Microarray analysis identifies a common set of cellular genes modulated by different HCV replicon clones

<p>Abstract</p> <p>Background</p> <p>Hepatitis C virus (HCV) RNA synthesis and protein expression affect cell homeostasis by modulation of gene expression. The impact of HCV replication on global cell transcription has not been fully evaluated. Thus, we analysed the expression profiles of different clones of human hepatoma-derived Huh-7 cells carrying a self-replicating HCV RNA which express all viral proteins (HCV replicon system).</p> <p>Results</p> <p>First, we compared the expression profile of HCV replicon clone 21-5 with both the Huh-7 parental cells and the 21-5 cured (21-5c) cells. In these latter, the HCV RNA has been eliminated by IFN-α treatment. To confirm data, we also analyzed microarray results from both the 21-5 and two other HCV replicon clones, 22-6 and 21-7, compared to the Huh-7 cells. The study was carried out by using the Applied Biosystems (AB) Human Genome Survey Microarray v1.0 which provides 31,700 probes that correspond to 27,868 human genes. Microarray analysis revealed a specific transcriptional program induced by HCV in replicon cells respect to both IFN-α-cured and Huh-7 cells. From the original datasets of differentially expressed genes, we selected by Venn diagrams a final list of 38 genes modulated by HCV in all clones. Most of the 38 genes have never been described before and showed high fold-change associated with significant p-value, strongly supporting data reliability. Classification of the 38 genes by Panther System identified functional categories that were significantly enriched in this gene set, such as histones and ribosomal proteins as well as extracellular matrix and intracellular protein traffic. The dataset also included new genes involved in lipid metabolism, extracellular matrix and cytoskeletal network, which may be critical for HCV replication and pathogenesis.</p> <p>Conclusion</p> <p>Our data provide a comprehensive analysis of alterations in gene expression induced by HCV replication and reveal modulation of new genes potentially useful for selection of antiviral targets.</p

IFN-α Regulates Blimp-1 Expression via miR-23a and miR-125b in Both Monocytes-Derived DC and pDC

Type I interferon (IFN-I) have emerged as crucial mediators of cellular signals controlling DC differentiation and function. Human DC differentiated from monocytes in the presence of IFN-alpha (IFN-alpha DC) show a partially mature phenotype and a special capability of stimulating CD4+ T cell and cross-priming CD8+ T cells. Likewise, plasmacytoid DC (pDC) are blood DC highly specialized in the production of IFN-alpha in response to viruses and other danger signals, whose functional features may be shaped by IFN-I. Here, we investigated the molecular mechanisms stimulated by IFN-alpha in driving human monocyte-derived DC differentiation and performed parallel studies on peripheral unstimulated and IFN-alpha-treated pDC. A specific miRNA signature was induced in IFN-alpha DC and selected miRNAs, among which miR-23a and miR-125b, proved to be negatively associated with up-modulation of Blimp-1 occurring during IFN-alpha-driven DC differentiation. Of note, monocyte-derived IFN-alpha DC and in vitro IFN-alpha-treated pDC shared a restricted pattern of miRNAs regulating Blimp-1 expression as well as some similar phenotypic, molecular and functional hallmarks, supporting the existence of a potential relationship between these DC populations. On the whole, these data uncover a new role of Blimp-1 in human DC differentiation driven by IFN-alpha and identify Blimp-1 as an IFN-alpha-mediated key regulator potentially accounting for shared functional features between IFN-alpha DC and pDC

An integrated approach identifies IFN-regulated microRNAs and targeted mRNAs modulated by different HCV replicon clones

Abstract Background Infections with hepatitis C virus (HCV) progress to chronic phase in 80% of patients. To date, the effect produced by HCV on the expression of microRNAs (miRs) involved in the interferon-β (IFN-β) antiviral pathway has not been explored in details. Thus, we compared the expression profile of 24 selected miRs in IFN-β-treated Huh-7 cells and in three different clones of Huh-7 cells carrying a self-replicating HCV RNA which express all viral proteins (HCV replicon system). Methods The expression profile of 24 selected miRs in IFN-β-treated Huh-7 cells and in HCV replicon 21-5 clone with respect to Huh-7 parental cells was analysed by real-time PCR. To exclude clone specific variations, the level of 16 out of 24 miRs, found to be modulated in 21-5 clone, was evaluated in two other HCV replicon clones, 22-6 and 21-7. Prediction of target genes of 3 miRs, confirmed in all HCV clones, was performed by means of miRGator program. The gene dataset obtained from microarray analysis of HCV clones was farther used to validate target prediction. Results The expression profile revealed that 16 out of 24 miRs were modulated in HCV replicon clone 21-5. Analysis in HCV replicon clones 22-6 and 21-7 indicated that 3 out of 16 miRs, (miR-128a, miR-196a and miR-142-3p) were modulated in a concerted fashion in all three HCV clones. Microarray analysis revealed that 37 out of 1981 genes, predicted targets of the 3 miRs, showed an inverse expression relationship with the corresponding miR in HCV clones, as expected for true targets. Classification of the 37 genes by Panther System indicated that the dataset contains genes involved in biological processes that sustain HCV replication and/or in pathways potentially implicated in the control of antiviral response by HCV infection. Conclusions The present findings reveal that 3 IFN-β-regulated miRs and 37 genes, which are likely their functional targets, were commonly modulated by HCV in three replicon clones. The future use of miR inhibitors or mimics and/or siRNAs might be useful for the development of diagnostic and therapeutic strategies aimed at the recovering of protective innate responses in HCV infections.</p

IFN-α Regulates Blimp-1 Expression via miR-23a and miR-125b in Both Monocytes-Derived DC and pDC

<div><p>Type I interferon (IFN-I) have emerged as crucial mediators of cellular signals controlling DC differentiation and function. Human DC differentiated from monocytes in the presence of IFN-α (IFN-α DC) show a partially mature phenotype and a special capability of stimulating CD4+ T cell and cross-priming CD8+ T cells. Likewise, plasmacytoid DC (pDC) are blood DC highly specialized in the production of IFN-α in response to viruses and other danger signals, whose functional features may be shaped by IFN-I. Here, we investigated the molecular mechanisms stimulated by IFN-α in driving human monocyte-derived DC differentiation and performed parallel studies on peripheral unstimulated and IFN-α-treated pDC. A specific miRNA signature was induced in IFN-α DC and selected miRNAs, among which miR-23a and miR-125b, proved to be negatively associated with up-modulation of Blimp-1 occurring during IFN-α-driven DC differentiation. Of note, monocyte-derived IFN-α DC and <i>in vitro</i> IFN-α-treated pDC shared a restricted pattern of miRNAs regulating Blimp-1 expression as well as some similar phenotypic, molecular and functional hallmarks, supporting the existence of a potential relationship between these DC populations. On the whole, these data uncover a new role of Blimp-1 in human DC differentiation driven by IFN-α and identify Blimp-1 as an IFN-α-mediated key regulator potentially accounting for shared functional features between IFN-α DC and pDC.</p> </div

pDC and IFN-α DC share miRNA expression signatures: down-regulation of miR-125b by IFN-α.

<p><b>A</b>. Box plot representing expression of 10 miRNAs in peripheral pDC and IFN-α DC analyzed by qRT-PCR. Data represent fold change values of miRNA modulation in pDC and IFN-α DC, with respect to monocytes treated with GM-CSF alone, obtained from 5 and 10 different healthy donors, respectively. <b>B</b>. Expression levels of miRNAs targeting Blimp-1 in untreated and IFN-α-treated pDC with respect to IFN-α DC. Median fold-changes are indicated.</p

IFN-α DC and peripheral pDC share phenotypic, molecular and functional features.

<p><b>A</b>. Flow cytometry analysis of the expression of plasmacytoid or myeloid DC markers in IFN-α DC, untreated or IFN-α-treated pDC and GM-CSF-treated monocytes. Broken line histograms represent isotype controls. Representative data of 1 experiment out of 4 are shown. <b>B</b>. Expression of specific pDC molecular markers evaluated by qRT-PCR in the same DC populations indicated in panel <b>A</b>. The data are presented as the means ± SD of 3 independent experiments. <b>C</b>. Production of IFN-I in IFN-α DC and pDC after in vitro infection with NDV. DC populations were infected with NDV for 1 h. Virus was then washed out and the supernatant was harvested after 18 h incubation and assayed for IFN-I bioactivity, as described in Materials and Methods. Data are representative of 2 independent experiments. Statistical analyses were performed by using Mann-Whitney test, except for IFN-α DC and GM-CSF-treated monocytes comparison for which Wilcoxon test was performed (*<i>p</i>≤0.006, **<i>p</i>≤0.0005, ns = not significant).</p

IFN-α-driven Blimp-1 expression is under the control of miR-23a and miR-125b.

<p><b>A</b>. Blimp-1α protein reduction upon transfection of miR-23a and miR-125b in HeLa cells, measured by western blot. As a control, HeLa cells transfected with empty plasmid or unrelated miRNAs (unrelated1: miR-1, unrelated2: miR-159sense) were used. Intensities of bands were measured and numbers indicate the values expressed as fold induction with respect to Gapdh. 1 representative experiment out of 3 is shown. <b>B</b>. Blimp-1 expression in HeLa cells transfected or not with single or combined miR-23a and miR-125b and treated for 24 or 48 hours with the indicated doses of IFN-α. HeLa cells transfected with empty plasmid were used as a control. Numbers indicate Blimp-1α isoform quantification expressed as fold induction with respect to Gapdh. 1 representative experiment out of 3 is shown.</p