Hepatocellular carcinoma (HCC) is the major primary liver cancer. Glypican-3 (GPC3), one of the most abnormally expressed genes in HCC, participates in liver carcinogenesis. Based on data showing that GPC3 expression is posttranscriptionally altered in HCC cells compared to primary hepatocytes, we investigated the implication of microRNAs (miRNAs) in GPC3 overexpression and HCC. To identify GPC3-regulating miRNAs, we developed a dual-fluorescence FunREG (functional, integrated, and quantitative method to measure posttranscriptional regulations) system that allowed us to screen a library of 876 individual miRNAs. Expression of candidate miRNAs and that of GPC3 messenger RNA (mRNA) was measured in 21 nontumoral liver and 112 HCC samples. We then characterized the phenotypic consequences of modulating expression of one candidate miRNA in HuH7 cells and deciphered the molecular mechanism by which this miRNA controls the posttranscriptional regulation of GPC3. We identified five miRNAs targeting GPC3 3 0 -untranslated region (UTR) and regulating its expression about the 876 tested. Whereas miR-96 and its paralog miR-1271 repressed GPC3 expression, miR-129-1-3p, miR-1291, and miR-1303 had an inducible effect. We report that miR-1271 expression is down-regulated in HCC tumor samples and inversely correlates with GPC3 mRNA expression in a particular subgroup of HCC. We also report that miR-1271 inhibits the growth of HCC cells in a GPC3-dependent manner and induces cell death. Conclusion: Using a functional screen, we found that miR-96, miR-129-1-3p, miR-1271, miR-1291, and miR-1303 differentially control GPC3 expression in HCC cells. In a subgroup of HCC, the up-regulation of GPC3 was associated with a concomitant down-regulation of its repressor miR-1271. Therefore, we propose that GPC3 overexpression and its associated oncogenic effects are linked to the down-regulation of miR-1271 in HCC. (HEPATOLOGY 2013;57:195-204) H epatocellular carcinoma (HCC) is the most common form of primary liver cancer. 1 It usually develops in an affected liver with cirrhosis due to viral infection (hepatitis B virus, HBV; hepatitis C virus, HCV), alcohol abuse, metabolic disorders, or a carcinogenic agent. 1-3 HCC is a very heterogeneous class of tumors characterized by multiple types of genomic damages associated with its various

Because some metabolic intermediates are involved in more than one pathway, crosstalk between pathways is crucial to maintaining homeostasis. AMP and histidine biosynthesis pathways are coregulated at the transcriptional level in response to adenine availability. 5Ј-Phosphoribosyl-4-carboxamide-5-aminoimidazole (AICAR), a metabolic intermediate at the crossroads between these two pathways, is shown here to be critical for activation of the transcriptional response in the absence of adenine. In this study, we show that both AMP and histidine pathways significantly contribute to AICAR synthesis. Furthermore, we show that upregulation of the histidine pathway clearly interferes with regulation of the AMP pathway, thus providing an explanation for the regulatory crosstalk between these pathways. Finally, we revisit the histidine auxotrophy of ade3 or ade16 ade17 mutants. Interestingly, overexpression of PMU1, encoding a potential phosphomutase, partially suppresses the histidine requirement of an ade3 ade16 ade17 triple mutant, most probably by reducing the level of AICAR in this mutant. Together our data clearly establish that AICAR is not just a metabolic intermediate but also acts as a true regulatory molecule.

In the past 10 years, transcriptome and proteome analyses have provided valuable data on global gene expression and cell functional networks. However, when integrated, these analyses revealed partial correlations between mRNA expression levels and protein abundance thus suggesting that post-transcriptional regulations may be in part responsible for this discrepancy. In the present work, we report the development of a functional, integrated, and quantitative method to measure post-transcriptional regulations that we named FunREG. This method enables (i) quantitative measure of post-transcriptional regulations mediated by selected 3-untranslated regions and exogenous small interfering-RNA or micro-RNAs and (ii) comparison of these regulatory processes in physiologically relevant systems (e.g. cancer versus primary untransformed cells). We applied FunREG to the study of liver cancer, and we demonstrate for the first time the differential regulatory mechanisms controlling gene expression at a post-transcriptional level in normal and tumoral hepatic cells. As an example, translation efficiency mediated by heparin-binding epidermal growth factor 3-untranslated region was increased 3-fold in liver cancer cells compared with normal hepatocytes, whereas stability of an mRNA containing a portion of Cyclin D1 3-untranslated region was increased more than 2-fold in HepG2 cells compared with normal hepatocytes. Consequently we believe that the method presented herein may become an important tool in fundamental and medical research. This approach is convenient and easy to perform, accessible to any investigator, and should be adaptable to a large number of cell type, functional and chemical screens, as well as genome scale analyses. Finally FunREG may represent a helpful tool to reconcile transcriptome and proteome data.