Profound differences of microRNA expression patterns in hepatocytes and hepatoma cell lines commonly used in hepatitis C virus studies (2011)

In their recent letter to the editor, Bensadoun et al. describe a divergent IL-28B (interleukin-28B) genotype of hepatoma cell lines including Huh7 and its derivatives.1 Because these cell lines are commonly used in cell culture studies of hepatitis C virus (HCV) and the IL-28B gene polymorphism is a predictive marker for the outcome of HCV infection, their observations emphasize the importance of cautiously interpreting infection studies performed in vitro. Here, we extend their conclusions and report profound differences in the microRNA (miRNA) expression between primary human hepatocytes (PHHs) and Huh7 cell lines. Cellular miRNAs are key regulators in posttranscriptional regulation of gene expression. They can also directly participate in virus replication or act indirectly by determining the expression level of replication cofactors. To analyze whether the commonly used Huh7 cell lines differ in their miRNA expression patterns, we screened 883 human miRNAs using the Geniom Biochip miRNA Homo sapiens (febit holding gmbh, Heidelberg, Germany). PHHs isolated from human liver resections of two different patients were used as reference. Figure 1 and Supporting Table 1 show the profound differences not only between PHHs and hepatoma cell lines but also between the cell lines. Remarkably, the liver-specific miRNA122 that directly influences HCV replication both in cell culture and in infected chimpanzees2-4 was one of the most differentially expressed miRNAs. Furthermore, predictions of the gene targets of the miRNAs from Fig. 1A by the Genetrail program (freely accessible at http://genetrail.bioinf.uni-sb.de) identified a number of host proteins whose differential expression is known or expected to influence HCV replication (see Supporting Table 2 for the complete target list). Because studies on host factor requirements for virus replication nowadays involve small interfering RNA–mediated down-regulation of candidate proteins, and the level of knockdown is influenced by protein abundance and turnover and thus miRNA composition, one would expect that the respective experimental results would be influenced by the host cells used. The recent observations of nonoverlapping screening results for HCV host factors5 may well be related to corresponding miRNA differences in the host cells. MicroRNA patterns of primary human hepatocytes and HCV-permissive hepatoma cell lines. (A) Heat map of a hierarchical clustering of miRNAs and samples. The analyzed cell type is indicated on the x-axis and the 19 most different human miRNAs are indicated on the y-axis. Red color represents a high expression whereas green corresponds to a low expression of miRNAs. Relative expression levels are given in a relative scale from 0 to 10,000. Samples can mainly be separated into two clusters. The cluster on the left includes PHHs from two different donors without liver disease (PHH1 and PHH2), and the cluster on the right includes the three analyzed hepatoma cell lines Huh7, Huh7.5, and Huh7 Lunet. (B) Color-coded correlation of the overall miRNA expression pattern of PHHs and three hepatoma cell lines. Strong resemblance is indicated in yellow (1.0 equals 100%); no resemblance is given in dark blue. Additional Supporting Information may be found in the online version of this article. Please note: The publisher is not responsible for the content or functionality of any supporting information supplied by the authors. Any queries (other than missing content) should be directed to the corresponding author for the article. Michael Ehrhardt*, Petra Leidinger Ph.D. , Andreas Keller Ph.D. , Thomas F. Baumert Ph.D. §, Juana Díez Ph.D.¶, Eckart Meese Ph.D. , Andreas Meyerhans Ph.D.* **, * Departments of Virology, Saarland University, Homburg, Germany, Human Genetics, Saarland University, Homburg, Germany, Institut National de la Santé et de la Recherche Médicale, Unité 748, § Université de Strasbourg, Strasbourg, France, ¶ Molecular Virology Group, Department of Experimental and Health Sciences, Universitat Pompeu Fabra, Barcelona, Spain, ** ICREA Infection Biology Group, Department of Experimental and Health Sciences, Universitat Pompeu Fabra, Barcelona, Spain.