Project description:Hepatocellular carcinoma (HCC) is the leading cause of cancer-related deaths worldwide, reflecting the need for a better understanding of hepatocarcinogenesis. In this study, we examined the genome-wide expression profiles of both miRNAs and mRNAs from paired tumor and adjacent non-tumor tissues from a cohort of 96 HCC patients in Hong Kong where hepatitis B virus (HBV) is endemic. The comprehensive analysis of the coordinate expression of miRNAs and mRNAs reveals that miR-122 is under-expressed in HCC and that it is an important regulator for normal mitochondrial metabolism. A decreased level of miR-122 leads to an increase in expression of miR-122 seed-matched genes, a loss of mitochondrial metabolic function, and a decrease in the expression of many miR-122 secondary targets. These secondary targets are prognostic markers for HCC patients. Transcriptome profiling data from an additional 180 HCC and 40 liver cirrhotic patients in the same cohort were used to confirm the anti-correlation of miR-122 primary and secondary target gene sets. To test the HCC findings in normal mouse liver, we used an antagomir against miR-122 and identified altered gene expression profiling by microarray analysis in mouse in vivo experiments. The mouse data recapitulated the human HCC findings. Our anti-miR122 data further provided a direct link between increases in the mRNA levels of miR-122 controlled genes and impairments of mitochondrial metabolism. In summary, we find miR-122 loss may be detrimental to mitochondrial-related metabolisms in sustaining critical liver function and contribute to the morbidity and mortality of liver cancer patients. Mouse liver profiling: In one treatment regimen, four animals were given four doses of 100mg ASO/kg body weight, administered every other day, and sacrificed one day after the last dose. Four animals treated for four weeks were treated twice weekly with 50mg ASO/kg body weight, and sacrificed two days after the last dose. Mice were sacrificed in the morning, and liver was removed for further analysis. Samples from oligonucleotide-treated mice were referenced against samples from saline-treated mice in fluor-reversed pairs. Human liver profiling: For human HCC patient samples, 192 samples -- 96 resected tumor and 96 adjacent non-tumor liver tissues -- were collected from 96 patients who had undergone hepatectomy for curative treatment of HCC at Queen Mary Hospital, Pokfulam, Hong Kong between 1990 and 2007. Informed consents were obtained from patients regarding the use of the liver specimens for research. Samples were hybridized to Affymetrix arrays for mRNA profiling and were analyzed by quantitative PCR for microRNA profiling.

Project description:Pancreatic ductal adenocarcinoma (PDAC) is a nearly uniformly lethal malignancy, with most patients facing an adverse clinical outcome. Given the pivotal role of aberrant Notch signaling in the initiation and progression of PDAC, we investigated the effect of MRK-003, a potent and selective γ-secretase inhibitor, in preclinical PDAC models. We used a panel of human PDAC cell lines, as well as patient-derived PDAC xenografts, to determine whether pharmacological targeting of the Notch pathway could inhibit pancreatic tumor growth and potentiate gemcitabine sensitivity. In vitro, MRK-003 treatment downregulated the canonical Notch target gene Hes-1, significantly inhibited anchorage independent growth, and reduced the subset of CD44+CD24+ and aldehyde dehydrogenase (ALDH)+ cells that have been attributed with tumor initiating capacity. Ex vivo pretreatment of PDAC cells with MRK-003 in culture significantly inhibited the subsequent engraftment in immunocompromised mice. In vivo, MRK-003 monotherapy significantly blocked tumor growth in 5 of 9 (56%) patient-derived PDAC xenografts. Moreover, a combination of MRK-003 and gemcitabine showed enhanced antitumor effects compared to gemcitabine alone in 4 of 9 (44%) PDAC xenografts. Baseline gene expression analysis of the treated xenografts indicated that upregulation of nuclear factor kappa B (NFκB) pathway components was associated with the sensitivity to single MRK-003, while upregulation in B-cell receptor (BCR) signaling and nuclear factor erythroid-derived 2-like 2 (NRF2) pathway correlated with response to the combination of MRK-003 with gemcitabine. The preclinical findings presented here provide further rationale for small molecule inhibition of Notch signaling as a therapeutic strategy in PDAC. Pancreatic ductal adenocarcinoma xenografts were grown in Athymic Nude-Foxn1nu mice. RNA was extracted and profiled in Affymetrix platform to identify genes correlating with sensitivity to MRK-003

Project description:Deregulated developmental processes in the cerebellum cause medulloblastoma, the most common malignant tumor of the central nervous system. About 20-30% of cases are caused by mutations increasing the activity of the Sonic hedgehog (Shh) pathway, a critical mitogen in cerebellar development. The proto-oncogene Smoothened is a key transducer of the Shh pathway. Activating mutations in Smoothened that lead to constitutive activity of the Shh pathway have been identified in human medulloblastoma. To understand the molecular and cellular effects of Smoothened variants in normal development and medulloblastoma genesis, we generated the SmoA2 transgenic mouse model which expresses the transgene exclusively in granule neuron precursors. In this study, we demonstrate how two point mutations in a single molecule can produce starkly different phenotypes as seen in comparison to our previous model, ND2:SmoA1. The SmoA2 mice have severe aberrations in cerebellar development whereas the SmoA1 mice are largely normal during development. Medulloblastomas in the SmoA2 mice develop in the dysplastic cerebellar milieu. Intriguingly, despite disruptions in the stereotypic organization of the cerebellum, the SmoA2 mice do not exhibit any overt abnormalities in motor coordination. The differences in the global transcriptional profiles downstream of SmoA1 and SmoA2 further demonstrate the distinctiveness of the two oncogenic Smoothened mutations. The SmoA2 model serves as a unique spatiotemporal tool to investigate the functional significance of the reiterative cerebellar circuitry as well as to further understand Shh pathway mechanics in cerebellar development and oncogenesis. We previously generated a SmoA1 transgenic mouse medulloblastoma model, which expresses the SmoA1 transgene driven by the GNP-specific fragment of the promoter of ND2 transcription factor leading to constitutively active Shh signaling exclusively in the cerebellum. In this study, we characterize the ND2:SmoA2 transgenic mouse model with a similarly designed transgene expressing the SmoA2 mutation. To assess transcriptional changes downstream of SmoA1 and SmoA2, we evaluated global gene expression profiles of P5 SmoA1, SmoA2 and Wt age-matched cerebella. We chose this specific developmental stage because (1) the phenotypes of SmoA1 and SmoA2 are robust and distinct at P5; (2) at P5 GNPs undergo proliferation, migration and differentiation and therefore expression profiling could capture key differences in multiple processes.

Project description:Cancer cells exhibit the ability to proliferate indefinitely, but paradoxically, overexpression of cellular oncogenes in primary cells can result in a rapid and irreversible cell cycle arrest known as oncogene-induced senescence (OIS). However, we have shown that constitutive overexpression of the oncogene c-MYC in primary human foreskin fibroblasts results in a population of cells with unlimited lifespan; these immortalized cells are henceforth referred to as iMYC. Here, in order to further elucidate the mechanisms underlying the immortalization process, a gene expression signature of three independently established iMYC cell lines compared to matched early passage c-MYC overexpressing cells was derived. Network analysis of this "iMYC signature" indicated that a large fraction of the down-regulated genes were functionally connected and major nodes centered around the TGFb, IL-6 and IGF-1 signaling pathways. Here, we focused on the functional validation of the alteration of TGFb response during c-MYC-mediated immortalization. The results demonstrate loss of sensitivity of iMYC cells to activation of TGFb signaling upon ligand addition. Furthermore, we show that aberrant regulation of the p27 tumor suppressor protein in iMYC cells is a key event that contributes to loss of response to TGFb. These findings highlight the potential to reveal key pathways contributing to the self-renewal of cancer cells through functional mining of the unique gene expression signature of cells immortalized by c-MYC. Cell Cycle. 2011 Aug 1;10(15). Cell culture. Human foreskin fibroblast cell lines expressing empty vector pBABE or vector with gene sequence encoding c-MYC were previously described in PMID 17982115. Cells were cultured in Dulbecco’s modified eagle medium (DMEM) supplemented with 10% fetal bovine serum and penicillinstreptomycin. For cell growth assays, equal number of cells per cell line were plated and counted on the specified days after plating. Microarray analysis. Total RNA was purified using an RNeasy kit (Qiagen, Valencia, CA). Genome-scale expression analysis was performed using custom microarrays (Affymetrix, Santa Clara, CA) containing oligonucleotide probes corresponding to approximately 22,000 human genes. Microarray analysis was performed as described in PMID 12925520. Data were analyzed using Rosetta Resolver(TM) software. We determined a p-value cutoff of 0.01 for genes in all three samples.

Project description:FGF21 is a novel secreted protein with robust anti-diabetic, anti-obesity, and anti-atherogenic activities in preclinical species. In the current study, we investigated the signal transduction pathways downstream of FGF21 following acute administration of the growth factor to mice. Focusing on adipose tissues, we identified FGF21-mediated downstream signaling events and target engagement biomarkers. Specifically, RNA profiling of adipose tissues and phosphoproteomic profiling of adipocytes, following FGF21 treatment revealed several specific changes in gene expression and post-translational modifications, specifically phosphorylation, in several relevant proteins. Affymetrix microarray analysis of white adipose tissues isolated from both C57BL/6 (fed either regular chow or HFD) and db/db mice identified over 150 robust potential RNA transcripts and over 50 potential secreted proteins that were changed greater than 1.5 fold by FGF21 acutely. Phosphoprofiling analysis identified over 130 phosphoproteins that were modulated greater than 1.5 fold by FGF21 in 3T3-L1 adipocytes. Bioinformatic analysis of the combined gene and phosphoprotein profiling data identified a number of known metabolic pathways such as glucose uptake, insulin receptor signaling, Erk/Mapk signaling cascades, and lipid metabolism. Moreover, a number of novel events with hitherto unknown links to FGF21 signaling were observed at both the transcription and protein phosphorylation levels following treatment. We conclude that such a combined "omics" approach can be used not only to identify robust biomarkers for novel therapeutics but can also enhance our understanding of downstream signaling pathways; in the example presented here, novel FGF21-mediated signaling events in adipose tissue have been revealed that warrant further investigation. Three mouse strains (C57BL6 on chow diet, C57BL6 on high fat diet, and db/db on chow diet) were treated with either vehicle, wild-type FGF21, or pegylated FGF21 acutely or for several days and three white adipose tissues (IWAT, EWAT, RPWAT) and brown adipose tissue (BAT) were profiled on custom Affymetrix microarrays. The primary goal was to identify robust and consistent acute target engagement biomarkers of FGF21 activation in white adipose tissues.

Project description:Hypertension is a condition with major cardiovascular and renal complications, affecting nearly a billion patients worldwide. Few validated gene targets are available for pharmacological intervention, so there is a need to identify new biological pathways regulating blood pressure and containing novel targets for treatment. The genetically hypertensive âblood pressure highâ (BPH), normotensive âblood pressure normalâ (BPN), and hypotensive "blood pressure low" (BPL) inbred mouse strains are an ideal system to study differences in gene expression patterns that may represent such biological pathways. We profiled gene expression in liver, heart, kidney, and aorta from BPH, BPN, and BPL mice and determined which biological processes are enriched in observed organ-specific gene signatures. As a result, we identified multiple biological pathways linked to blood pressure phenotype that could serve as a source of candidate genes causal for hypertension. In order to distinguish causal genes from responsive genes in the kidney gene signature we integrated phenotype associated genes into Genetic Bayesian networks, identifying several novel candidate genes causal for hypertension. The integration of data from gene expression profiling and genetics networks is a valuable approach to identify novel potential targets for the pharmacological treatment of hypertension. Animals: BPH/2J, BPL/1J and BPN/3J (142 Â± 5 mmHg, 69 Â± 1.7 mmHg, 94 Â± 6 mmHg, SBP respectively) male mice (Jackson Laboratory) were maintained on a 12:12-h light-dark cycle and fed with standard chow ad libitum in facilities accredited by the Association for Assessment and Accreditation of Laboratory Animal Care. All procedures were in conformance with the National Research Council's Guide for the Care and Use of Laboratory Animals. Tissue collection: Mice (12 weeks old) were euthanized with CO2. Liver, heart, and kidney were flash frozen in liquid nitrogen. Aorta from the aortic root to the left renal artery was rinsed with PBS, immersed in 300ul of RNeasy Lysis buffer (Qiagen), and flash frozen in liquid nitrogen. RNA preparation: Tissues (~100mg, 5 mice per strain) were collected independently and homogenized in 2ml of Trizol (Invitrogen). After extraction with 0.4 ml of chloroform, RNA was extracted with SV Total RNA extraction kit (Promega) followed by DNase I treatment and purification using the RNeasy Kit (Qiagen). RNA was assayed for quality (Agilent Bioanalyzer) and yield (Ribogreen). Kidney, heart and liver RNA was amplified and labeled using a custom automated version of the RT/IVT protocol and reagents provided by Affymetrix. Aorta RNA was amplified and labeled using a custom automated version of the NuGEN Ovation WB protocol (NuGEN). Hybridization, labeling and scanning were according to Affymetrix. All samples were processed independently. Microarray analysis: Merck/Affymetrix mouse 1.0 custom arrays monitoring 38384 individual transcripts (25846 Entrez genes) were used. Raw intensity was normalized using the RMA algorithm.

Dataset Information

Study of gene expression of human liver Hepatocellular carcinoma

Similar Datasets

OmicsDI is part of the ELIXIR infrastructure