Project description:Interferons (IFNs) are key players in the antiviral response. IFN sensing by the cell activates transcription of IFN-stimulated genes (ISGs) able to induce an antiviral state by affecting viral replication and release. IFN also induces the expression of ISGs that function as negative regulators to limit the strength and duration of IFN response. The ISGs identified so far belong to coding genes. However, only a small proportion of the transcriptome corresponds to coding transcripts and it has been estimated that there could be as many coding as long non-coding RNAs (lncRNAs). To address whether IFN can also regulate the expression of lncRNAs, we analyzed the transcriptome of HuH7 cells treated or not with IFNα2 by expression arrays. Analysis of the arrays showed increased levels of several well-characterized coding genes that respond to IFN both at early or late times. Furthermore, we identified several IFN-stimulated or -downregulated lncRNAs (ISRs and IDRs). Further validation showed that ISR2, 8, and 12 expression mimics that of their neighboring genes GBP1, IRF1, and IL6, respectively, all related to the IFN response. These genes are induced in response to different doses of IFNα2 in different cell lines at early (ISR2 or 8) or later (ISR12) time points. IFNβ also induced the expression of these lncRNAs. ISR2 and 8 were also induced by an influenza virus unable to block the IFN response but not by other wild-type lytic viruses tested. Surprisingly, both ISR2 and 8 were significantly upregulated in cultured cells and livers from patients infected with HCV. Increased levels of ISR2 were also detected in patients chronically infected with HIV. This is relevant as genome-wide guilt-by-association studies predict that ISR2, 8, and 12 may function in viral processes, in the IFN pathway and the antiviral response. Therefore, we propose that these lncRNAs could be induced by IFN to function as positive or negative regulators of the antiviral response. HuH7 cells were treated with 10000 units/ml of IFN a2 and RNA was isolated 3 days post-treatment
Project description:It has been recently shown that the transcription factor p53 induces the expression of multiple lincRNAs. However, relatively little is known about the role that lincRNAs play in this pathway. Here we characterize a lincRNA named PINT (p53 Induced Noncoding Transcript). We show that PINT is a ubiquitously expressed lincRNA that is finely regulated by p53. In mouse cells, PINT promotes cell proliferation and survival by regulating the expression of genes of TGF-beta, MAPK and p53 pathways. PINT is a nuclear lincRNA that directly interacts with Polycomb Repressive Complex 2 (PRC2), being required for PRC2 targeting of specific genes for repression. Furthermore, PINT functional activity is dependent on PRC2 expression, representing a connection between the p53 pathway and epigenetic regulation by PRC2. We have also identified PINT human ortholog (hPINT), which presents suggestive analogies with the mouse lincRNA. hPINT is similarly regulated by p53, and its expression correlates significantly with the same cellular pathways as the mouse ortholog, including the p53 pathway. Interestingly, hPINT is significantly downregulated in colon cancer, representing a novel tumor suppressor candidate. Our results not only help our understanding of the role of p53 and lincRNAs in cancer, but also contribute to the open debate regarding the utility of mouse models for the study of lincRNAs. Normal tissue located at least 20 cm away from a tumor was obtained through surgical resection from patients of colorectal cancer. Total RNA was extracted and subjected to microarray analysis.
Project description:Proteomic analysis of differentially expressed proteins in MDA-MB-231 and MCF-10A cell lines when miR-200c and miR-203 were transiently expressed or inhibited, respectively.
Project description:Postmenopausal hormone therapy (HT) is associated with many diseases and conditions, but the underlying molecular mechanisms involved are incompletely understood. The aim of the current study was to investigate the effect of 4 types of HT on gene transcription. 24 women (6 women in 4 treatment groups) received 2 mg 17M-NM-2-estradiol combined with 1 mg noresthisterone acetate (NETA), 1 mg 17M-NM-2-estradiol combined with 0.5 mg NETA, tibolone, or raloxifene hydrochloride. RNA was isolated from whole blood before treatment (baseline) and after 6 weeks on treatment. The changes in mRNA from baseline to 6 weeks were assessed with a microarray chip. 4 treatment groups with 6 women in each group were blood sampled before treatment (baseline) and after 6 weeks on treatment, that is a total of 48 samples. The gene expression data at 6 weeks were compared to the expression data at baseline for each treatment.
Project description:Dermal fibroblasts represent a heterogeneous population of cells with diverse features that remain largely undefined due to a lack of functional subclasses. Here we reveal the presence of multiple lineages of dermal fibroblasts within the dorsal back. Genetic lineage tracing and transplantation assays demonstrate that the bulk of connective tissue deposition during embryonic development, cutaneous wound healing, radiation fibrosis, and cancer stroma formation is carried out by a single, somitic-derived fibroblast lineage. Reciprocal transplantation of distinct fibroblast lineages between the dorsal back and oral cavity induced ectopic dermal architectures that mimic their place–of-origin. These studies demonstrate that intra and inter-site diversity of dermal architectures are set embryonically and maintained postnatally by distinct lineages of fibroblasts. Lineage-specific cell ablation using transgenic-mediated expression of the simian diphtheria toxin receptor in conjunction with localized administration of diphtheria toxin led to diminished connective tissue deposition in wounds and significantly reduced melanoma growth in the dorsal skin of mice. Using flow cytometry and in silico approaches, we identify CD26/DPP4 as a surface marker that allows for the isolation of this fibrogenic, scar-forming lineage. Small molecule-based inhibition of CD26/DPP4 enzymatic activity during wound healing results in diminished cutaneous scarring. The identification and prospective isolation of these lineages holds promise for translational medicine aimed at in vivo modulation of their fibrogenic behavior. Dermal fibroblasts were harvested for FACS from the oral dermis and cranial dermis of Wnt1Cre; R26mTmG mice, and from ventral and dorsal dermis of En1Cre; R26mTmG mice as previously described. For all microarray analysis, EPFs and ENFs where harvested from backskin of mice at 30 days of age. Positivity for GFP or RFP allowed for the separation of EPFs from ENFs and WPFs from WNFs. RNA was precipitated via chloroform-phenol extraction. Samples were processed for cleanup and concentration using Rneasy MinElute cleanup kit (cat. 74204, QIAGEN). RNA yield was typically 0.5-1 µg RNA/sorted subpopulations. RNA samples from all sorted populations were converted to cDNA using SuperScript III first strand synthesis system for RT-PCR (cat. 18080-051, Invitrogen), and hybridized to Affymetrix Mouse Genome 430 2.0 arrays. Microarrays were normalized by robust multichip average (RMA) and quantile normalization in R. Cluster analysis was performed with AutoSOME (33), using the following settings for gene expression clustering: p-value threshold of 0.05, 100 ensemble iterations, unit variance normalization of arrays, median centering of genes, and sum of squares=1 normalization for both genes and arrays.
Project description:Low phosphate concentrations are frequently a constraint for maize growth and development, and therefore, enormous quantities of phosphate fertilizer are expended in maize cultivation, which increases the cost of planting. Low phosphate stress not only increases root biomass but can also cause significant changes in root morphology. Low phosphate availability has been found to favor lateral root growth over primary root growth by dramatically reducing primary root length and increasing lateral root elongation and lateral root density in Arabdopsis. While in our assay when inbred line Q319 subjected to phosphate starvation, The numbers of lateral roots and lateral root primordia were decreased after 6 days of culture in a low phosphate solution (LP) compared to plants grown under normal conditions (sufficient phosphate, SP), and these differences were increased associated with the stress caused by phosphate starvation. However, the growth of primary roots appeared not to be sensitive to low phosphate levels. This is very different to Arabidopsis. To elucidate how low phosphate levels regulate root modifications, especially lateral root development, a transcriptomic analysis of the 1.0-1.5 cm lateral root primordium zone (LRZ) of maize Q319 treated after 2 and 8 days by low phosphate was completed respectively. The present work utilized an Arizona Maize Oligonucleotide array 46K version slides, which contained 46,000 maize 70-mer oligonucleotides designated by TIGR ID, and the sequence information is available at the website of the Maize Oligonucleotide Array Project as the search item representing the >30,000 identifiable unique maize genes (details at http://www.maizearray.org). Keywords: low phosphate, Lateral Root Primordium Zone, maize Two-condition experiment, low phosphate treated lateral root primordium zone of maize root vs. normal cultrued lateral root primordium zone. Biological replicates: 9 control, 9 treated, independently grown and harvested. One replicate per array.
Project description:Maize plants were subjected to foliar herbivory and both whorl and root tissue were collected at 0 h and 24 h. microarray was performed to compare transcriptional profiles and interpret the signaling molecules mediating systemic responses
Project description:Viral hepatitis, obesity, and alcoholism all represent major risk factors for hepatocellular carcinoma (HCC). Although these conditions also lead to integrated stress response (ISR) or unfolded protein response (UPR) activation, the extent to which these stress pathways influence the pathogenesis of HCC has not been tested. Here we provide multiple lines of evidence demonstrating that the ISR-regulated transcription factor CHOP promotes liver cancer. We show that CHOP expression is upregulated in liver tumors in human HCC and two mouse models thereof. Chop-null mice are resistant to chemical hepatocarcinogenesis, and these mice exhibit attenuation of both apoptosis and cellular proliferation. Chop-null mice are also resistant to fibrosis, which is a key risk factor for HCC. Global gene expression profiling suggests that deletion of CHOP reduces the levels of basal inflammatory signaling in the liver. Our results are consistent with a model whereby CHOP contributes to hepatic carcinogenesis by promoting inflammation, fibrosis, cell death, and compensatory proliferation. They implicate CHOP as a common contributing factor in the development of HCC in a variety of chronic liver diseases. 24 samples were analyzed from 9 month old male C57BL/6J or backcrossed Chop-/- male mice, injected with either PBS or a single dose of 25 mg/kg diethylnitrosamine (DEN) at 15 d.o. Samples are either from whole liver or liver tumors.
Project description:Neurotrophic factors (NTFs) are a relevant group of secreted proteins that modulate growth, differentiation, repair, and survival of neurons; playing a role in the maintenance of the synaptic unions, dendrites and axons; also, being crucial for peripheral nervous system development and regulating plasticity in the adult central nervous system. On the other hand, insulin-like growth factor 1 (IGF-1) has been ascertained multiple beneficial actions in the brain: neuro-development, -protection, -genesis and plasticity. To further investigate the possible mechanisms underlying IGF-1 deficiency in the establishment of neurological disease, microarray and RT-qPCR gene expression analyses coupled with in silico processing were performed in an experimental model of partial IGF-1 deficiency. Results show that the mere IGF-1 deficiency seems to be responsible for an altered expression of genes coding for neurotrophic factors (particularly ciliary neurotrophic factor and mesencephalic astrocyte-derived neurotrophic factor), their receptors and signaling pathways (specially RET). The presented findings support that IGF-1 deficiency might be involved in the establishment and progression of neurodegenerative disorders.