MELK and UBE2C gene knockdown in prostate cancer cell line PC3
Ontology highlight
ABSTRACT:
ORGANISM(S): Homo sapiens
SUBMITTER: Ruprecht Kuner
PROVIDER: E-GEOD-32873 | biostudies-arrayexpress |
REPOSITORIES: biostudies-arrayexpress
Publications
The maternal embryonic leucine zipper kinase (MELK) is upregulated in high-grade prostate cancer.
Kuner Ruprecht R Fälth Maria M Pressinotti Nicole Chui NC Brase Jan C JC Puig Sabrina Balaguer SB Metzger Jennifer J Gade Stephan S Schäfer Georg G Bartsch Georg G Steiner Eberhard E Klocker Helmut H Sültmann Holger H
Journal of molecular medicine (Berlin, Germany) 20120904 2
Loss of cell cycle control is a prerequisite for cancer onset and progression. In prostate cancer, increased activity of cell cycle genes has been associated with prognostic parameters such as biochemical relapse and survival. The identification of novel oncogenic and druggable targets in patient subgroups with poor prognosis may help to develop targeted therapy approaches. We analyzed prostate cancer and corresponding benign tissues (n = 98) using microarrays. The comparison of high- and low-gr ...[more]
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Project description:In order to address the putative role of MELK and UBE2C in prostate cancer development and progression, we performed functional analysis upon siRNA-based knockdown, and searched for downstream genes and processes by microarray experiments. RNAi-based inhibition of MELK and UBE2C was efficient in PC3 prostate cancer cells and decreased transcriptional level down to about 30% remaining expression level.
2012-10-19 | GSE32873 | GEO
Project description:Siomycin A treatment in prostate cancer cell lines resulted in a concurrent inhibition of specific cell cycle genes and strongly impaired cell viability. Illumina microarray experiments were done using PC3 and LNCaP cell lines treated with 0.5 microM Siomaycin A either 24h or 48h in triplicates.
2012-10-19 | E-GEOD-36531 | biostudies-arrayexpress
Project description:The epidemiologic association between statin use and decreased risk of advanced prostate cancer suggests that statins may inhibit prostate cancer development and/or progression. Studies were performed to determine the effects of a model statin, atorvastatin (ATO), on the proliferation and differentiation of prostate cancer cells, and to identify possible mechanisms of ATO action. ATO inhibited the in vitro proliferation of both LNCaP and PC3 human prostate cancer cells in dose-dependent fashion. The greater inhibitory activity of ATO in PC3 cells was associated with induction of autophagy in that cell line, as demonstrated by increased expression of LC3-II. miR-182 was consistently upregulated by ATO in PC3 cells, but not in LNCaP cells. ATO upregulation of miR-182 in PC3 cells was p53-independent and was reversed by geranylgeraniol. Transfection of miR-182 inhibitors decreased expression of miR-182 by >98% and attenuated the antiproliferative activity of ATO. miR-182 expression in PC3 cells was also increased in response to stress induced by serum withdrawal, suggesting that miR-182 upregulation can occur due to nutritional stress. Bcl2 and p21 were identified to be potential target genes of miR-182 in PC3 cells. Bcl2 was downregulated and p21 was upregulated in PC3 cells exposed to ATO. These data suggest that miR-182 may be a stress-responsive miRNA that mediates ATO action in prostate cancer cells. Gene and miRNA expression in two prostate cancer cell lines treated with Atorvastatin vs. untreated control.
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2017-12-31 | E-MTAB-4858 | biostudies-arrayexpress
Project description:The epidemiologic association between statin use and decreased risk of advanced prostate cancer suggests that statins may inhibit prostate cancer development and/or progression. Studies were performed to determine the effects of a model statin, atorvastatin (ATO), on the proliferation and differentiation of prostate cancer cells, and to identify possible mechanisms of ATO action. ATO inhibited the in vitro proliferation of both LNCaP and PC3 human prostate cancer cells in dose-dependent fashion. The greater inhibitory activity of ATO in PC3 cells was associated with induction of autophagy in that cell line, as demonstrated by increased expression of LC3-II. miR-182 was consistently upregulated by ATO in PC3 cells, but not in LNCaP cells. ATO upregulation of miR-182 in PC3 cells was p53-independent and was reversed by geranylgeraniol. Transfection of miR-182 inhibitors decreased expression of miR-182 by >98% and attenuated the antiproliferative activity of ATO. miR-182 expression in PC3 cells was also increased in response to stress induced by serum withdrawal, suggesting that miR-182 upregulation can occur due to nutritional stress. Bcl2 and p21 were identified to be potential target genes of miR-182 in PC3 cells. Bcl2 was downregulated and p21 was upregulated in PC3 cells exposed to ATO. These data suggest that miR-182 may be a stress-responsive miRNA that mediates ATO action in prostate cancer cells. Gene and miRNA expression in two prostate cancer cell lines treated with Atorvastatin vs. untreated control.
2013-09-30 | E-GEOD-46374 | biostudies-arrayexpress
Project description:MELK and UBE2C gene knockdown in prostate cancer cell line PC3
| PRJNA146599 | ENA
Project description:Following androgen ablation treatment for advanced prostate cancer, almost all men relapse after a period of initial response to therapy, which eventually is life threatening. We have previously found that purine-rich element binding protein, PUR alpha, was significantly repressed in androgen-independent prostate cancer cell lines in comparison to an androgen-dependent line. Moreover, over-expressing PURa in androgen-independent prostate cancer cells attenuated their cell proliferation. The aim of the studies described here was to uncover some of the mechanisms by which over-expression of PURa attenuates cell proliferation. A set of common genes induced by over-expressing PURa both in PC3 and LNCaP cells was analyzed by DNA microarray. The results were then validated utilizing quantitative reverse transcription-PCR. Using a 5.3-kb region of the PSA promoter containing androgen response elements, the participation of PURa in androgen regulated gene expression was determined. Genes involved in stress response and cell differentiation were induced in cells over-expressing PURa. Some of the genes that are targets of androgen regulation are also induced. Most strikingly, ectopic expression of PURa induced transcriptional activity of the 5.3-kb PSA promoter containing androgen response elements, without androgen stimulation. Based upon the consideration that some of the genes involved in cell stress and differentiation are also regulated by androgens our data suggest that PURa shares some common pathway regulated by the androgen receptor. These findings suggest that regulation of PURa expression in prostate cancer cells may serve as a therapeutic target for hormone refractory prostate cancer. Experiment Overall Design: PC3 cells (low in PURa expression) was stably over-expressed PURa. Total RNA of the cells was collected simultaneously with that of PC3 transfected with mock vector. Experiment Overall Design: LNCaP cells (Express PURa more than PC3 cells) was PURa-over-expressed with retrovirus vector (MSCV-PIG) and selected with puromycin for 72hr. Total RNA of the cells was collected simultaneously with that of LNCaP cells infected with mock vector. Experiment Overall Design: One set of RNA derived from PC3 cells and two independent RNA derived from LNCaP cells retrovirus infection were used for DNA microarray. Quality and concentration of total RNA was verified using Agilent Bioanalyzer and Nanodrop Spectrophotometer, respectively. T7 oligo(dT) primer (Sigma-Proligo, Boulder, CO) and 5ug total RNA are combined for first strand cDNA synthesis. Following second strand cDNA synthesis and cDNA cleanup (Phase Lock Gel Light, Eppendorf, Hamburg, Germany) an in vitro transcription reaction was performed overnight using T7 RNA transcript labeling kit provided by Enzo Life Sciences, Inc. (Farmingdale, NY). IVT reactions were cleaned using RNeasy Mini Kit (Qiagen) and concentration determined by Nanodrop Spectrophotometer. 15ug cRNA was fragmented using 5X fragmentation buffer (made in-house). Hybridization cocktail was made in accordance with Affymetrix eukaryotic expression array protocol (Affymetrix, Santa Clara, CA) and combined with fragmented cRNA. 10ug cRNA was loaded onto Human U133Plus 2.0 genome arrays (Affymetrix, Santa Clara, CA) and hybridized overnight for 16 hours. After hybridization, staining and washing of the arrays was performed following the Affymetrix eukaryotic expression array protocol, including staining with streptavidin-phycoerythrin, antibody stain, and a second streptavidin-phycoerythrin stain. After washing and staining, all arrays were scanned with the Affymetrix GeneChip Scanner and data collected with GeneChip Operating System 1.4 (GCOS, Affymetrix, Santa Clara, CA). Experiment Overall Design: The quality of the microarray experiments was assessed with affyPLM and Affy, two Bioconductor packages for statistical analysis of microarray data. To estimate the gene expression signals, data analysis was conducted on the chipsâ CEL file probe signal values at the Affymetrix probe pair (perfect match (PM) probe and mismatch (MM) probe) level, using the statistical algorithm Robust Multiarray Analysis (RMA) expression measure (Irizarry RA et al. 2003) with Affy. This probe level data processing includes a normalization procedure utilizing the quantile normalization method (Bolstad BM et al. 2003) to reduce the obscuring variation between microarrays, which might be introduced during the processes of sample preparation, manufacture, fluorescence labeling, hybridization and/or scanning. Exploratory data analysis (EDA) was performed with the preprocessed data above. Between-treatment and between-replicate variations were examined with the pair-wise MvA plots, in which the base 2 log ratios (M) between two samples are plotted against their averaged base 2 log signals (A). With the signal estimates, Principal Component analysis (PCA) was also performed to assess sample variability. Experiment Overall Design: With the signal intensities obtained above, an empirical Bayes method with the Gamma-Gamma modeling, as implemented in the bioconductor package EBarrays, was used to estimate the posterior probabilities of the differential expression of genes between PURA and Vector only sample conditions (Newton MA et al. 2001, Kendziorski Cm et al. 2003, Newton MA et al. 2004). The criterion of the posterior probability > 0.5, which means the posterior odds favoring change, was used to produce the differentially expressed gene list. Experiment Overall Design: All Bioconductor packages are available at http://www.bioconductor.org and all computation was performed under R environment (http://www.r-project.org).
2009-02-07 | E-GEOD-14464 | biostudies-arrayexpress
Project description:Expression profiles of aggressive versus non-aggressive ovarian, breast, melanoma, and prostate cancer cell lines Expression profiles of aggressive versus non-aggressive ovarian, breast, melanoma, and prostate cancer cell lines was determined. 231MFP, C8161, SKOV3, DU145, and PC3 are aggressive and MCF7, MUM2C, OVCAR3, and LNCaP are non-aggressive cancer cells. We are not comparing across all of the cell lines--just between C8161 and MUM2C, SKOV3 and OVCAR3, 231MFP and MCF7, and LNCaP/DU145/PC3. Therefore the normalization strategies used are different. We have not used the same normalization strategy
2011-08-09 | E-GEOD-28846 | biostudies-arrayexpress
Project description:Analysis of genes regulated by miR-23b/-27b overexpression in aggressive PC3-ML cells, confirmed by antagomiR inhibition of miR-23b and miR-27b in the relatively indolent cell line LNCaP. Genes that were downregulated in PC3-ML overexpression and upregulated with LNCaP inhibition were further explored as downstream targets of miR-23b/-27b. PC3-Ml cells were transduced with miR-23b/-27b or a scrambled miRNA control, and only cells expressing greater than 95% transduction efficiency were used for array. LNCaP cells were transfected with antagomiRs to miR-23b and miR-27b, or a non-coding control.
2016-02-10 | E-GEOD-76620 | biostudies-arrayexpress
Project description:BACKGROUND: Cancer stem-like cells are proposed to sustain solid tumors by virtue of their capacity for self-renewal and differentiation to cells that comprise the bulk of the tumor, and have been identified for a variety of cancers based on characteristic clonal morphologies and patterns of marker gene expression. METHODS: Single cell cloning and spheroid culture studies were used to identify a population of cancer stem-like cells in the androgen-independent human prostate cancer cell line PC3. RESULTS: We demonstrate that, under standard culture conditions, ~10% of PC3 cells form holoclones with cancer stem cell characteristics. These holoclones display high self-renewal capability in spheroid formation assays under low attachment and serum-free culture conditions, retain their holoclone morphology when passaged at high cell density, exhibit moderate drug resistance, and show high tumorigenicity in scid immunodeficient mice. PC3 holoclones readily form spheres, and PC3-derived spheres yield a high percentage of holoclones, further supporting their cancer stem cell-like nature. We identified one gene, FAM65B, whose expression is consistently up regulated in PC3 holoclones compared to paraclones, the major cell morphology in the parental PC3 cell population, and two genes, MFI2 and LEF1, that are consistently down regulated. This molecular profile, FAM65Bhigh/MFI2low/LEF1low, also characterizes spheres generated from parental PC3 cells. The PC3 holoclones did not show significant enriched expression of the putative prostate cancer stem cell markers CD44 and integrin M-NM-12M-NM-21. PC3 tumors seeded with holoclones showed dramatic down regulation of FAM65B and dramatic up regulation of MFI2 and LEF1, and unexpectedly, a marked increase in tumor vascularity compared to parental PC3 tumors, suggesting a role of cancer stem cells in tumor angiogenesis. CONCLUSIONS: These findings support the proposal that PC3 tumors are sustained by a small number of tumor-initiating cells with stem-like characteristics, including strong self-renewal and pro-angiogenic capability and marked by the expression pattern FAM65Bhigh/MFI2low/LEF1low. These markers may serve as targets for therapies designed to eliminate cancer stem cell populations associated with aggressive, androgen-independent prostate tumors such as PC3. (Mol Cancer. 2010 Dec 29;9:319. doi: 10.1186/1476-4598-9-319; PMID: 21190562; PMCID: PMC3024252). Four PC3 cell-derived holoclones, selected based on their clear and unambiguous holoclone morphology and designated holoclones 2H10, 2G7, 1A8 and 5A2, were used for global transcriptome/microarray analysis in direct comparison to parental PC3 cells. Total RNA was prepared from each of four sequential cell passages for each PC3-derived holoclone, and from four passages of parental PC3 cells, 48 h after seeding the cells at 100,000 cells per well of a 6-well plate. For each sample (holoclones or parental PC3 cells), a pool of RNA was prepared by combining equal amounts of RNA from each passage to minimize the effects of passage number and inter-sample variability on gene expression profiles. All RNAs had an RNA integrity number >8.0, determined using an Agilent Bioanalyzer 2100 instrument. cDNAs transcribed from pools of RNA for each holoclone, and for parental PC3 cells, were labeled with Alexa 647 or Alexa 555 dyes in a fluorescent reverse pair (dye swap) design for competitive hybridization to Agilent Whole Human Genome Microarrays.
2013-11-07 | E-GEOD-52161 | biostudies-arrayexpress