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【Objective】 To investigate the expression profile of circRNA in nuclear receptor NURR1 overexpressed prostate cancer (PCa) cells, so as to provide reference for revealing the mechanism of PCa progression. 【Methods】 The expression of NURR1 in PCa was analyzed with UALCAN and TNMplot. The distinct circRNAs in NURR1 overexpressed PCa cells were screened with RNA-sequencing. The functions and signal pathways of differentially expressed circRNA molecules were analyzed with GO and KEGG. 【Results】 The circ_0000915 was significantly downregulated in DU145, LNCaP and PC3 cells. In NURR1 overexpressed DU145 cells, circ_0005991 was up-regulated, while circ_0001460 and circ_0001315 were down-regulated. In NURR1 overexpressed LNCaP cells, circ_0040729 and circ_0000722 were significantly up-regulated. In NURR1 overexpressed PC3 cells, circ_0001577, circ_0000854 and circ_0018168 were up-regulated, while circ_013035, circ_0003028, circ_0082096 and circ_0005320 were down-regulated. KEGG analysis revealed that the differentially expressed circRNAs were significantly associated with dorsal/ventral neural tube patterns, protein folding chaperones, disordered domain specific binding, positive regulation of BMP signaling pathways, and neural tube patterning functions. 【Conclusion】 CircRNAs play an important role in NURR1 mediated PCa progression, but there are certain differences among different prostate cancer cell types. The regulatory mechanism between NURR1 and circ_0000915 in the progression of PCa needs further investigation.
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Objective To propose a method for determing cell Poisson’s ratio based on micropipette aspiration technique. Methods Based on the assumption of deformation symmetry, the analytical expression between Poisson’s ratio and the amount of deformation was derived by extracting the extrusion deformation characteristics of the cells under micropipette aspiration according to the generalized Hooke’s law. The accurate determination of Poisson’s ratio of cells was realized according to position changes of markers on the surface of cell membrane. ResultsThe Poisson’s ratio of LNCaP cells in prostate cancer cells was measured. The result showed that the Poisson’s ratio of LNCaP cells was between 0.44 and 0.46, with an average value of 0.45. The influence of the location of the same cell feature points on calculation results of Poisson’s ratio was within the error range of 1.6%. Conclusions This method is simple and feasible, can improve the measurement accuracy of Poisson’s ratio of cells, and is helpful for cell detection and screening by using cell mechanical properties in clinic.
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Objective@#To investigate the effect of the down-regulated expression of pituitary tumor-transforming gene 1 (PTTG1) on the senescence of human castration-resistant prostate cancer LNCaP-AI cells.@*METHODS@#Human castration-resistant prostate cancer LNCaP-AI cells were induced in vitro and transfected with siRNA targeting PTTG1 (the siRNA-PTTG1 group), the reagent lip3000 only (the mock group) or siRNA negative control vector (the NC group). All the cells were cultured in fetal bovine serum (FBS) or charcoal-stripped bovine serum (CSS) and counted with the cell counting chamber. The senescence characteristics of the transfected LNCaP-AI cells were examined by senescence-associated β-galactosidase (SA-β-Gal) staining, and the expressions of the senescence-related β-galactosidase-1-like proteins (Glb1), the cyclin-dependent kinase inhibitors p-21CIP1 and p-27Kip1, and the chromatin-regulating heterochromatin protein 1γ (HP1γ) were detected by Western blot.@*RESULTS@#The expression of PTTG1 in the human prostate cancer LNCaP-AI cells was significantly reduced in the siRNA-PTTG1 group compared with those in the mock and NC groups (0.21 ± 0.01 vs 0.56 ± 0.02 and 0.61 ± 0.02, P < 0.05). Culture with FBS markedly increased while that with CSS decreased the number of LNCaP-AI cells transfected with siRNA, but both FBS and CSS enhanced the proliferation of the LNCaP-AI cells in the mock and NC groups. SA-β-Gal staining revealed that reducing the expression of PTTG1 induced a remarkably higher positive rate of the LNCaP-AI cells in the siRNA-PTTG1 than in the mock and NC groups ([63.5 ± 2.35]% vs [11.3 ± 1.24]% and [12.4 ± 1.15]%, P < 0.05). The siRNA-PTTG1 group, in comparison with the mock and NC groups, showed a significantly down-regulated expression of PTTG1 (0.21 ± 0.01 vs 0.56 ± 0.02 and 0.61 ± 0.02, P < 0.05), but up-regulated expressions of p-21CIP1 (0.32 ± 0.03 vs 0.20 ± 0.02 and 0.21 ± 0.03, P < 0.05), p-27Kip1 (0.38 ± 0.02 vs 0.20 ± 0.03 and 0.22 ± 0.01, P < 0.05), Glb1 (0.24 ± 0.01 vs 0.13 ± 0.01 and 0.15 ± 0.01, P < 0.05), and HP1γ (0.41 ± 0.01 vs 0.26 ± 0.01 and 0.27 ± 0.02, P < 0.05) in the LNCaP-AI cells.@*CONCLUSIONS@#Down-regulated expression of PTTG1 induces senescence of human castration-resistant prostate cancer LNCaP-AI cells.
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Objective@#To investigate the effects of long non-coding RNA RP1-90L14.1 on the proliferation, migration and invasion of prostate cancer LNCaP cells and the expressions of GRIN2A and BACE2.@*METHODS@#Using RT-PCR, we detected the expression of RP1-90L14.1 in LNCaP and LNCaP-AI cells, transiently transfected the RP1-90L14.1 overexpression plasmid (the RP1-90L14.1 group) and vector plasmid (the LNCaP-NC group) into the LNCaP cells, and cultured the two groups of cells with ordinary medium and phenol red-free activated carbon adsorption medium (PRF-ACA). Then we examined the proliferation, migration and invasiveness of the cells by CCK-8 and Transwell, and determined the mRNA and protein expressions of GRIN2A and BACE2 by RT-PCR and Western blot.@*RESULTS@#The expression of RP1-90L14.1 was significantly higher in the LNCaP-AI than in the LNCaP cells (8.49 ± 0.43 vs 2.53 ± 0.95, P < 0.05), and so was that of LNCaP-RP1-90L14.1 in the RP1-90L14.1 than in the LNCaP-NC group after transfection (0.71 ± 0.22 vs 0.02 ± 0.01, P < 0.05). The optical densities (OD) of the cells were 51.95% and 50.69% higher in the RP1-90L14.1 than in the LNCaP-NC group after 72 hours of culture with ordinary medium and phenol red-free ACA (1.22 ± 0.08 vs 0.08 ± 0.05, P < 0.05; 0.79 ± 0.02 vs 0.53 ± 0.05, P < 0.05), and 51.72% and 60.23% higher in the former than in the latter after 96 hours (1.72 ± 0.07 vs 1.13 ± 0.05, P < 0.05; 1.18 ± 0.05 vs 0.73 ± 0.08, P < 0.05). The numbers of the migrating cells cultured with common medium and PRF-ACA were markedly higher in the RP1-90L14.1 than in the LNCaP-NC group after transfection (682.0 ± 42.7 vs 422.0 ± 37.1, P < 0.05; 419.0 ± 42.9 vs 251.0 ± 25.9, P < 0.05), and so were those of the invading cells (507.0 ± 22.2 vs 274.0 ± 19.6, P < 0.05; 352.0 ± 14.1 vs 216.0 ± 14.3, P < 0.05). Statistically significant differences were observed between the RP1-90L14.1 and LNCaP-NC groups in the mRNA and protein expressions of GRIN2A (5.13 ± 0.89 vs 2.09 ± 0.54, P < 0.05; 5.88 ± 0.29 vs 2.03 ± 0.22, P < 0.05) and BACE2 (5.82 ± 0.50 vs 2.53 ± 0.30, P < 0.05; 4.89 ± 0.19 vs 3.37 ± 0.13, P < 0.05).@*CONCLUSIONS@# lncRNA RP1-90L14.1 may play important roles in the proliferation, migration and invasiveness of prostate cancer cells. RP1-90L14.1 can promote the expressions of GRIN2A and BACE2 and may have an endogenous competitive relation with GRIN2A and BACE2.
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@# Objective: To investigate the effects of prostate cancer exosomes on the migration and invasion ability of stromal cells (WPMY-1), and to explore its mechanism. Methods: Exosomes in LNCaP-AI+F prostate cancer cell supernatant were isolated by ultracentrifugation and the typical structure of exosome was captured by electron microscope. The particle size distribution was analyzed by Zetaview, and Wb was used to identify the marker proteins and other proteins.After co-incubation of WPMY-1 cells and prostate cancer exosomes (40 µg/ml), laser confocal microscope was used to observe the uptake of PKH67-labeled exosomes by WPMY-1 cells; Transwell assay was used to detect the migration and invasion ability of WPMY-1 cells; qPCR was performed to detect the expression of three cancer-associated fibroblast (CAF)-related molecules (IL-8, PDGFB and MMP9) at mRNA level; and the phosphorylation of EGFR and ERK1/2 was analyzed by Wb. Results: Typical cup-shaped structure of exosomes was observed under electron microscope. The Zetaview results showed that the particle size distribution was concentrated at about 100 nm. The expression of exosome marker proteins CD63 andALIX further verified that the isolated particles were exosomes. Besides, EGFR, HER2 and SRC, which were related to the progression of prostate cancer, were also enriched in exosomes. After co-incubation, confocal microscope imaging showed a number of PKH67 labeled exosomes in recipient WPMY-1 cells. Transwell experiments showed that exosomes could significantly enhance the migration and invasion ability of WPMY-1 cells (all P<0.01). Compared with the control group, increased secretion of IL-8, PDGFB and MMP9 was observed after exosome treatment (40 µg/ml) (P<0.05 or P<0.01). Wb indicated that exosomes could promote the phosphorylation of EGFR and ERK1/2 of WPMY-1 cells (P<0.01). Conclusion: Prostate cancer cell exosomes could act on the stromal cell WPMY-1 to highly express multiple CAF-related molecules, promote the phosphorylation of EGFR and ERK1/2 and enhance the migration and invasion ability of WPMY-1 cells.
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Objective@#To establish enzalutamide-resistant human prostate cancer cell lines and screen out the lncRNA and mRNA expression profiles associated with enzalutamide resistance.@*METHODS@#Human prostate cancer cell lines LNCAP and C4-2B were cultured with 10 μmol/L enzalutamide for 6 months in vitro for the establishment of enzalutamide-resistant subclones LNCAP-ENZA and C4-2B-ENZA. The IC50 value and enzalutamide resistance index of each cell line were examined by MTT assay, the expressions of enzalutamide-related genes FL-AR, AR-V7 and HnRNPA1 were determined by Western blot, and the lncRNA and mRNA differential expressions of C4-2B and C4-2B-ENZA were detected by high-throughout lncRNA microarray.@*RESULTS@#Compared with LNCAP and C4-2B, the IC50 values of enzalutamide-resistant subclones LNCAP-ENZA (60.83 μmol/L) and C4-2B-ENZA (88.32 μmol/L) were increased significantly (P < 0.05) and the enzalutamide-resistance indexes of the LNCAP-ENZA and C4-2B-ENZA cells were 4.94 and 4.67, respectively. The expressions of AR-V7 and HnRNPA1 were markedly up-regulated in the LNCAP-ENZA and C4-2B-ENZA cells as compared with those in the LNCAP and C4-2B cells, but that of FL-AR showed no significant change. A total of 1 440 lncRNAs and 1 236 mRNAs were identified as differentially expressed in the C4-2B-ENZA cells.@*CONCLUSIONS@#Enzalutamide -resistant human prostate cancer cell subclones LNCAP-ENZA and C4-2B-ENZA were successfully established and enzalutamide resistance-associated lncRNA and mRNA were identified, which may provide some molecular evidence for the management of enzalutamide-resistant human prostate cancer.
Subject(s)
Humans , Male , Cell Line, Tumor , Drug Resistance, Neoplasm , Phenylthiohydantoin , Pharmacology , Prostatic Neoplasms , Drug Therapy , Genetics , Pathology , RNA, Long Noncoding , Metabolism , RNA, Messenger , Metabolism , RNA, Neoplasm , Metabolism , Receptors, AndrogenABSTRACT
Objective To construct the lentiviral expression vector of SOX9 gene and establish a LNcap cell strain with stable expression of SOX9 . Methods SOX9 gene was amplified by PCR and cloned into lentiviral expression vector pLVX-IRES-Puro. pLVX-IRES-FLAG-SOX9 recombinant plasmid was verified by restriction enzyme digestion and DNA sequencing. Then we gained recombinant virus particles in packaging cell HEK 293 and infected LNcap cell. The monoclonal LNcap cell strain stably expressed were obtained through puromycin screening. The mRNA level and protein level in the infected LNcap cell were detected by qRT-PCR and Western blot respectively. Results Restriction enzyme digestion and sequencing demonstrated that SOX9 cDNA was successfully cloned into pLVX-IRES-FLAG lentiviral vector. After the transfection to LNCaP cells, the monoclonal cell strain of stably expressed SOX9 were obtained by puromycin screening, which showed expression of SOX9 mRNA detected by qRT-PCR. Western blot analysis revealed that SOX9 protein expressed markedly. Conclusion The recombinant lentiviral vector bearing human SOX9 cDNA has been successfully constructed, and the exogenous expression of SOX9 in LNcap cells was achieved.
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Objective@#To investigate the anti-prostate cancer (PCa) effect of roemerine in vitro and in vivo in the mouse model of PCa.@*METHODS@#We detected the effects of roemerine on the proliferation, apoptosis and migration of PCa cells DU145, LNCaP, PC-3 and 22RV1, screened out the sensitive cell line and constructed a tumor-bearing model in mice for verification of the antitumor efficacy of roemerine in vivo.@*RESULTS@#Roemerine inhibited the proliferation and migration of the DU145, LNCaP, PC-3 and 22RV1 cells and induced their apoptosis in different degrees, particularly those of the LNCaP cells. The average tumor weight was less in the roemerine intervention group ([1.99±0.95] g) than in the control ([2.95±1.04] g), the least in the high-dose roemerine (30 mg/kg) plus paclitaxel intervention group ([0.90±0.16] g). The mean heart, liver, and kidney indexes were markedly lower in the roemerine (0.58±0.06, 6.20±0.42 and 1.49±0.33) than in the paclitaxel group (0.66±0.04, 6.99±0.72 and 1.95±0.34), while the mean spleen and thymus indexes were remarkably higher in the former (0.54±0.11 and 0.06±0.01) than in the latter (0.41±0.09 and 0.05±0.01). Pathological staining showed a lower degree of malignancy and metastasis in both the roemerine and the roemerine + paclitaxel intervention group than in the control, as well as a lower degree of visceral injury in the roemerine and roemerine + paclitaxel groups than in the paclitaxel group.@*CONCLUSIONS@#Roemerine has some anti-PCa effect and alleviates adverse reactions in paclitaxel combination administration.
Subject(s)
Animals , Male , Mice , Alkaloids , Therapeutic Uses , Antineoplastic Agents, Phytogenic , Therapeutic Uses , Apoptosis , Cell Line, Tumor , Cell Movement , Cell Proliferation , Disease Models, Animal , Drug Therapy, Combination , Methods , Drugs, Chinese Herbal , Therapeutic Uses , Mice, Nude , Paclitaxel , Therapeutic Uses , Prostatic Neoplasms , Drug TherapyABSTRACT
Objective@#To investigate the effects of down-regulation of PTTG1 expression on the proliferation, invasiveness and apoptosis of androgen-independent human prostate cancer LNCaP-AI cells and their sensitivity to androgen antagonists.@*METHODS@#Human prostate cancer LNCaP-AI cells were transfected with siRNA targeting the PTTG1 gene using the Lipofectamine 2000 transfection reagent. The proliferation, invasiveness and apoptosis of the cells were detected by MTT, Transwell assay and flow cytometry, respectively. The protein expressions of PTTG1, p-Akt, and p-ERK were determined by Western blot and the mRNA expression of PTTG1 measured by agarose gel electrophoresis.@*RESULTS@#The siRNA expression vector markedly down-regulated the expression of PTTG1, which effectively suppressed the proliferation of the LNCaP-AI cells, with the inhibition rates of (19.47 ± 2.12), (24.01 ± 2.13) and (48.02 ± 2.22)% at 24, 48 and 72 hours, respectively, after transfection, with statistically significant differences among the three groups (P <0.05). The number of the cells passing through the polycarbonate film was remarkably decreased at 24, 48 and 72 hours (74.67 ± 9.85, 56.44 ± 8.66 and 37.33 ± 6.14) as compared with the baseline (111.11 ± 13.47) (P <0.01), while the apoptosis rate of the cells was significantly increased at 24, 48 and 72 hours (18.32 ± 0.94), (19.94 ± 1.30) and (21.73 ± 1.88)% in comparison with the baseline ([2.17 ± 0.49]%), (P <0.05). PTTG1 siRNA combined with androgen antagonist flumatide exhibited even more significant effects in inhibiting the proliferation and promoting the apoptosis of the LNCaP-AI cells than either used alone, and in a flumatide dose-dependent manner. The inhibition and apoptosis rates of the LNCaP-AI cells treated with 50 nmol/L flumatide were (27.13 ± 3.52) and (3.94 ± 0.48)%, and those treated with siRNA + 50 nmol/L flumatide were (67.51 ± 5.13) and (19.93 ± 1.72)%, respectively, both with statistically significant differences between the two groups (P <0.05). The inhibition and apoptosis rates of the cells treated with 100 nmol/L flumatide were (43.72 ± 3.90) and (5.33 ± 0.66)%, and those treated with siRNA + 100 nmol/L flumatide were (73.19 ± 4.78) and (23.43 ± 1.76)%, respectively, both with statistically significant differences between the two groups (P <0.05).@*CONCLUSIONS@#The siRNA expression vector can down-regulate the expression of PTTG1, which can inhibit the proliferation and invasiveness of LNCaP-AI cells, promote their apoptosis, and increase their sensibility to androgen antagonists. Suppressing the expression of PTTG1 may enhance the effect of androgen-deprivation therapy on advanced prostate cancer.
Subject(s)
Humans , Male , Androgen Antagonists , Pharmacology , Apoptosis , Cell Line, Tumor , Cell Proliferation , Down-Regulation , Neoplasm Invasiveness , Prostatic Neoplasms , Drug Therapy , Metabolism , Pathology , RNA, Small Interfering , Metabolism , Securin , Genetics , Metabolism , Time Factors , TransfectionABSTRACT
<p><b>OBJECTIVE</b>To evaluate whether mono (2-ethylhexyl) phthalate (MEHP) affects genomic DNA methylation and the methylation status of some specific genes such as patched gene (PTCH) and smoothened gene (SMO) in LNCaP cells.</p><p><b>METHODS</b>LNCaP cells were treated with MEHP (0, 1, 5, 10, and 25 μmol/L) for 3 days. An ELISA assay was preformed to detect genomic methylation, including 5-methylcytosine (5-mC) and 5-hydroxymethylcytosine (5-hmC) content. A pyrosequencing assay was applied to assess DNA methylation in PTCH and SMO gene promoters. The correlation between DNA methylation and gene expression was assessed.</p><p><b>RESULTS</b>The proportion of cytosines with 5-mC methylation in LNCaP cells was significantly decreased by MEHP (1, 5, 10, and 25 μmol/L) in a dose-dependent manner (P < 0.01). For genes in the Hedgehog pathway, there was no significant MEHP concentration-dependent difference in the DNA methylation of PTCH and SMO.</p><p><b>CONCLUSION</b>MEHP might affect the progression of prostate cancer through its effect on global DNA methylation.</p>
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Humans , Male , Antineoplastic Agents , Chemistry , Pharmacology , Cell Line, Tumor , DNA Methylation , Phthalic Acids , Chemistry , Prostatic Neoplasms , MetabolismABSTRACT
To determine whether PlncRNA-1 induces apoptosis in prostate cancer cells through the Her-2 pathway. The expression of PlncRNA-1, Her-2, and related cyclin proteins in 23 cases of prostate cancer and adjacent normal tissues was analyzed and compared. LNCaP cells were divided into a control group and an LNCaP-PlncRNA-1-siRNA experimental group. Normal prostate RWPE-1 cells were divided into an RWPE-1 control group and an RWPE-1-PlncRNA-1 experimental group. After PlncRNA-1 silencing and overexpression, changes in Her-2 and cyclinD1 expression levels were detected both in vivo and in vitro. In prostate cancer tissues, Her-2 and PlncRNA-1 were highly expressed and significantly correlated. In LNCaP cells, the expression of Her-2 and cyclinD1 decreased following the downregulation of PlncRNA-1 as assessed by real-time PCR and Western blotting. In RWPE-1 cells, the expression of Her-2 and cyclinD1 increased following PlncRNA-1 overexpression. Flow cytometry revealed that the proportion of LNCaP cells in G2/M phase was significantly increased after PlncRNA-1 silencing and that the proportion of RWPE-1 cells in G2/M phase was significantly decreased after PlncRNA-1 overexpression. Furthermore, animal experiments validated these results. In conclusion, in prostate cancer, PlncRNA-1 regulates the cell cycle and cyclinD1 levels and can also regulate proliferation and apoptosis in prostate cancer cells through the Her-2 pathway.
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[ ABSTRACT] AIM:To explore the role of SHARPIN in regulation of Rip1 in castration-resistant prostate cancer LNCaP-AI cells.METHODS:The LNCaP-AI cells were treated with TNF-α+Z-VAD ( an inhibitor of pan-caspase) to activate necroptosis, which were compared to the cells treated with TNF-α+Z-VAD+Nec-1 ( an inhibitor of Rip1 ) .A blank group and a TNF-α-treated group were set up as controls.The cell viability in each group was measured by MTS as-say.In addition, SHARPIN was knocked down by siRNA, and the inhibitory efficiency was evaluated by RT-qPCR.The expression of Rip1 at mRNA and protein levels after knocking down SHARPIN was determined by RT-qPCR and Western blot to explore the underlying mechanism of regulatory network of necroptosis in prostate cancer.RESULTS: Compared with blank control group and TNF-α-treated group, the viability of LNCaP-AI cells treated with TNF-α+Z-VAD decreased by 28%(P LNCaP-AI cells.CONCLUSION:Necroptosis is an important way of cell death .Inhibition of oncogenic factor SHARPIN enhances necroptosis via activating Rip1 in LNCaP-AI cells.
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<p><b>Objective</b>To explore the expression of pituitary tumor transforming gene 1 (PTTG1) during the transformation of prostate cancer from androgen-dependent (ADPC) to androgen-independent (AIPC).</p><p><b>METHODS</b>We established an AIPC cell model LNCaP-AI by culturing the androgen-dependent LNCaP cell line in the hormone-deprived medium for over 3 months. The cell model was verified and the PTTG1 expression in the LNCaP cells was detected by Western blot and RT-PCR during hormone deprivation.</p><p><b>RESULTS</b>The AIPC cell model LNCaP-AI was successfully established. The PTTG1 expression was gradually increased in the LNCaP cells with the prolonged time of hormone deprivation and the expressions of matrix metalloproteinases MMP-2 and -9 were elevated at the same time.</p><p><b>CONCLUSIONS</b>The expression of PTTG1 is increased gradually in AIPC, which may be a target of gene therapy for advanced prostate cancer.</p>
Subject(s)
Humans , Male , Blotting, Western , Cell Line, Tumor , Gene Expression Regulation, Neoplastic , Matrix Metalloproteinase 2 , Metabolism , Matrix Metalloproteinase 9 , Metabolism , Neoplasms, Hormone-Dependent , Prostatic Neoplasms , Genetics , Securin , GeneticsABSTRACT
OBJECTIVE@#To investigate the relationship between genetic factor and prostate cancer (Pca) risk and the possible cause in it.@*METHODS@#The polymorphisms of cytochrome P450 family 17 (CYPl7) rs743572, p27 V109G and androgen receptor (AR) gene CAG repeat length in peripheral blood from 70 cases and 70 controls were detected through the polymerase chain reaction-restriction fragment length polymorphism technique or short tandem repeat-polymerase chain reaction technique. Then, according to the results of case-control study, the recombinant plasmids containing the wild/mutant p27 gene were constructed and transfected Pca LNcap cells. After 24 and 72 h of transfection, the cell proliferative activity was determined by MTT method, cell cycle distribution and apoptosis was detected by flow cytometry, and the expression level of bcl-2, caspase-3 and p27 protein was determined by Western-blot.@*RESULTS@#In three target polymorphisms, only p27 V109G polymorphism was related to Pca risk (P = 0.030, OR = 0.202, 95% CI = 0.042-0.973). Pca risk of p27-109G allele was lower than -109V allele (P = 0.006, OR = 0.285, 95% CI = 0.110-0.737). Cells transfected with wild/mutant p27 gene both showed the higher cells apoptosis rate and the lower cell proliferative activity than mock cells (P < 0.05 or 0.01), the regulatory effect of mutant p27 on cell proliferation and apoptosis was stronger than the wild p27 (P < 0.05).@*CONCLUSIONS@#p27-109G allele that could cause higher p27 protein expression than -109V allele in LNcap cells, maybe is the protective factor of Pca.
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Objective: To investigate the relationship between genetic factor and prostate cancer (Pca) risk and the possible cause in it. Methods: The polymorphisms of cytochrome P450 family 17 (CYPl7) rs743572, p27 V109G and androgen receptor (AR) gene CAG repeat length in peripheral blood from 70 cases and 70 controls were detected through the polymerase chain reaction-restriction fragment length polymorphism technique or short tandem repeat-polymerase chain reaction technique. Then, according to the results of case-control study, the recombinant plasmids containing the wild/mutant p27 gene were constructed and transfected Pca LNcap cells. After 24 and 72 h of transfection, the cell proliferative activity was determined by MTT method, cell cycle distribution and apoptosis was detected by flow cytometry, and the expression level of bcl-2, caspase-3 and p27 protein was determined by Western-blot. Results: In three target polymorphisms, only p27 V109G polymorphism was related to Pca risk (P = 0.030, OR = 0.202, 95% CI = 0.042-0.973). Pca risk of p27-109G allele was lower than -109V allele (P = 0.006, OR = 0.285, 95% CI = 0.110-0.737). Cells transfected with wild/mutant p27 gene both showed the higher cells apoptosis rate and the lower cell proliferative activity than mock cells (P < 0.05 or 0.01), the regulatory effect of mutant p27 on cell proliferation and apoptosis was stronger than the wild p27 (P < 0.05). Conclusions: p27-109G allele that could cause higher p27 protein expression than -109V allele in LNcap cells, maybe is the protective factor of Pca.
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OBJECTIVE: To investigate the preparation of novel cationic liposomes encapsulating siRNA, and evaluate their silence efficiency on target mRNA in LNCap cells. METHODS: Reverse evaporation technique was used to prepare the cationic liposomes. Protamine and calf thymus DNA were added to increase the encapsulation efficiency. The physicochemical property of the liposomes was evaluated. Human prostate cancer cells, LNCap, were used as the cell model. The silence efficiency on target gene in LNCap cells was evaluated with lipofectamine 2000 as the positive control. RESULTS: The optimum liposomes were obtained when the ratio of DOTAP and cholesterol was 3:1 and the ratio of liposomes and siRNA was 300:1. The size distribution and Zeta potential of the liposomes were (117 ± 4.3) nm and (43 ± 3.6) mV, respectively. The liposomes significantly improved siRNA accumulation in cells. CONCLUSION: The novel cationic liposome showed high transfection efficiency and distinct silence efficiency for target gene, which are expected to become a highly effective drug delivery system for anti-cancer drugs.
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OBJECTIVE: To employ PEG-PCL diblock copolymers to prepare DTX-loaded polymeric micelles (PEG-PCL-DTX micelles, DTX-PMs) which addressed the issue of DTX's drug loading capacity, encapsulation efficiency and in vitro release. We also studied its effectiveness for the cytotoxicity on prostate cancer. METHODS: The polymeric micelles were screened by its shape using transmission electron microscope and were also characterized in terms of particle size, Zeta potential, drug loading efficiency, in vitro release and cytotoxicity by using laser particle size analyzer and HPLC. Cytotoxicity against LNCap-C4-2B prostate cancer cells of the DTX-PMs and commercial product of Duopafei® were evaluated by MTT assay. RESULTS: The average particle size and Zeta potential of DTX-PMs were found to be 25.1 nm and 0.64 mV. The micelles' drug loading and encapsulation efficiency were 8.72% and 98.1%, respectively. Cytotoxicity assay showed that DTX-PMs exerted significant anti-proliferation activity on LNCap-C4-2B prostate cancer cells. CONCLUSION: Slightly soluable DTX successfully formulated into the PEG-PCL micells, exhibiting small partical size and good stability. Delayed release in vitro and maintained quite a constant concentration in plasma for a long period, which was favorable for its clinic application. In conclusion, DTX-PMs developed here sufficiently solubilized DTX and increase the concentration of DTX in aqueous phase, offering a sustained in vitro release and effective cytotoxicity on LNCap-C4-2B prostate cancer.
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Aims: The homeoprotein TGIFLX (transforming growth factor-β-induced factor 2-like, Xlinked), which is essential in male reproduction and development and likely oncogenic when aberrantly expressed in prostate. We have previously shown an aberrant expression of TGIFLX in the majority of human prostate tumors. However, mechanism by which TGIFLX acts in prostate cancer is unknown. The aim of this study was to investigate the effects of overexpression of wild-type TGIFLX (wt-TGIFLX) on LNCaP, human prostate adenocarcinoma cells. Study Design: As a prospective study, we used adenovirus expression system for evaluation of TGIFLX expression effects on mammalian cells. Place and Duration of Study: Medical Genetics Department, Tehran University of Medical Sciences (TUMS), between December 2009 and July 2012. Methodology: We cloned entire coding sequence of TGIFLX gene into adenovirus and subsequently LNCaP cells were transfected with the recombinant virus harboring TGIFLX cDNA or control. The TGIFLX expression was confirmed by microscopic analysis and RT-PCR technique. Following molecular cloning and characterization of TGIFLX transcription factor, we then studied the effects of overexpression of TGIFLX in LNCaP cells on mRNA expression of BAX and BCL2 genes. Results: Our results showed that overexpression of TGIFLX downregulated BCL2 gene (P<0.05) and upregulated BAX gene (P<0.05) at transcript level. Our results suggested that TGIFLX could be a tumor suppressor gene and might be involved in initiation and/or development. Conclusion: TGIFLX can play a role as a transcriptional modulator of the genes involved in cell cycle pathway. But still more investigations are necessitated for clarifying this claim.
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Objective To compare profiles of microRNA between the LNCaP and LNCaP-AI cell lines, so as to futher elucidate the post-transcritional mechanism regulating the progression to androgen-independence. Methods The microRNA profiles of LNCaP and LNCaP-AI cell lines were examined by Agilent's microassay. The expression of six microRNAs was verified by RT-PCR. The functions of differentially expressed microRNAs were eludicated by a search with miRBase software (http://www.mirbase.org/). Results The Aglint's microRNA microassay showed that 11 microRNAs were up-regulated and 27 were down-regulated during the LNCaP progression to androgen-independence. RT-PCR results were consistent with those of the Agilent's microassay chips. By searching the targets of microRNAs in the miRBase software, we found that the differentically expressed microRNAs were mainly involved in regulation of matrix metalloproteinase 9 (MMP-9), Bcl-2, and epithelial growth factor receptor (EGFR) and genes mediating androgen metabolism. Conclusion There is alteration of microRNA during the progression of LNCaP to androgen-independence, which may involve androgen receptor related pathway, metalloenzyme, anti-apoptotic gene and genes related to androgen metabolism.
ABSTRACT
Objective: To establish and identify androgen-independent human prostate cancer cell line LNCap by culturing LNCaP cells with gradual deprivation of hormone. Methods: LNCaP cells were cultured in the medium with gradual deprivation of hormone (treated by active carbon to simulate androgen deprivation) for 10 days; and then the cells were cultured with complete deprivation of androgen for 3 months till the cell entered the rapid proliferation phase again. The cell growth and expression of PSA and androgen were examined by CCK-8, immunfluorescence and RT-PCR methods. Results: LNCaP cells grew slowly after deprivation of hormone and took on a neuroendocrine phenotype and cluster growth pattern. After 3 months' non-androgen culture,the cells regained original morphology and growth. CCK-8 indicated that LNCaP cells could grow in non-androgen condition; immunofluorescence assay indicated that LNCaP-AI cells could regain PSA-secreting activity in non-androgen condition; and RT-PCR suggested that androgen was highly expressed in LNCaP-AI cells. Conclusion: Androgen-independent LNCaP cell line can be established by culturing with gradual deprivation of hormone for 3 months.