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MicroRNA profiling in TGFβ signaling and development of fibrosis.

ABSTRACT: End stage liver disease due to Hepatitis C Virus (HCV) infection is a major health concern worldwide. Liver fibrosis following chronic HCV infection plays a pivotal role in loss of liver function and end stage liver disease. However the dynamics and molecular events that lead to fibrosis in HCV infection are poorly defined. Therefore, we determined the influence of HCV infection in altering the miRNA expression levels which can modulate immune responses to HCV leading to fibrosis. Analysis of the miRNA expression profiles of HCV infected liver biopsies revealed that 45 miRNAs were differentially expressed in the HCV infected liver when compared to normal livers. In silico target prediction of these differentially expressed miRNAs indicated that their targets include chemokine/cytokine signaling, cell cycle genes and extracellular matrix protein gene expression. Gene expression profiling using whole genome microarray demonstrated that 1320 genes were differentially expressed in chronic HCV liver when compared to normal. These genes could be functionally grouped into those involved in cell cycle regulation, cytokines and chemokines expression, cell adhesion, intracellular signaling and enzymes. Further pathway analysis using GeneGo software identified cell adhesion, cytoskeleton remodeling, cytokine signaling and metabolic pathways as the major pathways activated in chronic HCV. Combinatorial target prediction analysis of miRNA expression along with gene expression analysis indicated that differentially expressed microRNAs in HCV significantly impact transforming growth factor beta (TGF-β) signaling pathway, cell adhesion (integrin expression), chemokine signaling, Notch signaling and cell-cycle( Cyclin D,K) regulation. Overall these results demonstrate that chronic HCV infection induces specific miRNA signatures that will modulate genes involved in the cytoskeletal remodeling and cytokine signaling that can promote the development of fibrosis following HCV infection.

ORGANISM(S): Homo sapiens

PROVIDER: GSE38226 | GEO | 2012/06/15

SECONDARY ACCESSION(S): PRJNA168569

REPOSITORIES: GEO

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Project description:End stage liver disease due to Hepatitis C Virus (HCV) infection is a major health concern worldwide. Liver fibrosis following chronic HCV infection plays a pivotal role in loss of liver function and end stage liver disease. However the dynamics and molecular events that lead to fibrosis in HCV infection are poorly defined. Therefore, we determined the influence of HCV infection in altering the miRNA expression levels which can modulate immune responses to HCV leading to fibrosis. Analysis of the miRNA expression profiles of HCV infected liver biopsies revealed that 45 miRNAs were differentially expressed in the HCV infected liver when compared to normal livers. In silico target prediction of these differentially expressed miRNAs indicated that their targets include chemokine/cytokine signaling, cell cycle genes and extracellular matrix protein gene expression. Gene expression profiling using whole genome microarray demonstrated that 1320 genes were differentially expressed in chronic HCV liver when compared to normal. These genes could be functionally grouped into those involved in cell cycle regulation, cytokines and chemokines expression, cell adhesion, intracellular signaling and enzymes. Further pathway analysis using GeneGo software identified cell adhesion, cytoskeleton remodeling, cytokine signaling and metabolic pathways as the major pathways activated in chronic HCV. Combinatorial target prediction analysis of miRNA expression along with gene expression analysis indicated that differentially expressed microRNAs in HCV significantly impact transforming growth factor beta (TGF-?) signaling pathway, cell adhesion (integrin expression), chemokine signaling, Notch signaling and cell-cycle( Cyclin D,K) regulation. Overall these results demonstrate that chronic HCV infection induces specific miRNA signatures that will modulate genes involved in the cytoskeletal remodeling and cytokine signaling that can promote the development of fibrosis following HCV infection. Liver biopsies from chronic HCV patients and control liver biopsies from normal subjects (donor liver prior to transplantation) were used to analyze the miRNA and gene expression profile. Patients with HBV and/or HIV were excluded from the study. This Series represents the mRNA gene expression profiling data only.

Project description:Background & Aims: Chronic hepatitis C virus (HCV) infection is complicated by hepatic fibrosis. Hypothesizing that fibrogenic signals may originate in cells susceptible to HCV infection, gene expression of hepatocytes was analyzed from persons with chronic HCV at different stages of liver fibrosis. Methods: HCV-infected subjects with significant liver fibrosis (Ishak fibrosis M-bM-^IM-%3) were matched for age, race, and gender to subjects with minimal fibrosis (Ishak fibrosis 0-1). RNA from portal tracts and hepatic parenchyma was isolated from biopsies by laser capture and transcriptome profiling was performed using hybridization arrays. Results: Portal tracts from both groups were enriched for immune related genes when compared to hepatocytes but high fibrosis subjects showed a loss of this enrichment. Hepatocytes from persons with high fibrosis were depleted for genes involved in small molecule and drug metabolism, especially butyrylcholinesterase (BCHE), a gene involved in the metabolism of drugs of abuse. Differential expression of BCHE was validated in the same tissues using qPCR. Cross-sectional and longitudinal testing in an expanded cohort of HCV-infected individuals showed that serum BCHE activity decreased in advance of progression to fibrosis. Conclusion: Chronic HCV infection is associated with a loss of hepatocyte metabolic function, decreased enrichment of immune-related genes in portal tracts and downregulation of BCHE in hepatocytes. Our results indicate that BCHE may be involved in the progression of fibrosis during HCV infection among injection drug users and may serve as a useful marker for fibrosis progression. Liver tissues were chosen from five subjects with chronic HCV infection and Ishak fibrosis stage 3-5 who had sufficient tissue stored in OCT and no HIV infection. Tissues that were stored in TrizolM-BM-. or other lysis buffers were excluded to avoid homogenization of transcriptomes between cellular constituents. Five control tissues were selected from persons of the same race and gender with chronic HCV infection estimated to be of similar duration (by age matching) but whose baseline Ishak fibrosis score was 0-1. One high fibrosis case was later excluded because the subject was found to be HBsAg positive, leaving a total of nine subjects.

Project description:The impact of chronic hepatic infection on antigen non-specific immune cells, especially in association with liver fibrosis severity, remains poorly described. We previously reported lasting hyperfunction of bulk peripheral blood CD8 T cells in HCV-infected individuals with cirrhosis. RNA sequencing of blood CD8 T cells from HCV-infected individuals with minimal (Metavir F0-1, Fibroscan liver stiffness ≤ 7.0 kPa) or advanced fibrosis or cirrhosis (F4 ≥ 12.5 kPa), before and after direct-acting antiviral therapy, was performed. Principal component analyses determined robust differences in 362 genes expressed by CD8 T cells from HCV-infected individuals with minimal or advanced fibrosis and data suggests this remains relatively stable after viral clearance. Gene ontology analyses identified differential gene expression related to cellular metabolism, including upregulated phospholipase, phosphatidyl-choline/inositol activity and second-messenger-mediated signaling, while genes in pathways associated with nuclear processes, RNA transport and cytoskeletal dynamics were reduced. Gene Set Enrichment Analysis identified decreased expression of genes regulated by the cMyc and E2F transcription factors in cirrhotics, compared to the minimal fibrosis group, as well as reduced expression of genes linked to oxidative phosphorylation, mTOR signaling, and more. Upregulated gene sets in cirrhotics included IFN-α, -γ, TGF-β response genes, apoptosis, and apical surface pathways, among others. The Hedgehog (Hh) signaling pathway was the top featured gene set upregulated in cirrhotics, wherein hallmark genes GLI1 and PTCH1 ranked highly. This analysis of bulk CD8 T cell gene expression in chronic HCV infection suggests considerable reprogramming of the CD8 T cell pool in liver cirrhosis. Increased Hh signaling in cirrhosis may contribute to generalized CD8 T cell hyperfunction observed in chronic HCV infection. Understanding the lasting nature of immune cell dysfunction may help mitigate remaining clinical challenges after HCV clearance and more generally, improve long term outcomes for individuals with severe liver disease.

Project description:Hepatitis C virus (HCV)-induced chronic liver disease is one of the leading causes of hepatocellular carcinoma (HCC). However, the molecular mechanisms underlying HCC development following chronic HCV infection remain poorly understood. MicroRNAs (miRNAs) play an important role in cellular homeostasis within the liver and deregulation of the miRNome has been associated with liver disease including HCC. While host miRNAs are essential for HCV replication, viral infection in turn appears to induce alterations of intrahepatic miRNA networks. Although the cross-talk between HCV and liver cell miRNAs most likely contributes to liver disease pathogenesis, the functional involvement of miRNAs in HCV-driven hepatocyte injury and HCC remains elusive. Here, we combined a hepatocyte-like based model system, high-throughput small RNA-sequencing, computational analysis and functional studies to investigate HCV-miRNA interactions that may contribute to liver disease and HCC. Profiling analyses indicated that HCV infection differentially regulated the expression of 72 miRNAs by at least two-fold including miRNAs that were previously described to target genes associated with inflammation, fibrosis and cancer development. Further investigation demonstrated that miR-146a-5p was consistently increased in HCV-infected hepatocyte-like cells and primary human hepatocytes as well as in liver tissues from HCV-infected patients. Genome-wide microarray and computational analyses indicated that miR-146a-5p over-expression is related to liver disease and HCC development. Furthermore, we showed that miR-146a-5p positively impacts on late steps of the viral replication cycle thereby increasing HCV infection. Collectively, our data indicate that the HCV-induced increase in miR-146a-5p expression both promotes viral infection and is relevant for pathogenesis of liver disease. To explore the functional relevance of miR-146a-5p up-regulation, we performed a genome-wide transcriptomic analysis of hepatocyte-like cells upon ectopic miR-146a-5p expression.

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MicroRNA profiling in TGFβ signaling and development of fibrosis.

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