Annual report 2013
Division of Advanced Genome Medicine
Advanced Clinical Research Center
Institute of Medical Science
The University of Tokyo
Associate Professor: Naoya Kato, M.D., Ph.D.
Lecturer/Assistant Professor: Yasuo Matsubara, M.D., Ph.D.
Project Assistant Professor: Ryosuke Muroyama, M.D., Ph.D.
Research Scientist: Kaku Goto, Ph.D.
Our major goal is to establish personalized medicine for patients with infectious diseases or cancers, especially those of gastrointestinal and hepatic fields, based on human or pathogenic microbe genome information.
1. Identification of functional SNPs in the promoter region of MICA which alter transcriptional activity
Ryosuke Muroyama, Kaku Goto, Wenwen Li, Ryo Nakagawa, Norie Kowatari, Chisiro Wakabayashi, Yasuo Matsubara, Naoya Kato
Hepatocellular carcinoma (HCC) reveals a very high mortality rate in the world, and hepatitis B virus (HBV) or hepatitis C virus (HCV) is one of the major etiological factors for developing HCC. Previously, we reported SNP (rs2596542) located in the promoter region of MHC class I polypeptide-related chain A (MICA) was significantly associated with the risk of HBV/HCV-induced HCC and also with serum levels of soluble MICA. However, functional SNPs were not fully elucidated so far because SNPs in the MICA locus show strong linkage disequilibrium. In this study, we tried to identify functional SNPs which alter transcriptional activity of MICA. We constructed MICA promoter-reporter plasmids using the sequence from HLE cells (G allele of rs2596542) or Huh7 cells (A allele of rs2596542), and compared the transcriptional activity between them by luciferase assay. The result showed that the promoter sequence which had G allele of rs2596542 exhibited 3-4 folds higher transcriptional activity than that which had A allele of rs2596542. We further analyzed the difference of transcriptional activity between them by reporter plasmids which containing serial truncated promoter region of MICA, and found that two functional SNPs (rs6906175 and 2301750) in the promoter region of MICA altered transcriptional activity. Therefore, these functional SNPs might be associated with the expression of MICA and the risk of HBV/HCV-induced HCC.
2. Small molecules for MICA expression regulation
Kaku Goto1, Masahisa Jinushi2, Ryosuke Muroyama1, Wenwen Li1, Ryo Nakagawa1, Norie Kowatari1, Chisiro Wakabayashi1, Yasuo Matsubara1, Naoya Kato1: 1Division of Advanced Genome Medicine, IMSUT; 2Research Center for Infection-Associated Cancer, Institute for Genetic Medicine, Hokkaido University
A genome-wide association study (GWAS) identified an anti-tumor ligand MHC class I polypeptide-related sequence A (MICA) to be a genetic susceptibility factor for HCV-induced hepatocellular carcinoma (HCC). Lower levels of MICA expression were associated with the elevated risk of HCC development in patients, implying preventive effects of MICA expression induction on hepatocarcinogenesis. Hence we sought to find small molecules for regulation of MICA expression. Sodium butyrate (NaB), a well-known HDAC inhibitor and a reported MICA inducer in multiple cancer cell lines, significantly upregualted the MICA mRNA levels in hepatoma cells. After the construction of active luciferase reporters encoding MICA promoter sequences, stable hepatoma cell transformants harboring the reporters were established via antibiotics selection, which responded to the NaB treatment in a dose-dependent fashion likewise. Using the luciferase reporter cell system, a primary screen for a FDA-approved drug library discovered that multiple drugs including an anti-cancer agent induced the MICA expression significantly at mRNA and protein levels, and the mode and impact of the ligand modulation are currently investigated. Findings in this study would expectedly serve to develop anti-tumor immunotherapies in virus-induced HCC.
3. Identification of a Functional Variant in the MICA Promoter Which Regulates MICA Expression and Increases HCV-Related Hepatocellular Carcinoma Risk
Paulisally Hau Yi Lo1, Yuji Urabe1, 2, Vinod Kumar1, Chizu Tanikawa1, Kazuhiko Koike4, Naoya Kato3, Daiki Miki2, Kazuaki Chayama2, Michiaki Kubo5, Yusuke Nakamura1, Koichi Matsuda1: 1Laboratory of Molecular Medicine, Human Genome Center, IMSUT; 2Departments of Medical and Molecular Science, Division of Frontier Medical Science, Programs for Biomedical Research, Graduate School of Biomedical Sciences, Hiroshima University; 3Division of Advanced Genome Medicine, IMSUT; 4Department of Gastroenterology, Graduate School of Medicine, The University of Tokyo; 5Center for Genomic Medicine, RIKEN
Hepatitis C virus (HCV) infection is the major cause of hepatocellular carcinoma (HCC) in Japan. We previously identified the association of SNP rs2596542 in the 5' flanking region of the MHC class I polypeptide-related sequence A (MICA) gene with the risk of HCV-induced HCC. In the current study, we performed detailed functional analysis of 12 candidate SNPs in the promoter region and found that a SNP rs2596538 located at 2.8 kb upstream of the MICA gene affected the binding of a nuclear protein(s) to the genomic segment including this SNP. By electrophoretic mobility shift assay (EMSA) and chromatin immunoprecipitation (ChIP) assay, we identified that transcription factor Specificity Protein 1 (SP1) can bind to the protective G allele, but not to the risk A allele. In addition, reporter construct containing the G allele was found to exhibit higher transcriptional activity than that containing the A allele. Moreover, SNP rs2596538 showed stronger association with HCV-induced HCC (P = 1.82×10−5 and OR = 1.34) than the previously identified SNP rs2596542. We also found significantly higher serum level of soluble MICA (sMICA) in HCV-induced HCC patients carrying the G allele than those carrying the A allele (P = 0.00616). In summary, we have identified a functional SNP that is associated with the expression of MICA and the risk for HCV-induced HCC.
4. A genome-wide association study of HCV induced liver cirrhosis in the Japanese population identifies novel susceptibility loci at MHC region
Yuji Urabe1, 2, Naoya Kato3, Vinod Kumar1, Ryosuke Muroyama3, Motoyuki Otsuka4, Ryosuke Tateishi4, Paulisally Hau Yi Lo1, Chizu Tanikawa1, Masao Omata4, Kazuhiko Koike4, Michiaki Kubo5, Kazuaki Chayama2, Yusuke Nakamura1, Koichi Matsuda1: 1Laboratory of Molecular Medicine, Human Genome Center, IMSUT; 2Departments of Medical and Molecular Science, Division of Frontier Medical Science, Programs for Biomedical Research, Graduate School of Biomedical Sciences, Hiroshima University; 3Unit of Disease Control Genome Medicine, IMSUT; 4Department of Gastroenterology, Graduate School of Medicine, The University of Tokyo; 5Center for Genomic Medicine, RIKEN
We performed a genome-wide association study (GWAS) of hepatitis C virus (HCV)-induced liver cirrhosis (LC) to identify predictive biomarkers for the risk of LC in patients with chronic hepatitis C (CHC). A total of 682 HCV-induced LC cases and 1,045 CHC patients of Japanese origin were genotyped by Illumina Human Hap 610-Quad bead Chip. Eight SNPs which showed possible associations (P < 1.0 x 10(-5)) in the GWAS stage were further genotyped using 936 LC cases and 3,809 CHC patients. We found that two SNPs within the major histocompatibility complex (MHC) region on chromosome 6p21, rs910049 and rs3135363, were significantly associated with the progression from CHC to LC (P(combined) = 9.15 x 10(-11) and 1.45 x 10(-10), odds ratio (OR) = 1.46 and 1.37, respectively). We also found that HLA-DQA1∗0601 and HLA-DRB1∗0405 were associated with progression from CHC to LC (P = 4.53 x 10-4 and 1.54 x 10-4 with OR = 2.80 and 1.45, respectively). Multiple logistic regression analysis revealed that rs3135363, rs910049, and HLA-DQA1∗0601 were independently associated with the risk of HCV-induced LC. In addition, individuals with four or more risk alleles for these three loci have a 2.83-fold higher risk for LC than those with no risk allele, indicating the cumulative effects of these variations. Our findings elucidated the crucial roles of multiple genetic variations within the MHC region as prognostic/predictive biomarkers for CHC patients.
5. AMPK-related kinase SNARK in chronic HCV infection
Kaku Goto1,2, Raymond T. Chung2, Naoya Kato1: 1Division of Advanced Genome Medicine, IMSUT; 2GI Unit, Massachusetts General Hospital, Harvard Medical School
Host cellular cofactors for hepatitis C virus (HCV) infection are recognized as attractive antiviral targets due to their independence from viral sequence. Our genome-wide RNAi screen for host cellular cofactors (Tai AW et al., Cell Host Microbe 2009) identified that sucrose, non-fermenting 1/AMP-activated protein kinase-related kinase (SNARK) positively regulated HCV replication. We therefore sought to clarify the mechanisms of reciprocal regulation between SNARK and HCV. Knockdown of SNARK decreased levels of HCV replication in both OR6 replicon and JFH1 infection systems. Overexpressed siRNA-resistant wild type SNARK rescued the suppressed viral replication, which was abrogated by either a kinase deficiency or phospho-deficient mutation. Conversely, SNARK mRNA level was upregulated by HCV infection in patients and cell culture, deranging cellular signalings. These SNARK-mediated effects on both virus and host were cancelled by a SNARK kinase inhibitor. Hence viral induction of the proviral kinase was speculated to promote HCV pathogenesis. We are presently investigating substrates and signalings targeted by the kinase and their pharmacological regulation.
6. Mutations in hepatitis B Virus (HBV) X region are hepatocellular carcinoma risk factors for HBV genotype C infected patients
Wenwen Li1, Ryosuke Muroyama1, Kaku Goto1, Ryo Nakagawa1, Norie Kowatari1, Chishiro Wakabayashi1, Yasuo Matsubara1, Qiang Li2, Naoya Kato1: 1Division of Advanced Genome Medicine, IMSUT; 2Jinan Infectious Disease Hospital, Shandong University, Jinan, China
Chronic hepatitis B virus (HBV) infection accounts for approximately 50% of the hepatocellular carcinoma (HCC) cases worldwide and even 80-90% in the areas where HBV is highly prevalent. The role of virus mutation in the interaction between virus and host during carcinogenesis is still far from clear, especially in patients infected by special genotypes which were considered to be more oncogenic than other genotypes.
Aim: We sought to clarify potential HCC characteristic mutations in HBV genotype C infected patients. HBV genotype C sequences were downloaded from online global databases. Sequences were then screened according to below criteria: 1. Full length HBV X; 2. genotype C; 3. human sera origin; 4. with diagnosis information and thus could be attributed to Non-HCC or HCC group. Continuous data were expressed as Mean±SD and were compared by t-test. Categorical data were analyzed by Fisher’s exact test (SPSS 16.0). P < 0.05 was considered to have significant difference. Logistic regression was performed to evaluate the effects of mutations on HCC risk. 1) Four hundred and ninety-five HBV genotype C sequences (HCC: 153; Non-HCC: 342) were finally extracted out of the downloaded 5380 HBV X sequences. 2) Twenty nucleotide positions showed significant different distribution between HCC and Non-HCC groups. Six of them were also located in overlapped Enhancer 2 (Enh2) region and 14 in overlapped core promoter (CP) region. 3) Logistic regression showed that mutations A1383C (OR: 2.00, 95% CI: 1.08-3.71), A1479C/G/T (OR: 2.93, 95% CI: 1.49-5.79; OR: 2.79, 95% CI: 1.32-5.90; OR: 6.70, 95% CI: 2.81-15.99), C1485T (OR: 2.63, 95% CI: 1.50-4.60), C1653T (OR: 1.95, 95% CI: 1.15-3.31), and A1762T (OR: 1.85, 95% CI: 1.01-3.40) were independent risk factors for genotype C HBV-related HCC. Five point mutations in genotype C HBV X region were risk factors of HCC though these mutations seemed to be more associated with the disease progression than HCC specific. Further longitudinal studies are needed to verify the roles of these mutations in earlier disease stages and the procession of oncogenesis.
7. Novel drug screening system based on the essential role of HBx trans-activation activity in HBV replication
Wenwen Li1, Kaku Goto1, Ryosuke Muroyama1, Ryo Nakagawa1, Chishiro Wakabayashi1, Norie Kowatari1, Yasuo Matsubara1, Qiang Li2, Naoya Kato1: 1Division of Advanced Genome Medicine, IMSUT; 2Jinan Infectious Disease Hospital, Shandong University, Jinan, China
Current efficacy of anti-hepatitis B virus (HBV) drugs is limited in eradicating nuclear covalently closed circular DNA (cccDNA), which is a vital factor helping the establishment of persistent HBV infection, which necessitates the development of novel anti-HBV drugs for erasing cccDNA. One of the HBV proteins, HBx, has been proved to be essential for HBV replication probably through the interaction with cccDNA and the transactivation function. Therefore, we hypothesized that suppressing HBV cccDNA via inhibiting HBx transcriptional transactivity should be a promising therapeutic strategy against HBV. Hence, we constructed genotype C HBx encoding plasmids and then examined the effects of HBx on major signaling pathways reported ever (i.e., NF-kappaB, AP-1, SRE) via luciferase assay, out of which two most stimulated signalings were selected. Subsequently, those luciferase reporters were stably transfected in a hepatoma cell line, and clones with the lowest background and high sensitivity to stimuli were isolated. Meanwhile a Tet-on system controlled HBx-encoding plasmid is also under construction. We will establish a dual stable transfectant hepatoma cell line with two types of plasmid, a signaling pathway luciferase reporter and the HBx Tet-on system vector, using multiple antibiotics selection. Finally, drug libraries, an FDA-approved drug library and the one from the open innovation center for drug discovery in the University of Tokyo, will be used for new drug screenings. Potential molecular mechanism of the interaction between viral onco-protein and host signaling pathways will be further analyzed.
8. Specifically expressed miRNA in CD4+ T cells participates in the pathogenesis of primary biliary cirrhosis
Ryo Nakagawa1, 2, Ryosuke Muroyama1, Wenwen Li1, Kaku Goto1, Norie Kowatari1, Chisiro Wakabayashi1, Hiroki Takahashi2, Mikio Zeniya2, Naoya Kato1: 1Division of Advanced Genome Medicine, IMSUT; 2Department of Gastroenterology and Liver Diseases, The Jikei University School of Medicine
Primary biliary cirrhosis (PBC) is a chronic inflammatory autoimmune liver disease. Although detailed mechanisms of the pathogenesis of PBC remain unknown, CD4+ T cells are suggested to play an important role. Recently microRNA (miRNA) was reported to be involved in the pathogenesis of PBC.
Aim: We analyzed the expression profile of miRNA and their target genes in CD4+ T cells of PBC patients to reveal their participation in pathogenesis of PBC. Clinically and pathologically diagnosed 7 PBC patients and 7 healthy controls, who agreed to provide samples with written informed consent, were enrolled in this study. Total RNA, including miRNA, was extracted from CD4+ T cells purified from peripheral blood. The comprehensive analysis of miRNA was undergone using microarray and quantitative real-time PCR (qRT-PCR). We predicted the target genes of miRNA, which was expressed specifically in PBC, using bioinformatics. The dynamics of predicted target genes were analyzed by microarray and qRT-PCR. Then, luciferase assay and miRNA mimic assay were performed to examine the binding of the specific miRNA to 3´untranslated region (3´UTR) of target genes. Finally, we tested the potential role of specifically expressed miRNA against target genes by overexpressing miRNA in cultured cells. Microarray miRNA study showed 2 increased and 13 decreased miRNAs in PBC (p<0.05). Among them, 5 miRNAs were validated to be down-regulated in PBC (p<0.05) by qRT-PCR. A total of 4,855 target genes were predicted from 5 miRNAs by bioinformatics. In the mRNA microarray analysis, the expression of 2,565 genes was significantly different between PBC and control. Comparison of the target prediction and gene expression microarray study revealed 238 target genes were specifically expressed in PBC. Among 238 target genes, we analyzed 10 genes that were the target for more than 3 specifically expressed miRNAs to reveal the regulation of the target genes by miRNAs. The expression of 3 target genes, which were reported to be associated with T cell development and function, were validated by qRT-PCR. Luciferase assay and miRNA overexpression assay demonstrated that the specific miRNAs regulate these target genes by binding to their 3´UTR. We have identified PBC specific expression of miRNA and their target genes. These miRNA may participate in the immunological pathogenesis of PBC through the regulation of the target genes in CD4+T cells.
9. Novel zinc finger protein in gastrointestinal tract
Yasuo Matsubara1,2, Kazuaki Takahashi2, Masahiro Arai2, Jun Miwa2, Shunji Mishiro2, Ryosuke Muroyama1, Kaku Goto1, Wenwen Li1, Ryo Nakagawa1, Norie Kowatari1, Chisiro Wakabayashi1, Naoya Kato1: 1Division of Advanced Genome Medicine, IMSUT; 2Department of Medical Science, Toshiba General Hospital
The gastrointestinal tract has definite anatomical and functional boundaries between its contiguous segments. Because some human cancers arise in a background of tissue metaplasia, e.g. Barrett’s esophagus and intestinal metaplasia of the stomach, it is important to clarify the molecular and cellular basis of region formation and preservation. Some genetic markers that delimit gastrointestinal boundaries have been reported, but it is still unknown how such boundaries are established and maintained. The aim of this study is to find another factor involved in segmental formation of gastrointestinal tract. Crude extracts from gastrointestinal tract biopsy specimens: esophagus, gastric fundus, duodenum and colon were subjected to PAGE. Specific band (less than 40kDa) in the gastric fundus sample was analyzed by mass spectrometry, and ZNF-114-like hypothetical protein that deduced from genome sequence was identified. Its mRNA sequence was determined by RACE. Functional analysis will be executed using expression vector.
10. IL28B minor allele is associated with a younger age of onset of hepatocellular carcinoma in patients with chronic hepatitis C virus infection
Masaya Sato1, Ryosuke Muroyama2, Norie Kowatari2, Wenwen Li2, Kaku Goto2, Ryo Nakagawa2, Ryosuke Tateishi1, Motoyuki Otsuka1, Shuichiro Shiina1, Haruhiko Yoshida1, Masao Omata1, Kazuhiko Koike1, Naoya Kato2: 1Department of Gastroenterology, Graduate School of Medicine, University of Tokyo; 2Division of Advanced Genome Medicine, IMSUT
IL28B polymorphisms were shown to be associated with response to peg-interferon based treatment in chronic hepatitis C (CHC) and spontaneous clearance. However, little is known about how this polymorphism affects the course of CHC, including the development of hepatocellular carcinoma (HCC). The aim of this study is to evaluate the influence of IL28B polymorphisms on hepatocarcinogenesis in CHC patients. We genotyped the rs8099917 single-nucleotide polymorphism in 351 hepatitis C-associated HCC patients without history of IFN-based treatment, and correlated the age at onset of HCC in patients with each genotype. The mean ages at onset of HCC for TT, TG, and GG genotypes were 69.9, 67.5 and 66.8, respectively. In multivariate analysis, IL28B minor allele (TG and GG genotypes) was an independent risk factor for younger age at onset of HCC (p = 0.02) in males (p<0.001) with higher body mass index (BMI; p=0.009). IL28B minor allele was also associated with a lower probability of having aspartate aminotransferase-to-platelet ratio index (APRI) >1.5 (minor vs. major, 46.7% vs. 58.6%; p = 0.01), lower AST (69.1 vs. 77.7 IU/l, p = 0.02), lower ALT (67.8 vs. 80.9 IU/l, P = 0.002), higher platelet count (12.8 vs. 11.2 ×104/μl, p = 0.002), and higher prothrombin time (79.3% vs. 75.4%, p = 0.002). In conclusion, IL28B minor allele was associated with lower inflammatory activity and less progressed fibrosis of liver, however, constituted a risk factor for younger-age onset of HCC in CHC patients.
11. Impact of IL28B genetic variation on HCV-induced liver fibrosis, inflammation, and steatosis: a meta-analysis
Masaya Sato1, Kondo M1, Tateishi R1, Fujiwara N1, Kato N2, Yoshida H1, Taguri M3, Kioke K1: 1Department of Gastroenterology, Graduate School of Medicine, University of Tokyo; 2Division of Advanced Genome Medicine, IMSUT; 3Department of Biostatistics and Epidemiology, Yokohama City University Medical Center
IL28B polymorphisms were shown to be strongly associated with the response to interferon therapy in chronic hepatitis C (CHC) and spontaneous viral clearance. However, little is known about how these polymorphisms affect the natural course of the disease. Thus, we conducted the present meta-analysis to assess the impact of IL28B polymorphisms on disease progression. A literature search was conducted using MEDLINE, EMBASE, and the Cochrane Library. Integrated odds ratios (OR) were calculated with a fixed-effects or random-effects model based on heterogeneity analyses. We identified 28 studies that included 10,024 patients. The pooled results indicated that the rs12979860 genotype CC was significantly associated (vs. genotype CT/TT; OR, 1.122; 95%CI, 1.003–1.254; P = 0.044), and that the rs8099917 genotype TT tended to be (vs. genotype TG/GG; OR, 1.126; 95%CI, 0.988–1.284; P = 0.076) associated, with an increased possibility of severe fibrosis. Both rs12979860 CC (vs. CT/TT; OR, 1.288; 95%CI, 1.050–1.581; P = 0.015) and rs8099917 TT (vs. TG/GG; OR, 1.324; 95%CI, 1.110–1.579; P = 0.002) were significantly associated with a higher possibility of severe inflammation activity. Rs8099917 TT was also significantly associated with a lower possibility of severe steatosis (vs. TG/GG; OR, 0.580; 95%CI, 0.351–0.959; P = 0.034), whereas rs12979860 CC was not associated with hepatic steatosis (vs. CT/TT; OR, 1.062; 95%CI, 0.415–2.717; P = 0.901). IL28B polymorphisms appeared to modify the natural course of disease in patients with CHC. Disease progression seems to be promoted in patients with the rs12979860 CC and rs8099917 TT genotypes.
12. Patatin-like phospholipase-3 (rs738409 C>G) polymorphism is associated with the development of hepatocellular carcinoma in patients with chronic hepatitis C infection
Masaya Sato1, Ryosuke Muroyama2, Norie Kowatari2, Wenwen Li2, Kaku Goto2, Ryosuke Tateishi1, Motoyuki Otsuka1, Shuichiro Shiina1, Haruhiko Yoshida1, Masao Omata1, Kazuhiko Koike1, Naoya Kato2: 1Department of Gastroenterology, Graduate School of Medicine, University of Tokyo; 2Division of Advanced Genome Medicine, IMSUT
An isoleucine to methionine substitution at position 148 in the PNPLA3 gene (p.l148M, rs738409) has recently been identified as a susceptibility factor for liver damage in steatohepatitis. However, little is known about the influence of this polymorphism on hepatocarcinogenesis. The aim of this study is to assess the impact of PNPLA3 polymorphism on the development of hepatocellular carcinoma (HCC) which is thought to be one of the major steatosis-related complications in patients with chronic hepatitis C. We genotyped the rs738409 single-nucleotide polymorphism (SNP) in 358 hepatitis C-related HCC patients, and correlated the age at onset of HCC and the duration between the hepatitis C virus (HCV) infection and the development of HCC. The median age at onset of HCC for the GG genotype was significantly younger compared to for non-GG genotypes (67.81 vs. 69.87 years, P < 0.001), and the median interval between HCV infection and the development of HCC was significantly shorter in patients with the GG genotype (39.96 vs. 40.85 years, P = 0.008). PNPLA3 GG genotype was also associated with a higher AST level (69.5 vs. 59.0 IU/l, P = 0.02), lower prothrombin time (73.0% vs. 78.0%, P = 0.008), and a higher prevalence of histological steatosis (40.0% vs. 22.2%, P = 0.01) at the time of HCC onset. In conclusion, the PNPLA3 genotype GG may be associated with accelerated hepatocarcinogenesis in CHC patients through increased steatosis in the liver.
13. SOCS1 abrogates IFN’s antiviral effect on hepatitis C virus replication
Run-Xuan Shao1, Kaku Goto1,2, Raymond T. Chung1: 1GI Unit, Massachusetts General Hospital, Harvard Medical School; 2Division of Advanced Genome Medicine, IMSUT
Suppressor of cytokine signaling 1 (SOCS1) and suppressor of cytokine signaling 3 (SOCS3) have been thought to block type I interferon (IFN) signaling. We have previously reported that SOCS3 suppresses HCV replication in an mTOR-dependent manner. However, the relationship between SOCS1 and HCV replication remains unclear. Here, we found that overexpression of SOCS1 alone did not have an effect on HCV RNA replication. However, suppression of HCV replication by IFN-α was rescued by SOCS1 overexpression. The upregulation of HCV replication by SOCS1 overexpression in the presence of IFN is likely a result of the impairment of IFN signaling by SOCS1 and subsequent induction of ISGs. Knockdown of SOCS1 alone with specific shRNA enhanced the antiviral effect of IFN compared with negative control. Thus, SOCS1 acts as a suppressor of type I IFN function against HCV.
14. Kinetic differences in the induction of interferon stimulated genes by interferon-α and interleukin 28B are altered by infection with hepatitis C virus
Nikolaus Jilg1, Kaku Goto1,2, Raymond T. Chung1: 1GI Unit, Massachusetts General Hospital, Harvard Medical School; 2Division of Advanced Genome Medicine, IMSUT
Several genome-wide association studies (GWAS) have identified a genetic polymorphism associated with the gene locus for interleukin 28B (IL28B), a type III interferon (IFN), as a major predictor of clinical outcome in hepatitis C. Antiviral effects of the type III IFN family have previously been shown against several viruses, including hepatitis C virus (HCV), and resemble the function of type I IFN including utilization of the intracellular Janus kinase signal transducer and activator of transcription (JAK-STAT) pathway. Effects unique to IL28B that would distinguish it from IFN-α are not well defined. By analyzing the transcriptomes of primary human hepatocytes (PHH) treated with IFN-α or IL28B, we sought to identify functional differences between IFN-α and IL28B to better understand the roles of these cytokines in the innate immune response. Although our data did not reveal distinct gene signatures, we detected striking kinetic differences between IFN-α and IL28B stimulation for interferon stimulated genes (ISGs). While gene induction was rapid and peaked at 8 hours of stimulation with IFN-α in PHH, IL28B produced a slower, but more sustained increase in gene expression. We confirmed these findings in the human hepatoma cell line Huh7.5.1. Interestingly, in HCV-infected cells the rapid response after stimulation with IFN-α was blunted, and the induction pattern resembled that caused by IL28B. Conclusion: The kinetics of gene induction are fundamentally different for stimulations with either IFN-α or IL28B in hepatocytes, suggesting distinct roles of these cytokines within the immune response. Furthermore, the observed differences are substantially altered by infection with HCV.
Publications
Lo PH, Urabe Y, Kumar V, Tanikawa C, Koike K, Kato N, Miki D, Chayama K, Kubo M, Nakamura Y, Matsuda K. Identification of a functional variant in the MICA promoter which regulates MICA expression and increases HCV-related hepatocellular carcinoma risk. PLoS One 2013; 8: e61279
Urabe Y, Ochi H, Kato N, Kumar V, Takahashi A, Muroyama R, Hosono N, Otsuka M, Tateishi R, Lo PH, Tanikawa C, Omata M, Koike K, Miki D, Abe H, Kamatani N, Toyota J, Kumada H, Kubo M, Chayama K, Nakamura Y, Matsuda K. A genome-wide association study of HCV induced liver cirrhosis in the Japanese population identifies novel susceptibility loci at MHC region. J Hepatol 2013; 58: 875-882
Goto K, Lin W, Zhang L, Jilg N, Shao RX, Schaefer EA, Zhao H, Fusco DN, Peng LF, Kato N, Chung RT. The AMPK-related kinase SNARK regulates hepatitis C virus replication and pathogenesis through enhancement of TGF- Signaling. J Hepatol 2013; 59: 942-948
Sato M, Kato N, Tateishi R, Muroyama R, Kowatari N, Li W, Goto K, Otsuka M, Shiina S, Yoshida H, Omata M, Koike K. IL28B minor allele is associated with a younger age of onset of hepatocellular carcinoma in patients with chronic hepatitis C virus infection. J Gastroenterol. Springer; DOI 10.1007/s00535-013-0826-x, 2013
Sato M, Kondo M, Tateishi R, Fujiwara N, Kato N, Yoshida H, Taguri M, Koike K. Impact of IL28B genetic variation on HCV-induced liver fibrosis, inflammation, and steatosis: a meta-analysis. PLoS One 2014; 9: e91822
Sato M, Kato N, Tateishi R, Muroyama R, Kowatari N, Li W, Goto K, Otsuka M, Shiina S, Yoshida H, Omata M, Koike K. The Impact of PNPLA3 Polymorphisms on the Development of Hepatocellular Carcinoma in Patients with Chronic Hepatitis C Virus Infection. Hepatol Res 2013 (Epub ahead of print)
Shao RX, Zhang L, Hong Z, Goto K, Cheng D, Chen WC, Jilg N, Kumthip K, Fusco DN, Peng LF, Chung RT. SOCS1 abrogates IFN’s antiviral effect on hepatitis C virus replication. Antiviral Res 2013; 97: 101-107
Jilg N, Lin W, Hong J, Schaefer EA, Wolski D, Meixong J, Goto K, Brisac C, Chusri P, Fusco DN, Chevaliez S, Luther J, Kumthip K, Urban TJ, Peng LF, Lauer GM, Chung RT. Kinetic differences in the induction of interferon stimulated genes by interferon-α and IL28B are altered by infection with hepatitis C virus. Hepatology 2014; 59: 1250-1261