Profiling transcriptional and translational response to anoxia or hypoxia of genes in DNA damage response and repair pathways in human fibroblast and prostate cancer cells using NanoString nCounter.
Ontology highlight
ABSTRACT: Identification of genes in DNA damage response and repair pathways differentially transcribed or translated under anoxia or hypoxia in GM05757 normal human fibroblast cells and DU145 human prostate cancer cells. Comparison of mRNA abundance and translation efficiency of genes in DNA damage response and repair pathways in selected anoxia/hypoxia-treated cells with those in normoxia-treated controls.
Project description:Using outcome after long term follow-up to define risk at disease presentation high risk (n=9 who went on to require liver transplantation) and low risk (n=7 who responded fully to UDCA) patients were identified and their first liver biopsies retrieved. RNA was successfully extracted and analysed using nanostring® transcriptomics. Patients with progressive disease appear to have a distinct molecular signature. high risk (n=9 who went on to require liver transplantation) and low risk (n=7 who responded fully to UDCA) patient material was processed along with non-diseased control liver (n=8)
Project description:The freshwater fish crucian carp (Carassius carassius) are able to survive chronic anoxia for several months at low temperatures. Consequently, anoxia-related physiological and biochemical adaptations in this species have been studied for more than half a century. Still, despite for the well-known role of protein phosphorylation in regulating cellular processes, no studies have comprehensively characterized the phosphoproteome in crucian carp. In this study, we report the global phosphoproteome in crucian carp brain and liver during anoxia and reoxygenation. By applying a bottom-up proteomic approach on enriched phosphopeptides we found that the brain phosphoproteome show surprisingly few changes during anoxia-reoxygenation exposure with 110 out of 4316 phosphopeptides being differentially regulated. By contrast, in the liver 395 out of 1293 phosphopeptides were regulated. Although most changes occurred in the liver phosphoproteome, we found evidence for metabolic depression and decreased translation in both brain and liver. We also found regulated phosphoproteins involved in apoptotic regulation and reactive oxygen species handling in both tissues. In the brain, some of the most regulated phosphopeptides belonged to proteins involved in central nervous system development and neuronal activity at the synaptic cleft. Regulated phosphoproteins specific for liver tissue were related to glucose metabolism, including glycolytic flux and glycogenolysis, and the ubiquitin-proteasome system.
Project description:The environment inside even a small tumor is characterized by total (anoxia) or partial oxygen deprivation, hypoxia. It has been shown that radiotherapy and some conventional chemotherapies may be less effective in hypoxia, and therefore it is important to investigate how different drugs act in different microenvironments. In the associated study we performed a large screening of the effects of 19 clinically used or experimental chemotherapeutic drugs on four different cell lines in conditions of normoxia, hypoxia and anoxia. A panel of 19 commercially available drugs: 5-fluorouracil, acriflavine, bortezomib, cisplatin, digitoxin, digoxin, docetaxel, doxorubicin, etoposide, gemcitabine, irinotecan, melphalan, mitomycin c, rapamycin, sorafenib, thalidomide, tirapazamine, topotecan and vincristine were tested for cytotoxic activity on the cancer cell lines A2780 (ovarian), ACHN (renal), MCF-7 (breast), H69 (SCLC) and U-937 (lymphoma). Parallel aliquots of the cells were grown at different oxygen pressures and after 72 hours of drug exposure viability was measured with the fluorometric microculture cytotoxicity assay (FMCA). Sorafenib, irinotecan and docetaxel were in general more effective in an oxygenated environment, while cisplatin, mitomycin c and tirapazamine were more effective in a low oxygen environment. Surprisingly, hypoxia in H69 and MCF-7 cells mostly rendered higher drug sensitivity. In contrast ACHN appeared more sensitive to hypoxia, giving slower proliferating cells, and consequently, was more resistant to most drugs. Gene expression analysis was performed on MCF-7 cells after 90 hours in either anoxic or hypoxic conditions, and compared to cells grown in a regular cell incubator. The gene expression analysis was performed to validate that the cells were hypoxic/anoxic and showed the characteristic hypoxia response. Microarray based mRNA profiling was used to charactarize cells grown in hypoxia and anoxia. In the associated study we performed a large screening of the effects of 19 clinically used or experimental chemotherapeutic drugs on four different cell lines in conditions of normoxia, hypoxia and anoxia. We fin that hypoxia/anoxia render cancer cells both more resistant and more sensistive, depending of the type of drug used. The gene expression analysis was performed to validate that the cells really were hypoxic/anoxic and showed the characteristic hypoxia response. The cell line used for the gene expression analysis was MCF-7.
Project description:Neuronal or glial cell fates related gene expression from single cells with variety of sAPPa or total Aβ secretion profiles were presented here. Single living human iPSC-derived forebrain neuronal cells with variety of sAPPa or total Aβ secretion profiles were selected and examined for gene expression.
Project description:The freshwater fish crucian carp (Carassius carassius) can survive complete oxygen depletion (anoxia) for several months at low temperatures, achieved by a combination of reduced energy demand and increased glycolysis fueled by large hepatic glycogen stores. In crucian carp, the energy-requiring protein synthesis is controlled in a tissue-specific manner when oxygen levels decrease. During anoxia, translational rates are maintained at almost normoxic levels in brain, while heart and liver translation rates are strongly reduced. However, little is known about how the global proteome of these tissues are affected by oxygen variations. By applying mass spectrometry-based proteomics, 3304 proteins in brain, 3004 proteins in heart and 2516 proteins in liver were detected, of which 66 brain proteins, 243 cardiac proteins and 162 hepatic proteins were differentially expressed during the course of anoxia-reoxygenation compared to normoxic control. The brain proteome showed few differences in response to oxygen variations, indicating that anoxic survival is not regulated through protein expression in this tissue. Cardiac and hepatic adaptions to anoxia included enrichment of mitochondrial proteins involved in aerobic respiration and mitochondrial membrane integrity. We show that enzymes in the electron transport system (ETS) are regulated in a tissue-specific manner since no ETS components were regulated in brain, but were downregulated in heart and upregulated in liver during anoxia and reoxygenation. Furthermore, complement system activation was enriched in heart during anoxia. During reoxygenation, proteins involved in the cristae junction organization were regulated in the heart, possibly explaining how reactive oxygen species can be avoided when oxygen returns in this master of anoxic survival.
Project description:We compared transcriptomic profiles of ICC tumor specimens to hepatocellular carcinoma (HCC) specimens using Affymetrix mRNA array and the miRNA array platforms to search for unique gene signatures linked to patient prognosis. ICC and HCC share common stem-like molecular characteristics and stem-like tumor features associated with poor prognosis. Gene expression profiling of 16 intrahepatic cholangiocarcinoma (ICC), 7 mixed type of combined hepatocellular cholangiocarcinoma (CHC), 2 Hepatic adenoma, 3 focal nodular hyperplasia (FNH), 5 non-tumor liver tissues, and 2 CCA cell lines were performed.
Project description:Neuronal or glial cell fates related gene expression from single cells with variety of sAPPa or total Aβ secretion profiles were presented here. Single living human iPSC-derived forebrain neuronal cells with variety of sAPPa or total Aβ secretion profiles were selected and examined for gene expression.
Project description:Tumour hypoxia exhibits a highly dynamic spatial and temporal distribution and is associated with increased malignancy and poor prognosis. Assessment of time-dependent gene-expression changes in response to hypoxia may thus provide additional biological insights and help with patient prognosis. Transcriptome profiling was performed for 3 cell lines derived from diverse tumour-types after hypoxic exposure at 8 time-points.
Project description:Numerous studies have described the altered expression and the causal role of miRNAs in human cancer. However, to date efforts to modulate miRNA levels for therapeutic purposes have been challenging to implement. Here, we find that Nucleolin (NCL), a major nucleolar protein, post-transcriptionally regulates the expression of a specific subset of miRNAs, including miR-21, miR-221, miR-222, and miR-103, causally involved in breast cancer initiation, progression and drug-resistance. We also show that NCL is commonly overexpressed in human breast tumors, and its expression correlates with that of NCL-dependent miRNAs. Finally, this study indicates that NCL-binding guanosine-rich aptamers affect the levels of NCL-dependent miRNAs and their target genes, reducing breast cancer cell aggressiveness, both in vitro and in vivo. These findings illuminate a path to novel therapeutic approaches based on NCL-targeting aptamers for the modulation of miRNA expression in the treatment of breast cancer. HeLa cells were transfected with the control or anti-nucleolin siRNA. After 72hours total RNA was collected and analyzed by NanoString.