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

Abstract-Osteopontin (OPN), an RGD-containing extracellular matrix protein, is associated with arterial smooth muscle cell (SMC) activation in vitro and in vivo. Many cytokines and growth factors involved in vessel wall remodeling induce OPN overexpression. Moreover, we recently demonstrated that the extracellular nucleotide UTP also induces OPN expression and that OPN is essential for UTP-mediated SMC migration. Thus, we set out to investigate the mechanisms of OPN expression. The aim of this study was to identify transcription factors involved in the regulation of OPN expression in SMCs. First, we explored the contribution of mRNA stabilization and transcription in the increase of UTP-induced OPN mRNA levels. We show that UTP induced OPN mRNA increases via both OPN mRNA stabilization and OPN promoter activation. Then, to identify transcription factors involved in UTP-induced OPN transcription, we located a promoter element activated by UTP within the rat OPN promoter using a gene reporter assay strategy. The Ϫ96 to ϩ1 region mediated UTP-induced OPN overexpression (ϩ276Ϯ60%). Sequence analysis of this region revealed a potential site for AP-1 located at Ϫ76. When this AP-1 site was deleted, UTP-induced activation of the Ϫ96 to ϩ1 region was totally inhibited. Thus, this AP-1 (Ϫ76) site is involved in UTP-induced OPN transcription. A supershift assay revealed that both c-Fos and c-Jun bind to this AP-1 site. Finally, we demonstrate that angiotensin II and platelet-derived growth factor, two main factors involved in vessel wall pathology, also modulated OPN expression via AP-1 activation. S everal studies suggest that migration and proliferation of arterial smooth muscle cells (SMCs) play a prominent role in vascular pathologies such as atherosclerosis, restenosis, and hypertension. 1 SMC migration and proliferation can be induced by many factors, including growth factors and cytokines. [2][3][4] We have previously shown that extracellular nucleotides are also able to induce cell-cycle progression of SMCs 5,6 and their migration. 7 Thus, we were interested in understanding the mechanisms by which UTP induces SMC migration. Nucleotide-induced SMC activation is mediated via G protein-coupled P2Y receptors. Their activation leads to phospholipidase C activation and consequently to [Ca 2ϩ ] i increase and protein kinase C activation. Our previous work demonstrated that P2Y 2 , P2Y 4 , and P2Y 6 receptors are expressed in cultured SMCs 8 and that P2Y 2 is overexpressed in balloon-injured rat carotids. 9 Moreover, we have shown that these receptors are involved in UTP-induced migration. 8 We also demonstrated that UTP induces the expression of the extracellular matrix protein osteopontin (OPN) 6 and that UTP-induced migration is dependent on OPN expression and binding to ␣ v ␤ 3 integrin. 7 OPN is an RGD-containing extracellular matrix (ECM) phosphoprotein involved in cell attachment 10 and migration 11,12 and prevention of apoptosis. 13 OPN expression is induced by many growth factors, hormones, and cytokines invol...

Osteopontin (OPN) is an important chemokinetic agent for several cell types. Our earlier studies have shown that its expression is essential for uridine triphosphate (UTP)-mediated migration of vascular smooth muscle cells. We demonstrated previously that the activation of an AP-1 binding site located 76 bp upstream of the transcription start in the rat OPN promoter is involved in the induction of OPN expression. In this work, using a luciferase promoter deletion assay, we identified a new region of the rat OPN promoter (؊1837 to ؊1757) that is responsive to UTP. This region contains an NFB site located at ؊1800 and an Ebox located at ؊1768. Supershift electrophoretic mobility shift assay and chromatin immunoprecipitation assays identified NFB and USF-1/USF-2 as the DNA binding proteins induced by UTP, respectively, for these two sites. Using dominant negative mutants of IB kinase and USF transcription factors, we confirmed that NFB and USF-1/USF-2 are involved in the UTP-mediated expression of OPN. Using a pharmacological approach, we demonstrated that USF proteins are regulated by the extracellular signal-regulated kinase (ERK)1/2 pathway, just as the earlier discovered AP-1 complex, whereas NFB is up-regulated through PKC␦ signals. Finally, our work suggests that the UTPstimulated OPN expression involves a coordinate regulation of PKC␦-NFB, ERK1/2-USF, and ERK1/2/ NAD(P)H oxidase AP-1 signaling pathways. Osteopontin (OPN)1 is a multifunctional phosphoprotein secreted by many cell types such as osteoclasts, lymphocytes, macrophages, epithelial cells, and smooth muscle cells (SMCs).Its expression is induced during vessel remodeling, as well as wound healing and bone synthesis (1). Overexpression of OPN can be found in pathological settings, such as immunological disorders, neoplastic transformation, metastasis, and formation of urinary stones. Its role in cell migration has been established in vitro in various cell types, including osteoclasts (2), macrophages (3), endothelial cells (4), and SMCs (5, 6). In vivo studies have established that OPN contributes to SMC recruitment into atherosclerotic plaques (7) and neointimal thickening (8), to osteoclast recruitment during bone resorption, (2) and to macrophage recruitment at inflammatory sites (atherosclerotic plaque, granulomas) (3).In the context of vascular remodeling, soluble factors, such as angiotensin II, basic fibroblast growth factor, plateletderived growth factor, interleukin-1 (9), and the nucleotides ATP and UTP (10), all induce expression of OPN. Moreover, nucleotides also modulate contraction, proliferation, and migration of vascular SMCs (11). They act through G proteincoupled P2Y receptors (12) and activate phospholipase C, resulting in elevated levels of intracellular free Ca 2ϩ and activation of PKC. In addition, they induce ROS production through NAD(P)H oxidase (13). Several transcription factors, such as NFAT, AP-1, cAMP-responsive element-binding protein, serum-responsive factor, STAT, and NFB are induced in UTP-stimulated SMCs (14), contro...