MIA gene family-related signaling in oral squamous cell carcinoma cells
ABSTRACT: Melanoma inhibitory activity (MIA) gene family is novel tumor-associated molecules. Although MIA gene family has several tumor progressive and/or suppressive functions, the detailed relevant signaling partners are unclear and investigated. In this study, we investigated the detailed MIA gene family-associated signaling using human oral squamous cell carcinoma cells. Human oral squamous cell carcinoma-derived HSC3 cells were transfected with control, MIA, MIA2, TANGO, MATE2, or LEMD1 siRNA. The effect on gene knockdown was evaluated by cDNA microarray.
Project description:Helicobacter pylori (H. pylori) infection and excessive salt intake are known as important risk factors for stomach cancer in humans. However, interactions of these two factors with gene expression profiles during gastric carcinogenesis remain unclear. In this study, we established a mouse gastric tumor model combining a chemical carcinogen, H. pylori infection and a high-salt diet. The tumor incidence and multiplicity in N-methyl-N-nitrosourea-treated mice were significantly increased by combination of H. pylori with a high-salt diet. In addition, detailed examination indicated that excessive salt could regulate progression of gastric tumor. Global gene expression profiles in glandular stomach of the mouse model were investigated by cDNA microarray analysis, and 36 and 31 more than twofold up-regulated and down-regulated genes, respectively, were detected in the H. pylori-infection and high-salt diet combined group compared with the other groups. Quantitative RT-PCR confirmed significant over-expression of several candidate genes including Cd177, Reg3g, and Muc13. These results suggest that our mouse model combined with H. pylori infection and high-salt diet is useful for gene expression profiling in gastric carcinogenesis. Five- to six-week-old male C57BL/6J mice (CLEA Japan, Tokyo, Japan) were inoculated with Helicobacter pylori (Sydney strain 1) (Sample No. 2 and 4) or Brucella broth (Sample No. 1 and 3). The animals were administered 120 ppm N-methyl-N-nitrosourea (MNU) in their drinking water on alternate weeks (total exposure, 5 weeks). Mice were given basal diet (CE-2, CLEA Japan) (Sample No. 1 and 2) or high-salt diet containing 10% NaCl (Sample No. 3 and 4). At 40 weeks, the animals were subjected to deep anesthesia and laparotomy with excision of the stomach.
Project description:The ubiquitin-proteasome and autophagy-lysosome pathways are the two major routes for protein and organelle clearance. In skeletal muscle, both systems’ excessive activation induces severe muscle loss. Although altered proteasomal function has been observed in various myopathies, the specific role of proteasomal activity in skeletal muscle has not been determined by loss-of-function approaches. Here, we report that muscle-specific deletion of a crucial proteasomal gene, Rpt3, resulted in profound muscle atrophy and decrease in force. Rpt3 null muscles showed reduced proteasomal activity in early age, accumulation of basophilic structure, disorganization of sarcomere, and formation of vacuoles and concentric membranous structures in electronmicroscope. We also observed accumulation of ubiquitin, p62, LC3, TDP43, FUS and VCP proteins. Proteasomal activity is important to preserve muscle mass and to maintain myofiber integrity. Our results suggest that inhibition/alteration of proteasomal activity can contribute to myofiber degeneration and weakness in muscle disorders, such as inclusion body myositis, characterized by accumulation of abnormal inclusions. Tibialis anterior muscles from Rpt3 null and control mice. each 3 mice.
Project description:We recently revealed that myeloid master regulator PU.1 directly represses metallothionein (MT)-1G through its epigenetic activity, but the functions of MT-1G in myeloid differentiation remain unknown. To clarify this, we established MT-1G-overexpressing acute promyelocytic leukemia NB4 (NB4MTOE) cells, and investigated whether MT-1G functionally contributes to all-trans retinoic acid (ATRA)-induced NB4 cell differentiation. Real-time PCR analyses demonstrated that the inductions of CD11b and CD11c and reductions in myeloperoxidase and c-myc by ATRA were attenuated in NB4MTOE cells. Since G1 arrest is a hallmark of ATRA-induced NB4 cell differentiation, we observed a decrease in G1 accumulation, as well as decreases in p21WAF1/CIP1 and cyclin D1 inductions, by ATRA in NB4MTOE cells. Nitroblue tetrazolium (NBT) reduction assays revealed that the proportions of NBT-positive cells were decreased in NB4MTOE cells in the presence of ATRA. Microarray analyses showed that the changes in expression of several myeloid differentiation-related genes (GATA2, azurocidin 1, pyrroline-5-carboxylate reductase 1, defensin-4, C-X3-C motif receptor 3, matrix metallopeptidase -8, S100 calcium-binding protein A12, neutrophil cytosolic factor 2 and oncostatin M) induced by ATRA were disturbed in NB4MTOE cells. Collectively, overexpression of MT-1G disturbs the proper differentiation of myeloid cells. The present study provides evidence that expression analysis of MT-1G in acute promyelocytic leukemia patients may be a good prediction marker to estimate the efficacy of ATRA. Cell culture and generation of MT-1G-overexpressing cells: To generate MT-1G-overexpressing cells and their control cells, the MT-1G expression vector and its parental pcDNA 3.1/myc-His(-) version A vector (Invitrogen) were transfected using a CLB-Transfection device (Lonza, Basel, Switzerland). NB4 clones stably transfected with the vectors were isolated by limiting dilution and selection with 400 µg/ml of neomycin in RPMI (Gibco BRL, Rockville, MD) containing 10% heat-inactivated fetal bovine serum (HIFBS). Cells were cultured under 5% CO2 at 37°C in a humidified atmosphere. Microarray analyses: MT-1G-overexpressing (NB4MTOE) cells and their control cells were seeded at a density of 1×105 cells/ml and treated with 1 µM all-trans retinoic acid (ATRA). The cells were harvested after 72 h and total cellular RNA was isolated from control (NB4pcDNA4, 6, 7) cells and NB4MTOE (NB4MT22, 23, 25) cells using an RNA Mini Purification Kit (Qiagen, Miami, FL) according to the manufacturer’s protocol. Aliquots containing 10 µg of RNA from each sample of control cells were mixed and used as controls. Similarly, 10 µg of RNA from each sample of NB4MTOE cells were mixed and used as NB4MTOE cells. The samples were subjected to microarray analyses using a CodeLink Human 54K Whole Genome Bioarray (Filgen, Nagoya, Japan).
Project description:[Objectives]: Neuropsychiatric systemic lupus erythematosus (NPSLE) is often difficult to diagnose and distinguish from those of other diseases, because no specific antibodies have yet been detected. [Methods]: We developed a novel proteomic strategy for identifying and profiling antigens in immune complexes (ICs) in the cerebrospinal fluid (CSF) of 26 NPSLE patients. We performed in vitro experiments using astrocytes and analyzed functional change by microarray. [Results]: We identified ICs of suprabasin (SBSN), a molecule which is thought to play a role in epidermal differentiation. Microarray data showed that the senescence and autophagy pathways were significantly changed in astrocytes with anti-SBSN antibody exposure compared to normal immunoglobulin G (IgG) exposure. [Conclusions]: These findings indicate that SBSN could be a novel autoantibody for the evaluation of suspected NPSLE, and may help elucidate the pathogenesis underlying this disease. The cultured human astrocyte exposed to non IgG, control rabbit IgG and anti suprabasin antibodies with or without lipopolysaccharide (1μg/ml) for 24 h were collected for RNA isolation. We divided into the following 6 samples, Non IgG LPS(-), Non IgG LPS(+), Norm IgG LPS(-), Norm IgG LPS(+), SBSN LPS(-) and SBSN IgG LPS(+).
Project description:Background: Lymphotoxin signaling via the lymphotoxin-β receptor (LTβR) has been implicated in several biological processes, ranging from development of secondary lymphoid organs, maintenance of splenic tissue, host defense against pathogens, autoimmunity, and lipid homeostasis. The major transcription factor that is activated by LTβR crosslinking is NF-κB. Two signaling pathways have been described that result in the activation of classical p50-RelA and alternative p52-RelB NF-κB heterodimers. Results: Using microarray analysis, we investigated the transcriptional response downstream of the LTβR in mouse embryoni fibroblasts (MEF) and its regulation by the RelA and RelB subunits of NF-κB. We describe novel LTβR-responsive genes that are regulated by RelA and/or RelB. Interestingly, we found that the majority of LTβR-regulated genes require the presence of both RelA and RelB, suggesting significant crosstalk between the two NF-κB activation pathways. Gene Ontology (GO) analysis confirmed that LTβR-NF-κB target genes are predominantly involved in the regulation of immune responses. However, other biological processes, such as apoptosis/cell death, cell cycle, angiogenesis, and taxis were also regulated by LTβR signaling. Moreover, we show that activation of the LTβR inhibits the expression of a key adipogenic transcription factor, peroxisome proliferator activated receptor-γ (pparg), suggesting that LTβR signaling may interfere with adipogenic differentiation. Conclusions: Thus, microarray analysis of LTβR-stimulated fibroblasts revealed further insight into the transcriptional response of LTβR signaling and its regulation by the NF-κB family members RelA and RelB. Keywords: cell type comparison (wt vs relA-/- vs relB-/-) after genetic modification using a time course for each cell type (wt, relA-/-, relB-/-) two time points were analysed (0h as control and 10h) using 3 technical replicates resulting in 18 samples in total
Project description:Kidney podocytes and their slit diaphragms contribute to prevent urinary protein loss. T cell from patients with systemic lupus erythematosus display increased expression of calcium/calmodulin kinase IV (CaMKIV). Here we evaluated the functional role of CaMKIV in lupus nephritis (LN) using kidney biopsy specimens and human podocyte cell line (AB8/13). We found that exposure of podocytes to IgG from LN patients resulted in entry of IgG into the cytoplasm. CaMKIV expression was found to be increased in podocytes of LN kidney biopsy specimens and exposure to IgG from LN patients. IgG entered podocytes using the FcRn receptor because when podocytes where treated with FcRn siRNA less IgG was found in the cytoplasm. The DNA microarray studies of podocytes exposed to LN IgG revealed that genes that are related to the activation of immune cells or podocyte damage were upregulated. These genes included CD86, CaMKIV, PTPN22, PDE5A, CD47 and MALT1. Interestingly, CD86 expression decreased after silencing CaMKIV in podocytes. Also, in situ hybridization experiments showed that the expression of CD86 was reduced in podocytes from MRL/lpr.camkiv−/− mice. IgG from LN patients may enter podocytes through the FcRn and causes the upregulation of a distinct set of genes which may alter podocyte function. Upregulation of CaMKIV appears to precede that of genes known to be linked to podocyte damage such as CD86. These findings may indicate that inhibition of CaMKIV may prove of clinical use in patients with LN. IgG Purification Kits (Dojindo Molecular Technologies, Inc.) are used for isolation and purification of immunoglobulin G of healthy and normal individual according to the manufacturer’s protocol. Flow through the column was used for non IgG binding samples. Cultured human podocytes with IgG purified from sera of normal individuals and LN patient for 24 hr were collected for RNA.
Project description:Background: Constitutive activation of the alternative NF-κB pathway leads to marginal zone B cell expansion and disorganized spleen microarchitecture. Furthermore, uncontrolled alternative NF-κB signaling results in the development and progression of cancer. We aimed here to learn about the mechanisms how does the constitutively active alternative NF-κB pathway exert its effects in these malignant processes. Methodology/Principal Findings: To explore the consequences of constitutive alternative NF-κB activation on genome-wide transcription, we compared gene expression profiles of wild-type and NF-kB2/p100-deficient (p100-/-) primary mouse embryonic fibroblasts (MEFs) and spleens. Microarray experiments revealed 73 differentially regulated genes in p100-/- vs. wild-type MEFs. Chromatin immunoprecipitation (ChIP) assays showed in p100-/- MEFs direct binding of RelB and p52 to the promoter of the enpp2 gene encoding Enpp2/Autotaxin, a protein with an important role in lymphocyte homing and cell migration. Gene ontology analysis revealed upregulation of genes with anti-apoptotic/proliferative activity (enpp2, serpina3g, traf1, rrad), chemotactic/locomotory activity (enpp2, ccl8), and lymphocyte homing activity (enpp2, cd34). Most importantly, biochemical analyses of MEFs and gene expression analyses of mice indicated a crosstalk between classical and alternative NF-κB pathways. Conclusions/Significance: The present results show that uncontrolled alternative NF-κB signaling is further enhanced by classical NF-κB activation, indicating that p100 deficiency alone is insufficient for full induction of a subset of genes by the alternative NF-κB pathway. cell type comparison (wt vs p100-/-) after genetic modification
Project description:Indole is a bacterial signal secreted by pathogenic and commensal Escherichia coli in the stationary phase at high concentrations (~600 µM). Prior work from our lab has shown that indole decreases E. coli O157:H7 (EHEC) chemotaxis, motility, attachment to epithelial cells and biofilm formation. However, its effect on epithelial cells is not known. We hypothesized that indole induces gene expression changes in epithelial cells that lead to decreased pathogen colonization and infection. Changes in gene expression with the human enterocyte cell line HCT-8 exposed to indole suggested down-regulation of Toll-like receptor signaling and coordinated changes in Jak-STAT and p38 MAPK pathways. Corresponding changes in the expression of cytokines, chemokines, and their receptors also suggested that indole functions as a modulator of inflammation in intestinal epithelial cells. In addition, the expression of genes involved in tight junction organization (claudins, and tight junction proteins) and mucin production were also up-regulated by indole. Keywords: Inter-kingdom signaling interactions between human cells and bacterial signal indole Human intestinal epithelial cells were exposed to 1 mM indole for 4 h or 24 h. RNA was isolated from the control cells and cells exposed to indole for 4 h and 24 h. The experiments were performed in triplicate. A total of 9 microarrays were performed, three for each condition. The gene expression data was analyzed using significance analysis of microarrays (SAM) where a false discovery rate cut-off of 1% was used. The genes were then classified and importnat regulated pathways were reported.
Project description:Three stallions were treated with 0.1 mg/kg dexamethasone i.v. while three control stallions received an equivalent volume of saline. Serum testosterone dropped 60% in treated stallions at 12 h post-injection. Testes were collected 12 h post-injection and assessed for differential gene expression. Adult stallions were acclimated to the clinic over a 10 day period, then treated 12 h prior to collection of testes. Differential gene expression in treated vs. control stallion testes
Project description:We used DNA microarrays to identify discriminative gene signatures for the purpose of classifying n-3 PUFA-fed, carcinogen injected Sprague Dawley rats at the initiation and progression stages. Animals were assigned to three dietary treatments differeing only in the type of fat (corn oi/n-6 PUFA, fish oil/n-3 PUFA, or olive oli/n-9 monounsaturated fatty acid). The effects of diet on colonic mucosal gene expression signatures during tumor initiation with the progression of colon cancer. Each dietary lipid source exhibited its own unique transcriptional profile, as assessed by linear discriminant analysis. Applying this novel approach we identified the single genes and the two- to three-gene combinations that best distinguished the dietary treatment groups. For the chemoprotective fish oil diet, mediators of stem cell homeostasis, e.g., ephrin B1 and bone morphogenic protein 4, were the top-permorming gene classifiers. keywords: diet analysis 29 samples were analyzed. 10 samples had repeat arrays. No control or reference samples were included.