, MMP1, MMP3, MMP10, MMP14, CD44, MYH9, VEGF-A, and ACTR2mRNA was elevated (2) The Western blot results further showed that the expression of MMP9in PC-3cells transfected with GOLPH3RNAi at protein levels was significantly decreased, other proteins expression levels were not changed significantly.Conclusions:(1) GOLPH3promotes prostate cancer cell invasion and migration by increasing the expression of IGF1、MMP2、MMP9, and VIM at transcriptional levels.(2) GOLPH3promotes prostate cancer cell invasion and migration by increasing MMP9expression at protein levels.(3) Regulation of metastasis related genes by GOLPH3at protein levels have no consistent with transcriptional levels, suggesting that regulation of GOLPH3in prostate cancer cells may be the existence of a negative feedback mechanism.Part Ⅳ. The study on modulation of GOLPH3with epidermal growth factor receptor (EGFR) signaling pathway Objective:To explore modulation of the shRNA-mediated GOLPH3silencing with EGFR signaling pathway.Methods:The expression of key proteins of EGFR signaling pathway, i.e., EGFR, p-EGFR, Src, p-Src, FAK, p-FAK, Akt, p-Akt, mTOR, p-mTOR, p70S6K, p-p70S6K in the stable GOLPH3RNAi-transfection of prostate cancer cells and its negative control stable cell lines was investigated by the Western blot assay.Results:The Western blot analysis showed that the expression of p-EGFR、p-Src protein in PC-3cells transfected with GOLPH3RNAi was significantly decreased, other proteins, i.e., EGFR, Akt, of pAkt, mTOR, p-mTOR, Src, FAK, p-FAK, p70S6K, and p-p70S6K protein expression were elevated or not changed significantly. Blocking GOLPH3could inhibit EGFR-Src signaling pathway, but had little effect on Akt-mTOR-p70S6K signaling pathway and FAK signaling pathway.Conclusions:GOLPH3can regulate the expression of the EGFR protein phosphorylation and its downstream Src protein phosphorylation, but can not regulate the EGFR downstream Akt, mTOR, FAK, and p70S6K protein expression and phosphorylation. GOLPH3may regulate prostate cancer invasion and metastasis by modulating EGFR-Src signaling pathway and its substrate MMP9.