Apoptosis induction in rat mammary tumor cells by repression of Pla2g4c expression.
Ontology highlight
ABSTRACT: Although some phospholipase A2 forms, the initiator of the arachidonic acid cascade, contribute to carcinogenesis of many organs, the phospholipase A2 group IVc (Pla2g4c) remains to be clarified. When Pla2g4c expression in Rat mammary tumor-1 E4 (RMT-1) cells was knocked down by using specific siRNAs, cell counts were found to decrease to 40 % of the number of control and apoptotic cells were increased. Whole transcript profiling revealed the up-regulation of lipocalin 2 and down-regulation of epithelical marker genes. The expression of Pla2g4c was blocked by transient transfection with Pla2g4c siRNA in RMT-1 cells.
Project description:Although some phospholipase A2 forms, the initiator of the arachidonic acid cascade, contribute to carcinogenesis of many organs, the phospholipase A2 group IVc (Pla2g4c) remains to be clarified. When Pla2g4c expression in Rat mammary tumor-1 E4 (RMT-1) cells was knocked down by using specific siRNAs, cell counts were found to decrease to 40 % of the number of control and apoptotic cells were increased. Whole transcript profiling revealed the up-regulation of lipocalin 2 and down-regulation of epithelical marker genes.
Project description:We examined FASN knockdown LNCaP cells obtained by shRNA transduction with Mission lentiviral transduction particles (SHCLNV-NM 00410, TRCN3128, Sigma) (FASN-RNAi cells). In this study, we used cells transfected with non-targeting shRNA as a control (control-RNAi cells). The expression of genes related to cellular proliferation (phospholipase A2, group IVA, PLA2G4A; tensin 3, TNS3; glypican 4 GPC4), cell adhesion and extracellular matrix organization [peroxidasin homolog (Drosophila) PXDN; sarcoglycan epsilon, SGCE; von Willebrand factor, VWF; hydroxysteroid (17-beta) dehydrogenase 12, HSD17B12; cysteine-rich secretory protein LCCL domain containing 2, CRISPLD2], and cell motility (TNS3, RAP2B member of RAS oncogene family, RAP2B) were shown to be down-regulated by FASN inhibition with RNAi. FASN inhibition led to down-regulation of the PLA2G4A and HSD17B12 genes encoding phospholipase A2 and 17-beta hydroxysteroid dehydrogenase, respectively, which are the key enzymes related to production of an intracellular second messenger arachidonic acid and androgen hormones, both playing roles in promotion of tumor progression. We also found that the genes related to arachidonic acid signalling, including RGS2, SPAG16, VWF and RAP2B, were also suppressed with FASN inhibition. Gene expression profiling therefore demonstrated that FASN inhibition induces down-regulation of genes related to cell proliferation, cell adhesion, migration, and invasion, as well as the production of arachidonic acid and androgen hormones, both of which drive tumor progression. Total RNA isolation was performed with a Micro-to-Midi total RNA purification system (Invitrogen). The integrity of total RNAs was evaluated using an Agilent 2100 Bioanalyzer (Agilent Technologies). Low Input Quick Amp Labeling Kit, one-color (Agilent Technologies) was used to prepare Cy3-labelled target cRNA according to the manufacturer's instructions. Labeled cRNAs were hybridized with a SurePrint G3 Human GE 8M-CM-^W60K Microarrays (Agilent Technologies). Two separate hybridizations were performed for each sample. Array images were captured using a DNA Microarray Scanner (Agilent Technologies), and data were analyzed using Feature Extraction Software (Agilent Technologies) to obtain background-corrected signal intensities. The data were further analysed with GeneSpring GX Software (Version 11.0, Agilent Technologies). After filtering of data, mRNAs differentially expressed in target versus control were considered using the Fisher exact test, followed by multiple corrections using the Benjamini and Hochberg false discovery rate (FDR) method. Gene sets with a FDR q-value < 0.05 were considered significant.
Project description:In this study, we demonstrate that insulin is produced not only in the mammalian pancreas but also in adult neuronal cells derived from hippocampus and olfactory bulb. Paracrine Wnt3 plays an essential role in promoting the active expression of insulin in both hippocampus and olfactory bulb-derived neural stem cells. Our analysis indicates that the balance between Wnt3, which triggers the expression of insulin via NeuroD1 transcription factor, and IGFBP-4, which inhibits the original Wnt3 action, is regulated depending on the diabetic status. We also show that adult neural progenitors derived from diabetic animals retain the ability to give rise to insulin-producing cells and that grafting neuronal progenitors into the pancreas of diabetic animals reduces glucose levels. This study provides an example of a simple and direct use of adult stem cells from one organ to another, without introducing additional inductive genes. In this study, we demonstrate that insulin is produced not only in the mammalian pancreas but also in adult neuronal cells derived from hippocampus and olfactory bulb. Paracrine Wnt3 plays an essential role in promoting the active expression of insulin in both hippocampus and olfactory bulb-derived neural stem cells. Our analysis indicates that the balance between Wnt3, which triggers the expression of insulin via NeuroD1 transcription factor, and IGFBP-4, which inhibits the original Wnt3 action, is regulated depending on the diabetic status. We also show that adult neural progenitors derived from diabetic animals retain the ability to give rise to insulin-producing cells and that grafting neuronal progenitors into the pancreas of diabetic animals reduces glucose levels. This study provides an example of a simple and direct use of adult stem cells from one organ to another, without introducing additional inductive genes. Total four different samples, gene expressions in hippocampal derived neural stem cells (HPC NSC), that in Olfactory bulb-derived neural stem cells (OB NSC), that in neurons derived from the HPC NSCs (HPC Neu) and that in neurons derived from the OB NSCs (OB Neu) were independently analyzed. Three independent experiments were performed to prepare each cell sample, and the extracted total RNAs from each cell source were mixed to apply following microarray analysis (Four independent RNA sample; HPC NSC, OB NSC, HPC Neu and OB Neu).
Project description:We analyzed the comprehensive gene expression changes by nerve growth factor (NGF) in PC12 cells using Agilent microarrays. The genes of 1920 NGF treated were up-regulated and 2670 genes were down-regulated significantly compared with the control group. The expression level of genes involved in cell structure and proteolysis were up-regulated. On the other hand, the genes of cell cycle and some metabolism were rather down-regulated. Therefore, it is suggested that it is necessary to stop the cell cycle and down-regulate metabolism to neurite outgrowth. Gene regulation associated with the differentiation program in PC12 cells still needs to be elucidated. We analyzed the genes expression with Agilent microarrays after 6 hours of NGF treatment by the one-color method, and found 2099 genes were up-regulated and 2781 genes were down-regulated.
Project description:Loss of function of dystonin cytoskeletal linker proteins causes neurodegeneration in the sensory ataxia, dystonia musculorum (dt). While much investigation has focused on understanding dt pathology, divergent functions of dystonin isoforms are still unknown. Here, we highlight a novel function of the dystonin-a2 isoform in mediating microtubule (MT) stability, golgi organization and flux through the secretory pathway. Using dystonin-null mice combined with isoform-specific loss of function analysis, we find dystonin-a2 is bound to MAP1B in the area surrounding the centrosome, where it maintains MT acetylation. In dt, the absence of the MAP1B-dystonin-A2 interaction results in a loss of MAP1B perinuclear localization, leading to MT deacetylation and instability. Deacetylated MTs lead to golgi fragmentation and prevent anterograde trafficking of motor proteins. Maintenance of MT acetylation through TSA administration or MAP1B overexperssion in vitro, mitigates the observed defect. These aberrations are apparent in pre-phenotype dorsal root ganglia (DRG) and primary sensory neurons, suggesting they are causal in the dt disorder. P4 dorsal root ganglia (DRG) tissue from 3 WT and 3 dt27J animals from 2 separate litters was subjected to RNA extraction and analyzed.
Project description:Osteoarthritis (OA) is a complex degenerative joint disease, which is not only a cartilage but also a bone disease. A better understanding of the early molecular mechanism changes of subchondral bone in vivo may contribute to elucidating the pathogenesis of OA. We used microarray technology to investigate the time-course molecular changes of subchondral bone just beneath damaged cartilage in early stage of experimental osteoarthritis, and found 2,234 differentially expressed (DE) genes at 1 week, 1,944 at 2 weeks and 1,517 at 4 weeks postsurgery.Further analysis of dysregulated genes indicated that subchondral bone remodeling occurred sequentially and in a time-dependent manner at the gene expression level. Some known dysregulated genes suspected roles in influencing bone development or bone remodeling, such as Alp, Igf1, Tgf M-NM-21, Postn, Mmp3, Tnfsf11, Acp5, Bmp5, Aspn and Ihh, were confirmed by real-time PCR, and results indicated that our microarray data could accurately reflect gene expression patterns of early OA. Subsequently, to validate the results of our microarray analysis at protein level, immunohistochemistry staining was introduced to investigate the translational level of genes Mmp3 and Aspn in tissue sections, and results showed that the level of Mmp3 protein expression was totally matched the results of microarray and real-time PCR analysis. Nevertheless, the expression of Aspn protein was not observed differentially expressed at any time point. Ninety 10-week-old male Sprague-Dawley rats, weighing 300-325g, were used in the study. Animals were equally divided into two groups: experimental group (E-Group) and sham-operated group (S-Group). The E-Group rats underwent open surgery, involved in both medial meniscectomy and medial collateral ligament (MCL) transaction with micro-scissors. The S-Group rats were carried out with a sham operation, via a similar incision, without operations of the medial meniscus and the medial collateral ligament.Animals were killed at 1, 2, and 4 weeks postsurgery, and 15 animals were put into use per-timepoint in each treatment group. 5 animals were used for histological analysis and immunohistochemistry, and others were used for microarray study and Real-time polymerase chain reaction (PCR) analysis equally at each timepoint.
Project description:To further development of our gene expression approach to lithium pharmacodynamics, we have employed whole genome microarray expression profiling as a discovery platform to identify genes with the potential to distinguish lithium pharmacodynamics across an exposure range relevant for medical decision-making in lithium therapy. 8 healthy male subjects participated in this study. Lithium was prescribed for 2 weeks, enough to reach a therapeutic serum concentration (0.6-1.0mM). Leukocyte counts and serum lithium concentrations were determined at baseline (before medication), after 1 week and 2 weeks medication and post-2-weeks (2 weeks after stopping medication). Gene expression profiling was performed at each time point using Agilent G4112F Whole Human Genome arrays containing approximately 44,000 probe sets. Some of the candidate gene expressions were also determined by real-time PCR. Lithium-induced gene expression in human blood was measured at baseline, after 1 week and 2 weeks of treatment, and after 2 weeks after stopping medication. 4 independent experiments were performed (baseline, 1wk, 2wk, after 2wk) using mixed samples from each donor.
Project description:To obtain insight into the effect of starvation on heart ischemia/reperfusion injury, gene expression patterns in the hearts of satiated and fasted mice were examined. Gene expression was examined in the hearts of satiated (n=4) and 2-day-fasted C57BL/6J male mice.
Project description:Oral administration of an extract of compost fermented with thermophiles to pigs reduces the incidence of stillbirth and promotes piglet growth. However, the mechanism by which compost extract modulates the physiological conditions of the animals remains largely unknown. Here, we investigate the effects of compost extract on the gene expression in the intestine of the rat as a mammalian model. Gene expression analyses of the intestine indicated that several immune-related genes were upregulated following compost exposure. Thus, thermophile-fermented compost can contain microbes and/or substances that activate the gut mucosal immune response in the rat. In Male Wistar rats aged 3 weeks, tap water was supplemented with 1.0% (v/v) compost extract for the experimental rats, whereas water only was given to the control rats. The rats received water ad libitum for 12 weeks. Fresh gut samples were collected from individual rats at the end of the feeding test and stored at -80°C. The intestine were separated from the gut and used as samples for the isolation of total RNA. otal RNA was then subjected to microarray experiments using the Whole Rat Genome (4x44k) Oligo Microarray (Agilent Technologies, Inc.)
Project description:Rat osteoblasts ROB17/2.8 cells were cultured in the presence of high (1mg/ml) or low (0.01mg/ml) concentration of polyphosphates. Poly(P) induced gene expression in rat osteoblast ROS17/2.8 cells was measured after the treatment with high (1mg/ml) or low (0.01mg/ml) for 24 h.