Expression of microRNA miR-122 facilitates an efficient replication in nonhepatic cells upon infection with hepatitis C virus

Hepatitis C virus (HCV) is one of the most common etiologic agents of chronic liver diseases, including liver cirrhosis and hepatocellular carcinoma. In addition, HCV infection is often associated with extrahepatic manifestations (EHM), including mixed cryoglobulinemia and non-Hodgkin's lymphoma. However, the mechanisms of cell tropism of HCV and HCV-induced EHM remain elusive, because in vitro propagation of HCV has been limited in the combination of cell culture-adapted HCV (HCVcc) and several hepatic cell lines. Recently, a liver-specific microRNA called miR-122 was shown to facilitate the efficient propagation of HCVcc in several hepatic cell lines. In this study, we evaluated the importance of miR-122 on the replication of HCV in nonhepatic cells. Among the nonhepatic cell lines expressing functional HCV entry receptors, Hec1B cells derived from human uterus exhibited a low level of replication of the HCV genome upon infection with HCVcc. Exogenous expression of miR-122 in several cells facilitates efficient viral replication but not production of infectious particles, probably due to the lack of hepatocytic lipid metabolism. Furthermore, expression of mutant miR-122 carrying a substitution in a seed domain was required for efficient replication of mutant HCVcc carrying complementary substitutions in miR-122-binding sites, suggesting that specific interaction between miR-122 and HCV RNA is essential for the enhancement of viral replication. In conclusion, although miR-122 facilitates efficient viral replication in nonhepatic cells, factors other than miR-122, which are most likely specific to hepatocytes, are required for HCV assembly.     

MicroRNAs and hepatitis C virus: Toward the end of miR-122 supremacy

ABSTRACT: The most common etiologic agents causing chronic hepatitis are hepatitis C and B viruses(HCV and HBV, respectively). Chronic infection caused by HCV is considered one of themajor causative agents of liver cirrhosis and hepatocellular carcinoma worldwide. Incombination with the increasing rate of new HCV infections, the lack of a current vaccineand/or an effective treatment for this virus continues to be a major public health challenge.The development of new treatments requires a better understanding of the virus and itsinteraction with the different components of the host cell. MicroRNAs (miRNAs) are smallnon-coding RNAs functioning as negative regulators of gene expression and represent aninteresting lead to study HCV infection and to identify new therapeutic targets. Until now,microRNA-122 (miR-122) and its implication in HCV infection have been the focus ofdifferent published studies and reviews. Here we will review recent advances in therelationship between HCV infection and miRNAs, showing that some of them emerge inpublications as challengers against the supremacy of miR-122.     

microRNA-122与丙型肝炎病毒复制和肝细胞癌的相关研究进展

microRNA-122(miR-122)是一种肝脏特异性微小RNA,与丙型肝炎病毒(HCV)的复制和感染及肝细胞癌(HCC)的发生密切相关。近年来,科研人员关于miR-122的研究已取得了较大进展。在此,我们就miR-122与HCV复制和HCC的相关关系等方面的研究进展做简要综述。     

肝脏特异性miR-122的分子生物学特性及功能

miR-122是在肝脏特异高表达的一种microRNA。研究表明：生理状态下,miR-122 在调控肝脏的细胞发育、诱导细胞分化、调节细胞代谢、参与肝细胞应急应答等生命活动过程中发挥重要作用;而在病理状态下,miR-122 与丙型肝炎病毒（HCV）和肝细胞肝癌（HCC）密切相关,可能促进HCV RNA 复制,并在HCC发生、发展过程中发挥抑癌基因样作用,可能对HCC 临床诊断和预后具有重要价值。鉴于miR-122 参与调控肝脏生理及肝脏重大疾病的发生、发展等过程,文章详细阐述并讨论miR-122 在肝脏中的生物学特性和功能,以及可能的作用机制。肝脏特异性miR-122 有可能作为治疗人类肝脏疾病的关键靶点。     

MicroRNA in HCV infection and liver cancer

In the more than two-decades since hepatitis C virus (HCV) was identified, there has been considerable improvement in our understanding of virus life cycle due largely to the development of in vitro culture systems for virus replication. Still challenges remain: HCV infection is a major risk factor for chronic hepatitis, liver cirrhosis and hepatocellular carcinoma worldwide; yet mechanistic details of HCV infection-associated hepatocarcinogenesis remain incompletely understood. A protective vaccine is not yet available, and current therapeutic options result in sustained virus clearance only in a subset of patients. Recent interest has focused on small non-protein coding RNAs, microRNAs (miRNAs), the dependence of virus replication on miRNAs, and miRNA-regulated genes in liver cancer. Functional analysis of the miRNA-targeted genes in liver cancer has advanced our understanding of the "oncomiRs" and their role in hepatocarcinogenesis. This review focuses on the dependence of HCV replication on miRNA and role of miRNA-targeted tumor suppressor genes as molecular markers of and possible targets for developing oncomiR-targeted therapy of chronic hepatitis and HCC. This article is part of a Special Issue entitled: MicroRNAs in viral gene regulation.     

HepG2 cells expressing microRNA miR-122 support the entire hepatitis C virus life cycle

The liver-specific microRNA miR-122 is required for efficient hepatitis C virus (HCV) RNA replication both in cell culture and in vivo. In addition, nonhepatic cells have been rendered more efficient at supporting this stage of the HCV life cycle by miR-122 expression. This study investigated how miR-122 influences HCV replication in the miR-122-deficient HepG2 cell line. Expression of this microRNA in HepG2 cells permitted efficient HCV RNA replication and infectious virion production. When a missing HCV receptor is also expressed, these cells efficiently support viral entry and thus the entire HCV life cycle.     

Circulating microRNAs associated with liver fibrosis in chronic hepatitis C patients

A major challenge in hepatitis C research is the detection of early potential for progressive liver disease. MicroRNAs (miRNAs) are small RNAs that regulate gene expression and can be biomarkers of pathological processes. In this study, we compared circulating miRNAs identified in hepatitis C virus (HCV)-infected patients presenting two extremes of liver disease: mild/moderate fibrosis and cirrhosis. The patients in the cirrhosis group subsequently developed hepatocellular carcinoma (HCC). We identified 163 mature miRNAs in the mild/moderate fibrosis group and 171 in the cirrhosis group, with 144 in common to both groups. Differential expression analysis revealed 5 upregulated miRNAs and 2 downregulated miRNAs in the cirrhosis group relative to the mild/moderate fibrosis group. Functional analyses of regulatory networks (target gene and miRNA) identified gene categories involved in cell cycle biological processes and metabolic pathways related to cell cycle, cancer, and apoptosis. These results suggest that the differentially expressed circulating miRNAs observed in this work (miR-215-5p, miR-483-5p, miR-193b-3p, miR-34a-5p, miR-885-5p, miR-26b-5p and miR -197-3p) may be candidates for biomarkers in the prognosis of liver disease.     

Evaluation of miR-122 level in the plasma of chronically HCV infected patients

MicroRNAs (miRNAs) are small non-coding RNA molecules, which have an important function in regulating RNA stability and gene expression. They also can circulate in a cell-free form in the blood thatmakes them potential disease markers. The liver contains various classes of miRNAs in which miR-122 accounts for about 70% of all miRNAs and it has been proved that its level increases in case of liver damage. Here, we investigated plasma levels of miR-122 as a useful disease parameter in patients with chronic hepatitis C (CHC) infection. Thirty five hemophilia and thalassemia patients with CHC were studied. The total RNA was extracted from plasma samples, and miR-122 levels were measured by qPCR and then compared with the specific liver markers. The plasma levels of alanine transaminase (ALT) and aspartate transaminase(AST) were correlated with plasma miR-122 level in CHC patients, and the level of circulating miR-122 in healthy individual groups were rarely lower than those of patients with CHC. In our study, miR-122 levels correlated well with markers of liver inflammatory activity. Plasma miR-122 can be assumed to be another marker in liver similar to the currently used specific markers such as ALT and AST for evaluation of liver damage in hepatitis C virus (HCV) infected patients. Moreover, the correlation between miR-122 and ALT was shown to be higher than between miR-122 and AST.     

Kinetics of miR-122 Expression in the Liver during Acute HCV Infection

The relationships among micro RNA-122 (miR-122) expression in the liver, hepatitis C virus (HCV) replication and hepatic damage were analyzed in three chimpanzees observed for 180 days after inoculation with HCV genotype 1a. Levels of miR-122 in the liver and serum were measured by real-time RT PCR in serial liver biopsies and serum samples. Hepatic miR-122 levels were normalized separately for each of three chimpanzees with small RNAs and microRNAs that are endogenous to the liver and are stably expressed. Two- to 4-fold rise in hepatic miR-122 levels was observed at the onset of HCV infection (the first 4 weeks) when HCV titers in the liver and serum increased rapidly in all three chimpanzees in concordance with in vitro data indicating the miR-122 significance for HCV replication. Between 10 to 14 weeks after inoculation, when hepatic and serum HCV RNA titers exceeded 3 logs and alanine aminotransferase (ALT) activity was elevated, hepatic miR-122 levels were in decline. Cumulative data derived from all three chimpanzees from 180 days of observation documented an inverse (negative) correlation between hepatic miR-122 and HCV RNA in the liver and serum and positive correlation between level of serum miR-122 and HCV replication. Subsequent rise of miR-122 level during HCV clearance and ALT normalization suggested a tri-phasic occurrence of the relationship among hepatic miR-122 expression, HCV replication and hepatic destruction, which was the most apparent in one chimpanzee but less evident in two other animals. In vivo kinetics of hepatic and serum miR-122, HCV replication and hepatic destruction reflects complexities of the virus-host interaction during the acute phase of HCV infection.     

Differential stimulation of hepatitis C virus RNA translation by microRNA-122 in different cell cycle phases

Hepatitis C virus (HCV) replicates preferentially in the liver, and in most cases, the HCV infection becomes chronic and often results in hepatocellular carcinoma. When the HCV plus-strand RNA genome has been delivered to the cytosol of the infected cell, its translation is directed by the internal ribosome entry site (IRES) in the 5′-untranslated region (5′-UTR) of the viral RNA. Thereby, IRES activity is modulated by several host factors. In particular, the liver-specific microRNA-122 (miR-122) interacts with two target sites in the HCV 5′-UTR and stimulates HCV translation, thereby most likely contributing to HCV liver tropism. Here, we show that HCV IRES-dependent translation efficiency in the hepatoma cell line Huh7 is highest during the G₀ and G₁ phases of the cell cycle but significantly drops during S phase and even more in the G₂/M phase. The superimposed stimulation of HCV translation by ectopic miR-122 works best during G₀, G₁ and G₂/M phases but is lower during S phase. However, the levels of Ago2 protein do not substantially change during cell cycle phases, indicating that other cellular factors involved in HCV translation stimulation by miR-122 may be differentially expressed in different cell cycle phases. Moreover, the levels of endogenously expressed miR-122 in Huh7 cells are lowest in S phase, indicating that the predominant G₀/G₁ state of non-dividing hepatocytes in the liver facilitates high expression of the HCV genome and stimulation by miR-122, with yet-unknown factors involved in the differential extent of stimulation by miR-122.Key words: HCV, translation, miR-122, microRNA, miRNA, Ago, Ago2     

MicroRNA 122对肝癌细胞基因表达谱的影响

为研究microRNA（miR-122）对肝癌细胞Hep3B基因表达谱的影响,并探讨其在肝癌发过程中的可能作用,构建了miR-122稳定高表达的Hep3B细胞,利用基因表达谱芯片技术筛选得到和对照组细胞比较的差异表达基因.研究结果显示,2倍以上变化的差异表达基因有490个,其中上调的有345个,下调的有145个.这些基因中有16个与肿瘤发生相关,其它基因涉及细胞周期、信号转导、细胞凋亡和细胞增殖分化等众多生物学过程.这些结果提示,miR-122可能在肝癌发生的过程中发挥作用,并可能与这些差异表达基因密切相关.另外,还结合生物信息学方法,在下调表达的基因中预测了miR-122可能直接作用的靶基因.本研究初步探讨了miR-122在肝癌细胞中的生物学功能,为进一步研究miR-122在肝癌发生中的作用奠定了基础,同时也为miRNA的生物学功能及其作用机制的研究提供了一些参考.     

Circulating microRNAs in patients with chronic hepatitis C and non-alcoholic fatty liver disease

MicroRNAs miR-122, miR-34a, miR-16 and miR-21 are commonly deregulated in liver fibrosis and hepatocellular carcinoma. This study examined whether circulating levels of these miRNAs correlate with hepatic histological disease severity in patients with chronic hepatitis C infection (CHC) or non-alcoholic fatty-liver disease (NAFLD) and can potentially serve as circulating markers for disease stage assessment. We first used an in vitro model of hepatitis C virus (HCV) infection to measure the extracellular levels of these four miRNAs. Whereas miR-21 extracellular levels were unchanged, extracellular levels of miR-122, miR-34a and to a lesser extent miR-16, steadily increased during the course of HCV infection, independently of viral replication and production. Similarly, in CHC patients, serum levels of miR-122, miR-34a and miR-16 were significantly higher than in control individuals, while miR-21 levels were unchanged. There was no correlation between the serum levels of any of these microRNAs and HCV viral loads. In contrast, miR-122 and miR-34a levels positively correlated with disease severity. Identical results were obtained in an independent cohort of CHC patients. We extended the study to patients with NAFLD. As observed in CHC patients, serum levels of miR-122, miR-34a and miR-16 were significantly higher in NAFLD patients than in controls, while miR-21 levels were unchanged. Again, miR-122 and miR-34a levels positively correlated with disease severity from simple steatosis to steatohepatitis. In both CHC and NAFLD patient groups, serum levels of miR-122 and miR-34a correlated with liver enzymes levels, fibrosis stage and inflammation activity. miR-122 levels also correlated with serum lipids in NAFLD patients. CONCLUSION: Serum levels of miR-34a and miR-122 may represent novel, noninvasive biomarkers of diagnosis and histological disease severity in patients with CHC or NAFLD.