LNCaP Research Articles

Biosynthesis of gold nanoparticles using sansevieria plant extract and its biomedical application

The gold nanoparticles (Au NPs) are used for various medical applications including biosensors, bioassays, photo-thermal treatments, and targeted drug delivery. Au NPs are synthesized by various methods, most of them are expensive and environmentally hazardous. In this study, the biosynthesis of Au NPs was performed by Sansevieria extract. The synthesized nanoparticle was investigated by X-Ray Diffraction (XRD), field-emission scanning electron microscopes, Fourier transform infrared spectroscopy (FTIR), and the UV–visible spectroscopy. The results showed that Sansevieria extract could produce spherical Au NPs with a diameter of about 40-70 nm in an economical and environmentally friendly synthesis method. It was also indicated by the MTT assay that the Au-NPs had low cellular cytotoxicity and promoted the antiproliferation effect of Cisplatin on prostate cancer cells (LN Cap).

VASN promotes proliferation of prostate cancer through the YAP/TAZ axis.

The purpose of this study was to uncover the role of VASN in regulating proliferative ability of prostate cancer (PCa) cells through the yes-associated protein/transcriptional coactivator with PDZ-binding motif (YAP/TAZ) axis, thus influencing the progression of PCa. VASN, YAP, and TAZ levels in PCa tissues or in the serum were detected by quantitative Real Time-Polymerase Chain Reaction (qRT-PCR). The diagnostic value of VASN in PCa was assessed by introducing receiver operating characteristic (ROC) curves. Besides, the regulatory effects of VASN on viability, clonality, and expression levels of YAP/TAZ were evaluated by cell counting kit-8 (CCK-8), colony formation, and Western blot, respectively. Finally, rescue experiments were conducted to uncover the involvement of YAP in VASN-regulated proliferation of PCa. Results manifested that VASN, YA, and TAZ were upregulated in PCa patients, and VASN presented a certain diagnostic value. Knockdown of VASN in LNCaP and C4-2 cells suppressed viability and clonality, and downregulated protein levels of YAP and TAZ. Notably, overexpression of YAP abolished the attenuated viability and clonality in PCa cells with VASN knockdown. VASN promotes proliferative ability in PCa via regulating the YAP/TAZ axis, thus aggravating the progression of the disease.

Influence of human adipose stem cells on prostate cancer cell growth.

ABSTRACTIn a novel regenerative cell-based treatment developed by us for the patients with stress urinary incontinence, autologous adipose-derived stem cells (ASCs) are injected into the periurethral region and the external urethral sphincter. Since the candidates for this treatment included prostate cancer patients after radical prostatectomy, we investigated the effects of ASCs on prostate cancer cell proliferation in vitro and in vivo to confirm the feasibility of our therapeutic approach. The LNCaP (human prostate cancer cell line) cells and ASCs were co-cultured, and prostate-specific antigen (PSA) concentration in their culture medium supernatant was measured at 48 and 96 h. The PSA concentration significantly decreased in the coculture medium supernatant as compared to the culture medium with LNCaP cells alone. On the contrary, PSA concentrations in the culture medium of LNCaP cells were not affected by supplementation with ASC culture supernatant. After subcutaneous transplantation of LNCaP cells, with or without ASCs, in immunodeficient mice, tumor growth was compared. The growth of LNCaP xenograft tumor in immunodeficient mice was significantly suppressed by ASC addition. These results indicated that ASCs inhibit prostate cancer cell growth, without no proliferative effect on prostate cancer cells.

Palmitoylome profiling indicates that androgens promote the palmitoylation of metabolism-related proteins in prostate cancer-derived LNCaP cells

To explore potential therapeutic targets other than androgen-deprivation treatment for prostate cancer by screening the proteins induced by androgen at palmitoylation modification level in LNCaP cells. The LNCaP cells were treated with androgen (Methyltrienolone, R1881, 5 nmol/L) or dimethyl sulfoxide (DMSO) for 24 h, and then labeled with alkynyl palmitic acid Alk-C16 (100 μmol/L). After that, the cells were collected, lysed, the total protein was extracted, agarose beads labeled with azide (1 mmol/L) were added, and the click-chemistry reaction was carried out at room temperature for 1 h. The covalent bond formed by click-chemistry reaction of azide and alkynyl group was used to enrich the palmitoylated proteins on agarose beads. Label-free quantitation (LFQ) was used to compare the protein palmitoylation level of R1881 treated and untreated cells to screen the proteins induced by androgen at palmitoylation modification level. In this experiment, 907 potential palmitoylated proteins (mascot score>2, P<0.05) were identified, among which 430 proteins had LFQ values not zero at least twice. Among the 430 proteins, the palmitoylation levels of 92 candidates were increased by androgen treatment, and their LFQ values were significantly upregulated (>1.5-fold, P<0.05) in ≥2 samples of androgen-treated vs. untreated LNCaP cells. We also used the software of cytoscape to classify the 92 proteins, and found that the known functional proteins of them could be divided into three categories: metabolism related, protein folding related and translation initiation related. Among them, metabolism related proteins included lipid metabolism (6), glucose metabolism (7) and respiratory electron transport chain (8), and a small amount of amino acid metabolism (2) and other metabolism related proteins (2). Notably, the ratio of LFQ of cytochrome b-c1 complex subunit 2 (UQCRC2) was significantly (>3-fold, P<0.05) higher in androgen-treated cells compared with untreated cells, indicating that the palmitoylation level of UQCRC2 was enhanced by androgen most significantly than that of others. The second was long-chain acyl CoA dehydrogenase (ACADVL) related to lipid metabolism and glucose 6-phosphate dehydrogenase (PGD) related to glucose metabolism, but the LFQ ratio of them was less than 3-fold. The research on palmitoylation mechanism of metabolism, especially the proteins related to respiratory electron transport chain, will provide a new guidance for the diagnosis and treatment of prostate cancer and the development of targeted drugs.

Urinary PCA3 detection in prostate cancer by magnetic nanoparticles coupled with colorimetric enzyme-linked oligonucleotide assay.

PCA3 is one of the most prostate cancer-specific genes described to date. Of note, PCA3 expression is detectable at high level in the urine of prostate cancer (PCa) patients. Accordingly, PCA3 is an ideal biomarker for PCa diagnosis. Several techniques for the measurement of this biomarker in urine have been developed but there are still some drawbacks. In this study, magnetic nanoparticle-based PCR coupled with streptavidin-horseradish peroxidase and a substrate for colorimetric detection was established as a potential assay for urinary PCA3 detection. The method provided a high specificity for PCA3 gene in LNCaP prostate cancer cell line. Additionally, this technique could detect PCA3 at femtogram level which was approximately 1,000-fold more sensitive than the conventional RT-PCR followed by agarose gel electrophoresis. The effectiveness of the method was assessed by PCA3 detection in clinical specimens. The relative PCA3 expression of PCa patients determined by this assay was significantly greater than that of benign prostatic hyperplasia (BPH) patients and healthy controls. The results of our test were comparable with the results of qRT-PCR. The proposed method is promising to distinguish between cancerous and non-cancerous groups. Altogether, this simple assay is practicable and useful for prostate cancer diagnosis.

177Lu-PSMA-I&T automated radiolabeling and preclinical prostate cancer targeting research

Objective To synthesis 177Lu-prostate specific membrane antigen (PSMA)-I&T with automated module, evaluate the biodistribution and pharmacokinetics in mice and study the targeting property in human prostate cancer cell line LNCaP Clone FGC. Methods The iQS-TS automated module was applied in labeling 177Lu-PSMA-I&T. Radiochemical purity and stability were determined with high performance liquid chromatography (HPLC). The biodistribution was observed in normal ICR mice and U-SPECT/CT imaging was performed in LNCaP Clone FGC tumor-bearing mice. Independent-sample t test was used to analyze the data. Results 177Lu-PSMA-I&T was stable in vitro and in vivo, with the radiolabeled yield of (91.5±4.9)% and radiochemical purity >99%. The half maximal inhibitory concentration (IC50) of 177Lu-PSMA-I&T binding to LNCaP Clone FGC cells was (26.74±3.53) nmol/L. The uptake of 177Lu-PSMA-I&T by LNCaP Clone FGC cells increased with time and significantly decreased after the inhibitor addition (t values: 4.301-27.483, all P<0.05). 177Lu-PSMA-I&T was cleared from blood rapidly and predominantly excreted by kidneys. Significant radioactive uptake was observed in tumors with a long retention time. Conclusion 177Lu-PSMA-I&T can be produced in a convenient and efficient procedure using iQS-TS automated module, with good biological properties and excellent affinity and targeting property towards prostate cancer cells, which making it a potential radiopharmaceutical for prostate cancer therapy. Key words: Prostatic neoplasms; Prostate-specific membrane antigen; Isotope labeling; Lutetium; Chemical synthesis; Tumor cells, cultured; Mice, nude

Differential expression of microRNA-218 in hormone-dependent and hormone-independent prostate cancer cell lines

Objective To verify and analyze the expression of microRNA-218 in hormone-dependent and hormone-independent prostate cancer cell lines. Methods Specific SYBR Green quantitative real-time polymerase chain reaction(RT-PCR) was used to detect the relative expression of microRNA-218 in hormone-dependent prostate cancer LNCaP cell line and hormone-independent prostate cancer C4-2, PC-3 and DU-145 cell lines, and the differences were verified and analyzed. Results The expression of MicroRNA-218 was not different in C4-type, PC-3 and DU-145 three hormone-independent prostate cancer cell lines(P>0.05), but was significantly higher than that in hormone-dependent prostate cancer cell line LNCaP(P<0.05). Conclusions MicroRNA-218 is involved in the progression from steroid-dependent to hormone-independent prostate cancer and is expected to become a biomarker for predicting the progression of prostate cancer. Key words: Prostatic Neoplasms; Cell Line; MicroRNAs

Knockdown of mediator complex subunit 19 suppresses the proliferation in prostate cancer LNCaP cells

Objective To study the role of mediator complex subunit 19 (Med 19) in prostate cancer by analyzing the effects of lentivirus-mediated suppression of Med 19 expression on LNCaP prostate cancer cells in vitro and explore the mechanism of proliferation. Methods The lentivirus vectors containing small interfering RNA (siRNA) targeting Med 19 gene were constructed and transfected to LNCaP cells. Real-time quantitative reverse transcriptase-polymerase chain reaction (RT-qPCR) and Western blotting analysis were used to detect the Med 19 expression in the siRNA group (LNCaP-Med 19-siRNA cells)and the control group (LNCaP-Med 19-scRNA cells) at 72 h after the transfection. The influence of Med 19 on the proliferation of prostate cancer cells were assessed using methyl thiazol tetrazolium (MTT), Soft Agar Assay. Cell cycle was analyzed with flow cytometry assay. Results Med 19 relative mRNA level and Med19 protein expressing in the siRNA group were significantly inhibited (t=-69.1413, P<0.01). Compared with the control group, the siRNA group displayed a significant cell proliferation defect by MTT and the number of colonies (8.61±0.51) was significant decreased (t=-11.948, P<0.01; t=-98.775, P<0.01). The siRNA group displayed an increased proportion of cells (67.29±1.70)% in the G0/G1 phase compared with the control group [(56.23±1.46)%, t=-21.906, P<0.01]. Conclusion Knockdown of Med 19 gene increased the number of human prostate cancer LNCaP cells in G0/G1 phase, resulting in significantly inhibited proliferation and growth of prostate cancer. Key words: Mediator complex subunit 19; Prostate carcinoma; Proliferation

Steroid 5-alpha reductase 1 is negatively regulated by Homeobox gene D10 in prostate cancer LNCaP cells

Objective To investigate the relationship between homeobox gene D10 (HOXD10) and steroid 5-alpha reductase 1 (SRD5A1) and the effect of the former on the latter in prostate cancer cells. Methods Expression of HOXD10 and SRD5A1 and their correlation was analyzed basing on Taylor and TCGA database. Then HOXD10-overexpressing prostate cancer LNCaP cells was constructed, and the effect of HOXD10 on SRD5A1 was detected by real-time quantitative reverse transcriptase-polymerase chain reaction (RT-qPCR) and Western blotting. Immunohistochemistry was used to analyze the expression of HOXD10 and SRD5A1 in human prostate cancer tissues.Statisticalanalysis of data using the GraphPad Prism Version 6 software. Results mRNA expression of HOXD10 decreased gradually with the progression of prostate cancer (Taylor: Benign: 8.94±0.09, Primary: 7.93±0.05, Met: 7.66±0.11, F=43.72, P<0.05), while SRD5A1 increased gradually (Taylor: Benign: 7.75±0.02, Primary: 7.79±0.02, Met: 7.98±0.01, F=5.306, P<0.05), and demonstrated a significant negative correlation (TCGA: HOXD10: 80.72±1.60, SRD5A1: 231.60±2.48; r=-0.220, P<0.01). Moreover, the expression of SRD5A1 in prostate cancer cells or patients was significantly reduced in which with higher expression of HOXD10 (Immunohistochemistry: χ2=5.868, P<0.05). (RT-qPCR: overexpressing HOXD10-LNCaP SRD5A1: 0.66±0.02, vector-LNCaP SRD5A1: 0.99±0.00, t=11.31, P<0.01; Western blotting: overexpressing HOXD10-LNCaP SRD5A1: 0.58±0.00, vector-LNCaP SRD5A1: 0.82±0.02, t=11.31, P<0.01). Conclusion HOXD10 negatively regulates SRD5A1 in prostate cancer and may provide a new strategy for endocrine therapy of prostate cancer. Key words: Homeobox gene D10; Steroid 5-alpha reductase 1; Prostate cancer

MiR-451 suppresses the growth, migration, and invasion of prostate cancer cells by targeting macrophage migration inhibitory factor

Background: Metastasis is the most common cause of death of prostate cancer patients. Identification of key regulators in prostate cancer metastasis is essential for improved management of the disease. Accumulation of evidence suggests that miRNAs play important roles in cancer metastasis. Here, we investigated the role of miR-451 in prostate cancer progression and metastasis. Methods: In this study, we performed reverse transcription-quantitative PCR analysis to examine the expression of miR-451 in clinical prostate cancer samples and cell lines. TargetScan Release 7.0 programs, luciferase reporter assay, and a rescue experiment were used to validate the direct target of miR-451. The protein expression of the miR-451 target was detected by Western blot. MTS cell viability assay, transwell migration assay, and modified Boyden chamber invasion assay were used to demonstrate the effects of increased miR-451 expression on prostate cancer cell growth, migration and invasiveness. Results: We identified the decreased expression of miR-451 in clinical metastatic prostate cancer compared to localized primary prostate cancer and benign prostate. Consistently, we also observed the decreased expression of miR-451 in highly metastatic prostate cancer cell lines (C4-2 and PC3M) compared to their low metastatic counterparts (LNCaP and PC3). Furthermore, we confirmed the macrophage migration inhibitory factor (MIF) is a direct target of miR-451 in prostate cancer and demonstrated a negative correlation between miR-451 and MIF expression in clinical prostate cancer samples. Functional studies showed increased miR-451 expression significantly suppressed cell growth, migration and invasiveness via targeting MIF. The effect of increased miR-451 expression can be reversed by overexpressing MIF in prostate cancer cells. Conclusions: These findings suggest that miR-451 suppresses prostate cancer cell growth, migration and metastasis, which provides insight into the development of novel therapeutic approaches for prostate cancer treatment.

Differential expression of exosomal miRNAs between androgen-dependent prostate cancer cell and androgen-independent prostate cancer cell

Objective Studying the differential expression of exosomal miRNAs between androgen-dependent prostate cancer cell and androgen-independent prostate cancer cell, in order to further elucidate the mechanism of androgen-independent prostate cancer and find new targets for its treatment. Methods Prostate cancer androgen-dependent LNCaP cell and prostate cancer androgen-independent LNCaP-AI+F cell (induced by androgen and flutamide) were selected as the study subjects. Illumina HiSeqTM 2500 was used to perform high-throughput sequencing between the two groups. The differentially expressed exosomal miRNAs was verified by quantitative real-time PCR(qRT-PCR). The difference was statistically significant by t-test. Results Through the analysis of high-throughput sequencing results, thirteen molecules were screened increased in extracellular exosomes of the androgen-independent cell, including miR-7-5p, let-7a-5p, miR-375, miR-423-3p, miR-378a-3p, and miR-92a-3p, etc. Among them, miR-7-5p was verified by quantitative real-time PCR to be up-regulated by 19.52-fold (t=9.857, P=0.001). Conclusion Differentially expressed exosomal miRNAs may predict the development of androgen-independent prostate cancer and may further regulate the development of androgen-independent prostate cancer. Key words: Prostatic neoplasms; Exosomes; microRNAs; High-throughput nucleotide sequencing

Overexpressed deoxycytidine triphosphate pyrophosphatase 1 promotes cell-cycle in LNCaP

Objective To investigate the expression of human deoxycytidine triphosphate pyrophosphatase 1 (dCTP pyrophosphatase 1) in prostate cancer tissues and LNCaP cells, and observe the effect of high expression of dCTP pyrophosphatase 1 on LNCaP cell cycle. Methods The expression of dCTP pyrophosphatase 1 in prostate cancer and non-cancer tissue was analyzed by immunohistochemistry. The expression of dCTP pyrophosphatase 1 protein in LNCaP and RWPE-1 cells was detected by Western blotting (LNCaP: 1.26±0.13 vs. RWPE-1: 0.52±0.01, P=0.000). The dCTP pyrophosphatase 1 overexpressed LNCaP cell lines were constructed and cell cycle changes were detected by flow cytometry (LNCaP-dCTP pyrophosphatase 1: 48.23±0.66 vs. LNCaP-NC: 30.10±1.01, P=0.022). Results The expression of dCTP pyrophosphatase 1 in cancer tissues was up-regulated (IHC score: non-cancer: 2.00±1.55, cancer: 4.00±1.52, P=0.000), and the high expression of dCTP pyrophosphatase 1 increased the S+ G2 phase of LNCaP cells (P=0.000). Conclusion High expression of dCTP pyrophosphatase 1 in prostate cancer promotes the division of prostate cancer cells. Key words: Deoxycytidine triphosphate pyrophosphatase 1; Prostate cancer; Cell cycle

The effect of ErbB3-binding protein 1 on the proliferation of prostate cancer cells and the clinical significance of its expression level

Objective To investigate the expression of ErbB3-binding protein 1 (Ebp1) in prostate cancer and explore the mechanism and clinical significance of Ebp1 in the development of prostate cancer. Methods Immunohistochemical SP was employed to detect the expression of Ebp1 protein in 33 cases of prostate cancer tissues, 25 cases of benign prostatic hyperplasia, analyzing gene expression of Ebp1 in prostate cancer gene chip, and the relationship between Ebp1 expression and clinicopathological parameters of prostate cancer were statistically analyzed. Ebp1 overexpression vector was constructed and transfected into LNCaP prostate cancer cell line, and the expression level of Ebp1 protein was detected by Western blotting. The mechanism of Ebp1 affecting tumor cell growth was verified by observing the proliferation of transfected cells and analyzing the cell cycle changes by flow cytometry. Results Immunohistochemistry showed that the expression of Ebp1 protein in normal prostate hyperplasia were significantly increased (positive rate 84.0%) compared with the expression of prostate cancer (positive rate 69.7%) (P=0.001), the expression was negatively correlated with Gleason score and pathological stage (P=0.035); similarly, the gene expression level and Gleason score, pathological stage, tumor metastasis and postoperative biochemical recurrence of prostate specific antigen (PSA) were negatively correlated (P=0.032). Over-expressed of Ebp1 prostate cancer cells proliferate slowly, and flow cytometry showed that the proportion of cells in the G1 phase of experimental group (78.75%) was higher than that of the control group (53.47%). Conclusion Ebp1 can inhibit the proliferation and growth of prostate cancer cells by regulating the cell cycle, and its expression level is related to the progression and clinical prognosis of prostate cancer. It is expected to become a marker and a therapeutic target to reflect the degree of malignancy and prognosis of prostate cancer. Key words: Prostate cancer; Cell cycle; ErbB3-binding protein 1; Tumor progression; Prognosis

Pharmacologically relevant concentrations of berberine transiently stimulate dihydrotestosterone-inducible androgen receptor-mediated luciferase activity in human prostate cancer cells

Background: Herbal medicines containing berberine have a wide spectrum of usage. However, the effect on androgen receptor (AR) activity at plasmatic relevant concentrations is omitted. Therefore, the effect of berberine on AR activity in prostate cancer cell lines was investigated. Methods: We used the reporter gene assay (RGA) and polymerase chain reaction (qPCR) for monitoring the activity of AR. Moreover, we monitored the proliferation of two prostate cancer cell lines treated with Berberine. Results: Berberine significantly potentiated dihydrotestosterone (DHT)-inducible AR-dependent luciferase activity in RGA. It also significantly decreased basal KLK3 mRNA level in 22Rv1 cells, but DHT-inducible KLK3 mRNA was not affected in androgen-independent (22Rv1) or androgen-dependent (LNCaP) cells. Proliferation of both cell lines was significantly inhibited by 100 and 1,000 nM concentrations in similar manner and Berberine increased Annexin V staining, suggesting an apoptotic event. Conclusions: Berberine inhibits proliferation of prostatic cell lines at pharmacologically reachable concentrations but with no apparent link to androgen receptor-driven target gene expression.

The study of suppression and action mechanism of malignant phenotype by downregulating expression of histone deacetylase in prostate cancer cell

Objective Histone deacetylase (HDAC) promoted growth and metastasis of prostate cancer cell and it’s action mechanism, however, remain unclear. The aim of present study is investigate the suppression and action mechanism of malignant phenotype by downregulating expression of histone deacetylase (HDAC1 and HDAC2) in prostate cancer cell. Methods Prostate cancer cell lines, LNCaP and DU145 cells were transfected with siHDAC using liposome 2000 or treated with MHY219, named as LNCaP-siHDAC, DU145-siHDAC cells negative control LNCaP-NC small interfering RNA (siRNA), DU145-NCsiRNA cells, and the expressions of HDAC1, HDAC2, HDAC3, matrix metalloproteinase (MMP)-1, MMP-2, MMP-9 and TIMP-1 protein were determined by Western blotting and the levels of MMP-1, MMP-2, MMP-9 and TIMP-1 mRNA were detected using Real time RT-PCR. Cell viability, clone formation ability and metastasis were monitored using MTT, clone formation assay and Transwell respectively in LNCaP and DU145 cells. Results The relative expression levels of HDAC1 and HDAC2 protein was respectively 0.22±0.01, 0.25±0.02 in LNCaP-siHDAC cell and 0.15±0.01, 0.30±0.03 in DU145-siHDAC cell, and significantly lower than LNCaP-NCsiRNA (1.00±0.05, FLNCaP=289.440, P=0.008) and DU145--NCsiRNA (1.00±0.03, FDU145=336.125, P=0.002). The relative expression levels of HDAC1 and HDAC2 protein was respectively 0.53±0.04, 0.60±0.02 in LNCaP cell and 0.58±0.04, 0.66±0.05 in DU145 cell treated with MHY219, and significantly lower than Untreated group (FLNCaP=147.158, P=0.022; FDU145=134.573, P=0.035). The results from the MTT indicated that cell viability was respectively (40.38±4.16)% in LNCaP-siHDAC cell and (21.42±5.26)% in DU145-siHDAC cell with statistical differences compared with LNCaP-NCsiRNA cell [(98.47±6.70)%, FLNCaP=125.627, P=0.004] and DU145-NCsiRNA cell [(99.15±7.00)%, FDU145=137.560, P=0.000]. The cell viability was respectively (61.75±7.12)% and (45.00±5.53)% in LNCaP cell and DU145 cell treated with MHY219, and with statistical differences compared with Untreated group LNCaP [(99.34±7.12)%, FLNCaP=88.392, P=0.032] and DU145 [(98.58±8.05)%, FDU145=103.10, P=0.017]. The results from clone formation assay shown that clone formation rate was respectively (26.37±3.50)% in LNCaP-siHDAC cell and (19.48±2.33)% in DU145-siHDAC cell, and with statistical differences compared with LNCaP-NCsiRNA cell [(97.20±7.88)%, FLNCaP=287.653, P=0.000] and DU145-NCsiRNA cell [(99.23±9.52)%, FDU145=315.042, P=0.000]. The clone formation rate was respectively (43.88±4.12)% and (438.05±6.11)% in LNCaP cell and DU145 cell treated with MHY219, and with statistical differences compared with Untreated group LNCaP [(100.00±5.24)%, FLNCaP=203.540, P=0.018] and DU145 [(97.70±8.74)%, FDU145=264.110, P=0.010]. Transwell results indicated that the cell number of metastasis was respectively (102.5±7.5) in LNCaP-siHDAC cell and (95.2±4.7) in DU145-siHDAC cell and significantly lower LNCaP-NCsiRNA cell (236.8±12.7, FLNCaP=316.047, P=0.00) and DU145-NCsiRNA cell (278.5±18.6, FDU145=357.052, P=0.000). Western blotting and Real time RT-PCR results shown that downegulated HDAC1 or (and) HDAC2 expression notable suppressed the expression of MMP-1and MMP-2 in protein and mRNA levels, but TIMP-1 expression was significantly enhanced in LNCaP and DU145 cells. Conclusion The suppression of malignant phenotype by downregulating expression of HDAC1 and HDAC2 in prostate cancer cell. Downregulated HDAC expression can inhibited malignant phenotype and it’s action mechanism was achieved at least partly by regulating expression of MMP-1, MMP-2 and TIMP-1 expression in LNCaP and DU145 cells. Key words: Prostate cancer; Histone deacetylase; Matrix metalloproteinase; Malignant phenotype; Action mechanism

Regulation of prostate cancer cells invasiveness via microRNA-221 by the expression level of target gene suppressor of cytokine signaling 1

Objective To investigate microRNA (miRNA, miR)-221 expression in prostate cancer cells and its influence on prostate cancer cell invasiveness. Methods miR-221 expression levels in prostate cancer cell lines were measured by real-time quantitative reverse transcriptase-polymerase chain reaction (RT-qPCR). The miR-221 overexpression and silencing cell lines were constructed by cell transfection. Effects of the depletion on cell invasiveness were assessed in vitro with Transwell. Results RT-qPCR showed miR-221 was down-regulated in PC3, LNCaP and DU145 cells than in PrEC (F=235.852, P=0.000), in which pairwise comparison also had significant differences. Cell invasiveness assay showed that migration of LNCaP (P=0.000) and DU145 (P=0.000) cells overexpressing miR-221 was significantly slower than in the control group. Conclusion The negative regulation of suppressor of cytokine signaling 1 (SOCS1) by miR-221 can inhibit the invasiveness of prostate cancer cells. Key words: Prostate cancer; MicroRNA-221; Invasiveness; Suppressor of cytokine signaling 1

Effect of synthetic small molecule RNA on cell cycle and proliferation of prostate cancer cell

Objective To investigate the effect of dsP21-555 on cell cycle and proliferation of prostate cancer cell line PC-3 and LNCaP. Methods dsP21-555 (experimental group) and dsControl (negative control group) were transfected into PC-3 and LNCaP, respectively. Real-time PCR and Western Blotting were used to detect the expression of p21 mRNA and p21 protein in prostate cancer cells after transfection. The cell cycle distribution was detected by flow cytometry. Cell viability and proliferation were measured by MTT assay and colony forming assay. Results The level of p21 mRNA in PC-3 and LNCaP cells was increased to 2.90-fold (P<0.01) and 2.05 - fold (P<0.01) respectively after transfection with dsP21-555. Western Blotting results were consistent with this trend. Flow cytometry showed that the percentage of cells in S phase and G2 / M phase decreased after transfection with dsP21-555, and the proportion of cells in G0 / G1 phase increased. MTT assay showed that the viability of PC-3 and LNCaP cells significantly decreased after dsP21-555 transfection compared with dsControl group. The colony formation assay showed that the number of colonies in the dsP21-555 group was fewer and the cell proliferation ability decreased. Conclusions Synthesized dsP21-555 can significantly activate the expression of p21 gene in prostate cancer cells and significantly inhibit the progression of cell cycle and proliferation ability of prostate cancer cells. Key words: Prostatic Neoplasms; RNA; Cell Cycle; Cell Proliferation

MicroRNA-488 mediates the growth and glycolysis of human prostate cancer cells by targeting 6-phosphofructo-2-kinase 3 gene

Objective To investigate the effect of micrioRNA (miRNA, miR) -488 on glycolysis and proliferation of prostate cancer cells and the underlying mechanism. Methods Real-time quantitative polymerase chain reaction (Real-time PCR) was used to examine the expression of miR-488 in prostate cancer cells. Bioinformatics prediction and luciferase reporter gene assay were utilized for the identification of the target genes of miR-488. The expression levels of 6-phosphofructo-2-kinase 3 (PFKFB3) mRNA and protein were detected by Real-time PCR and Western blotting after the miR-488 mimic was transfected 24 h later. After miR-488 mimics was transfected, or co-transfected with PFKFB3 over-expression plasmid for 24 h, the capacity of proliferation and the glycolysis of prostate cancer cells (PC-3 and DU145) were determined by cell counting kit-8 (CCK-8), glucose uptake and lactate assay, respectively. Results Compared to the normal prostate epithelium cell RWPE-1, the expression of miR-488 in PC-3 and DU145 cell lines was 0.26±0.03 (P=0.031) and 0.19±0.04 (P=0.021), but there was no statistically significant difference in LNCaP and 22RV1 cells. The glucose uptake rate and the lactate secretion rate were 0.54±0.03 (P=0.042) and 0.52±0.01 (P=0.032), and 0.55±0.02 (P=0.037) and 0.61±0.17 (P=0.048) after up-regulating the expression of miR-488, which indicating the decline of glycolysis ability and the proliferation ability. Bioinformatics prediction and luciferase reporter gene assay showed that PFKFB3 was the target gene of miR-488. After the expression of miR-488 was up-regulated, the expression levels of PFKFB3 mRNA and protein were down-regulated. When miR-488 and PFKFB3 over-expression plasmids were co-transfected into the cells, the proliferation was elevated and the glucose uptake rate and lactate secretion rate were 0.79±0.12 and 0.82±0.11, and 0.77±0.07 (P=0.015) and 0.83±0.04 (P=0.026) respectively, indicating a higher glycolysis rate than the control. Conclusion MiR-488 can regulate the ability of proliferation and glycolsis of prostate cancer cells by targeting PFKFB3 gene. Key words: MicroRNA-488; 6-phosphofructo-2-kinase 3; Prostate cancer; Proliferation; Glycolysis

Synthesis, structural and thermal characterizations and in vitro cytotoxic activities of new cyclotriphosphazene derivatives

GRAPHICAL ABSTRACT

Long non-coding RNA-p21 induced by hypoxia promotes the invasion of prostate cancer by enhancing Warburg effect

Objective To explore the role of long noncoding RNA-p21 (lncRNA-p21) induced by hypoxia in the progression prostate cancer. Methods Oxyrase was used to mimic the hypoxia condition for prostate cancer cells. Expression of lncRNA-p21 as well as hypoxia inducible factor (HIF)-1α in LNCaP and DU145 cells under hypoxia is determined by using quantitative real time polymerase chain reaction. Small interference RNA (siRNA) was used to knock down the expression of lncRNA-p21. After silencing of lncRNA-p21, and cell migration, cell invasion and Warburg effect were examined. HIF-1α mRNA and protein were detected, and the change of epithelial mesenchymal transition (EMT) was observed. Results Under hypoxia, expressions of lncRNA-p21 mRNA (LNCaP: t=4.721, P=0.009, DU145: t=11.930, P=0.000) and HIF-1α mRNA were up-regulated in LNCaP and DU145 cells (LNCaP: t=4.882, P=0.008, DU145: t=11.953, P=0.000). Hypoxia treatment led to a strong increase in glucose uptake (LNCaP: t=12.850, P=0.000, DU145: t=5.376, P=0.006) and lactate production (LNCaP: t=33.320, P=0.000, DU145: t=26.610, P=0.000) in prostate cancer cells. However, those activities were greatly reversed by lncRNA-p21 knockdown. Expressions of Vimentin and Twist-1 were downregulated after lncRNA-p21 knockdown. Simultaneously, expression of E-cadherin was upregulated compared to control group. Reducing HIF-1α expression was observed in lncRNA-p21 knockdown cells under hypoxia. Conclusion lncRNA-p21 induced by hypoxia promotes the invasion of prostate cancer cell via enhancing Warburg effect by binding to HIF-1α. Key words: Prostate cancer; Long non-coding RNA-p21; Warburg effect; Invasion