Loss of Mir-122 Expression in Liver Cancer Correlates with Suppression of the Hepatic Phenotype and Gain of Metastatic Properties

Oncogene (2009) 28, 3526–3536 & 2009 Macmillan Publishers Limited All rights reserved 0950-9232/09 $32.00 www.nature.com/onc ORIGINAL ARTICLE Loss of miR-122 expression in liver cancer correlates with suppression of the hepatic phenotype and gain of metastatic properties

C Coulouarn, VM Factor, JB Andersen, ME Durkin and SS Thorgeirsson

Laboratory of Experimental Carcinogenesis, Center for Cancer Research, National Cancer Institute, National Institutes of Health, Bethesda, MD, USA

Growing evidence indicates that microRNAs have a alcoholic or nonalcoholic steatohepatitis, and aflatoxin significant role in tumor development and may constitute intoxication (Thorgeirsson and Grisham, 2002; El- robust biomarkers for cancer diagnosis and prognosis. In Serag, 2004). Although recent advances in functional this study, we evaluated the clinical and functional genomics provide an increasingly comprehensive por- relevance of microRNA-122 (miR-122) expression in trayal of hepatocarcinogenesis (Thorgeirsson et al., human hepatocellular carcinoma (HCC). We report that 2006; Villanueva et al., 2007), the molecular patho- miR-122 is specifically repressed in a subset of primary genesis of HCC remains poorly understood. Indeed, the tumors that are characterized by poor prognosis. We clinical heterogeneity of HCC and the lack of good further show that the loss of miR-122 expression in tumor diagnostic markers and treatment strategies have cells segregates with specific gene expression profiles rendered the disease a major challenge. linked to cancer progression, namely the suppression of MicroRNAs (miRNAs) belong to an abundant family hepatic phenotype and the acquisition of invasive proper- of endogenous, short and noncoding RNAs that are ties. We identify liver-enriched transcription factors as believed to be key post-transcriptional regulators of central regulatory molecules in the gene networks gene activity. It has been widely shown that miRNAs associated with loss of miR-122, and provide evidence have an important role in the control of numerous suggesting that miR-122 is under the transcriptional biological processes, such as development, differentia- control of HNF1A, HNF3A and HNF3B. We further tion, proliferation and apoptosis (Ambros, 2004). show that loss of miR-122 results in an increase of cell Therefore, it is not surprising that miRNAs have been migration and invasion and that restoration of miR-122 implicated in cancer as well. An altered miRNA reverses this phenotype. In conclusion, miR-122 is a expression was observed in a large variety of neoplasms marker of hepatocyte-specific differentiation and an (Lu et al., 2005), including HCC (Murakami et al., 2006; important determinant in the control of cell migration Braconi and Patel, 2008; Budhu et al., 2008; Ladeiro and invasion. From a clinical point of view, our study et al., 2008; Li et al., 2008; Wong et al., 2008). The emphasizes miR-122 as a diagnostic and prognostic significance of miRNAs in cancer onset and progression marker for HCC progression. has been supported by the experimental demonstrations Oncogene (2009) 28, 3526–3536; doi:10.1038/onc.2009.211; of their oncogenic and tumor-suppressive properties published online 20 July 2009 (Esquela-Kerscher and Slack, 2006). In addition, miR- NA genes have been found to be frequently located in Keywords: hepatocellular carcinoma; microRNA; dif- cancer-associated genomic regions, such as fragile sites, ferentiation; metastasis; microarray minimal regions of loss of heterozygosity and minimal regions of amplification (Calin et al., 2004). MicroRNA-122 (miR-122) is the most abundant miRNA in the liver accounting for approximately 70% Introduction of the total miRNA population (Lagos-Quintana et al., 2002; Chang et al., 2004). Although miR-122 has been Hepatocellular carcinoma (HCC) is the third most described as a liver-specific miRNA, slight expression common cause of death from cancer worldwide, has been also reported in the heart (Tang et al., 2007). accounting for at least 600 000 deaths annually (Parkin Several studies have emphasized the importance of miR- et al., 2005). The most prominent etiologies of HCC 122 in liver homeostasis. In vivo, miR-122 has notably include chronic viral hepatitis B and C (HBV, HCV), been implicated in fatty acid and cholesterol metabolism and HCV infection (Jopling et al., 2005; Krutzfeldt Correspondence: Dr SS Thorgeirsson, Laboratory of Experimental et al., 2005; Esau et al., 2006; Chang et al., 2008; Cheung Carcinogenesis, Center for Cancer Research, National Cancer et al., 2008; Elmen et al., 2008; Henke et al., 2008). Institute, National Institutes of Health, 37 Convent Drive, Building Expression of miR-122 has been found to be repressed 37, Room 4146A, Bethesda, MD 20892-4262, USA. E-mail: [email protected] in HCC (Kutay et al., 2006; Murakami et al., 2006; Received 25 January 2009; revised 8 June 2009; accepted 18 June 2009; Gramantieri et al., 2007; Braconi and Patel, 2008; published online 20 July 2009 Budhu et al., 2008; Girard et al., 2008; Ladeiro et al., miR-122 repression is a marker of tumor progression in HCC C Coulouarn et al 3527 2008; Lin et al., 2008) as well as in nonalcoholic the HCC cell lines. Some cell lines including PLC/PRF/5, steatohepatitis (Cheung et al., 2008). Recent works suggest Huh-1 and Hep40 showed very high levels of miR- that miR-122 deregulation is associated either with specific 122, whereas others, such as Hep3B and HepG2, clinical risk factors (HBV, HCV) or HCC metastasis expressed little or did not express miR-122 at all (for (Braconi and Patel, 2008; Budhu et al., 2008; Girard et al., example, SNU387 and SNU398). Specificity and repro- 2008; Ladeiro et al., 2008; Varnholt et al., 2008). ducibility of PCR assays were confirmed by modulating Given the important role of miR-122 in liver the expression of miR-122 in vitro (Supplementary pathology, we aimed (i) to evaluate the diagnostic and Figure 2). We conclude that quantitative reverse prognostic significance of miR-122 expression in human transcription–PCR is a reliable assay for quantification HCC, and (ii) to determine the functional implication of of the miR-122 expression and that HCC cell lines miR-122 deregulation in liver cancer development. By represent a suitable model for functional studies of investigating the expression of miR-122 in 64 human miR-122 in liver cancer. HCC and 28 matched nonneoplastic surrounding liver tissues, we show that miR-122 repression in HCC Loss of miR-122 expression in HCC is associated with correlates with clinically relevant parameters, such as poor prognosis etiology, survival, tumor size and differentiation Next, the expression of miR-122 was analysed in a panel status. Furthermore, we show that the loss of miR-122 of 64 primary liver tumors and 28 matched nontumor expression correlates with distinct gene expression surrounding liver tissues extensively characterized in profiles characteristic of tumor progression (that is, our laboratory earlier (Lee et al., 2004a, 2004b, 2006; suppression of hepatic differentiation phenotype and Kaposi-Novak et al., 2006; Coulouarn et al., 2008). gain of metastatic properties). Finally, we identify liver- HCC samples were representative of patients with enriched transcription factors as central functional various clinical outcomes and pathological features regulators in the gene networks associated with the loss (Table 1). Interestingly, miR-122 levels were highly of miR-122 in human liver cancer and show that miR- variable among the clinical samples (CV>2), suggesting 122 is an important factor in the control of cellular that miR-122 expression in primary tumors was as migration and invasion. heterogeneous as that found in cell lines. To further explore clinical relevance, the expression of miR-122 was analysed as a function of various clinical Results parameters and tumor features (Figure 2). First, the expression levels of miR-122 were compared between Heterogeneous expression of miR-122 in human HCC subtypes characterized earlier by the experimen- HCC-derived cell lines tally well-defined gene expression signatures (Lee et al., Natural properties of miRNAs, including their small 2004a, b, 2006; Kaposi-Novak et al., 2006; Coulouarn size and the lack of polyadenylated tail, have made their et al., 2008). As shown in Figure 2a, miR-122 was quantification challenging. Here, the expression of miR- specifically repressed in the tumors that showed a bad 122 was first determined by real-time quantitative prognosis signature. Accordingly, the overall survival reverse transcription–PCR in 21 HCC-derived cell lines time of the patients with low and high miR-122 (Lee and Thorgeirsson, 2002) using specific sets of expression in HCC was 30.3±8.0 and 83.7±10.3 primers (Figure 1 and Supplementary Figure 1). Inter- months, respectively (Po0.001). MiR-122 was also estingly, the levels of miR-122 were extremely variable repressed in a subset of HCC, which harbored a with more than 1000-fold expression differences among hepatoblast signature as well as a c-Met and a late

10000

1000

100

expression 10 Relative miR-122

1 HLF HLE 7703 Huh-1 Huh-6 Hep40 Hep3B HepG2 FOCUS THLE-2 THLE-3 CHANG SNU423 SNU449 SNU182 SNU475 SNU387 SNU398 SK-Hep1 Hep3B-TR PLC/PRF/5 Figure 1 Relative expression of miR-122 in human hepatocellular carcinoma (HCC)-derived cell lines. Expression of miR-122 was evaluated in 21 human HCC-derived cell lines by quantitative reverse transcription–PCR and normalized to the level of 5s RNA. Data are represented as fold induction relative to the lowest miR-122 expressing SNU-398 cell line using the 2ÀDDCt method. The great heterogeneity of miR-122 expression indicated that HCC-derived cell lines represent excellent natural models for functional studies of miR-122 in liver cancer.

Oncogene miR-122 repression is a marker of tumor progression in HCC C Coulouarn et al 3528 Table 1 Clinical and pathological features of HCC cases with subsets of tumors with poor prognosis and aggressive phenotype (Kaposi-Novak et al., 2006; No. of cases Coulouarn et al., 2008). The loss of miR-122 expression Gender was correlated with a high proliferation and ubiquitina- Female 16 tion index along with a low apoptotic index (Figure 2b). Male 48 Supporting a recent observation (Varnholt et al., 2008), Age the expression of miR-122 was clearly etiology-depen- Median (range) 62 (34–85) dent as miR-122 was repressed only in HCC arising in o60 27 HBV-infected livers (Figure 2c). No significant correla- >60 37 tions (Po0.05) were observed between the status of Survival (months) HBV infection and the presence of hepatoblast, trans- Mean±s.e.m. 34.8±4.9 forming growth factor-b or c-Met signatures. Finally, miR-122 repression was found to be associated with Cirrhosis poor differentiation status and large tumor size No 33 (Figure 2c). Taken together, these data show that Yes 31 miR-122 expression in human HCC correlates with Plasma a-fetoprotein clinically relevant parameters and that the loss of miR- o300 ng/ml 41 122 is a marker of poor prognosis. >300 ng/ml 23 Etiology miR-122 levels define specific gene expression profiles HBV 18 in HCC HCV 13 HBV þ HCV 3 To get insights into the functional significance of miR- ALD 13 122 expression, tumors were divided into two groups. ALD þ viral infectiona 4 High and low miR-122-expressing tumors (15 and 17 Other 2 cases, respectively) were defined by >2-fold changes in Unknown 11 the median miR-122 expression across the samples. Tumor size Gene expression profiling identified 509 nonredundant o5cm 34 genes that were differentially expressed between the >5 cm 30 tumors with high versus low miR-122 expression (Supplementary Table 1). As expected, clustering Edmondson–Steiner grade Grade 1 1 analysis divided HCC into two distinct clusters (C1 Grade 2 26 and C2) defined by the level of miR-122 (Figure 3a). Grade 3 33 Differentially expressed genes were similarly organized Grade 4 4 in two clusters and included genes that displayed either positive (cluster A) or negative (cluster B) correlation Vascular invasion No 14 with miR-122 expression (Figure 3a). Supporting our Yes 29 approach, cluster B included numerous predicted targets for miR-122 (for example, ALDOA, CLIC1 and Gene signaturesb RHOA). The specific contribution of miR-122 to the Good prognosis 40 Bad prognosis 24 HCC transcriptome was further validated by testing the enrichment of the genes embedded in clusters A and B in Hepatocyte 49 the gene expression profiles derived from the experi- Hepatoblast 15 mental modulation of miR-122 levels (Krutzfeldt et al., 2005). As shown in Figure 3b, genes highly expressed in c-Met negative 45 c-Met positive 19 clusters A and B (high and low miR-122 expressing HCC) were significantly enriched in the gene expression TGF-b early 12 profiles corresponding to either control or mice treated TGF-b late 14 with antagomir-122, respectively (Krutzfeldt et al., 2005). In addition, the restoration of miR-122 expres- Abbreviations: ALD, alcoholic liver disease; HBV, hepatitis B; HCC, hepatocellular carcinoma; HCV, hepatitis C; TGF-b, transform- sion in vitro to a large extent was able to shift the ing growth factor-b. expression patterns of cluster A and B genes from C2 aEither HBV or HCV infection. (Low miR-122) toward C1 (High miR-122) (Supple- bFrom our previously published HCC stratification (Lee et al., 2004a, mentary Figure 3). These results show that the expres- 2006; Kaposi-Novak et al., 2006; Coulouarn et al., 2008). sion of genes embedded in clusters A and B is highly dependent on miR-122 expression. transforming growth factor-b signature (Figure 2a). The hepatoblast signature, derived from hepatic progenitor Loss of miR-122 in HCC coincides with suppression of the cells, is specific for tumors with a poor differentiation hepatic phenotype status (Lee et al., 2006), whereas c-Met and late As shown in Figure 3a, the gene expression profiles were transforming growth factor-b signatures are associated remarkably homogeneous within each subgroup of

Oncogene miR-122 repression is a marker of tumor progression in HCC C Coulouarn et al 3529 Prognosis signature Cell origin signature c-Met signature TGF-β signature

Good Hepatocyte Negative Early Bad Hepatoblast Positive Late ns 0.001 ns 0.001 ns 0.001 ns 0.003 2 2 2 2 1.5 1.5 1.5 1.5 1 1 1 1 0.5 0.5 0.5 expression 0.5

Relative miR-122 0 0 0 0 ST HCC ST HCC ST HCC ST HCC

Proliferation index Apoptotic index Ubiquitination index

Low High Low High Low High ns 0.001 ns 0.001 ns 0.039 1.5 1.5 2

1 1 1.5 1 0.5 0.5 0.5 expression

Relative miR-122 0 0 0 ST HCC ST HCC ST HCC

Etiology Edmonson Grade Tumor size

HBV HCV1+2 3+4< 7cm > 7cm ns 0.007 ns 0.112 ns 0.011 2.5 2.5 2.5 2 2 2 1.5 1.5 1.5 1 1 1

expression 0.5 0.5 0.5

Relative miR-122 0 0 0 ST HCC ST HCC ST HCC Figure 2 Repression of miR-122 correlates with clinically relevant parameters in human hepatocellular carcinoma (HCC). Expression of miR-122 was evaluated by quantitative reverse transcription–PCR in 64 human HCC and 28 nontumor surrounding liver tissues (ST). (a) Expression of miR-122 was evaluated in ST and HCC tissues as a function of experimentally well-deﬁned gene expression signatures (described by Lee et al., 2004a, 2006; Kaposi-Novak et al., 2006; Coulouarn et al., 2008). Data indicated that miR-122 was speciﬁcally repressed in HCC with bad prognosis that harbors hepatoblast, c-Met and late transforming growth factor (TGF)-b gene expression signatures. (b) The assessment of miR-122 expression as a function of biochemical parameters indicated that miR-122 was particularly repressed in HCC with a high proliferation and ubiquitination index, and low apoptotic index (Lee et al., 2004b). (c) The evaluation of miR-122 expression as a function of etiology, Edmonson grade and tumor size indicated that miR-122 was repressed in HCC arising from HBV-infected livers and was associated with poorly differentiated and large-size tumors.

HCC, suggesting that miR-122 deregulation represents global activation of an extensive regulatory network an important event during the course of HCC progres- centered on HNF4A (Figure 4a). Enrichment of sion. To reveal the dominant signaling networks HNF4A targets among miR-122-positive HCC suggests associated with the loss of miR-122 expression in the involvement of miR-122 in maintaining hepatocyte HCC, systematic pathway and gene ontology analyses differentiation, thereby implying that the loss of miR- were performed using genes that either positively 122 contributes to suppression of the hepatic phenotype (cluster A) or negatively (cluster B) correlated with in HCC with poor prognosis (Figure 2). miR-122 expression. Ingenuity pathway analysis showed a clear relationship between the miR-122 associated gene expression signature and the functional categories Relationship between miR-122 and liver-enriched linked to liver cancer and liver metabolism (Table 2). transcription factors Genes embedded in cluster A were implicated in As a paradigm, HCC-derived cell lines were divided into molecular and cellular functions characteristic of well- two groups based on the miR-122 expression levels. differentiated hepatocytes (Table 2 and Supplementary Gene expression profiling identified 300 genes that were Table 1). These genes were involved in lipid metabolism differentially expressed as a function of miR-122 (APO-A1, -C4, -F), acute phase response (ITIH3, expression (Supplementary Figure 4). Consistent with SERPIN-C1, -F2), detoxification (CYP-2C9, -3A4, our findings in primary tumors, high levels of miR-122 4A11) and coagulation (F12, F13B). Moreover, high in HCC-derived cell lines were correlated with high expression of miR-122 correlated significantly with the expression of genes involved in the pathways character-

Oncogene miR-122 repression is a marker of tumor progression in HCC C Coulouarn et al 3530

Figure 3 MicroRNA-122 (miR-122) expression defines specific gene expression profiles in human hepatocellular carcinoma (HCC). (a) Hierarchical cluster analysis of 509 genes differentially expressed in liver primary tumors as a function of miR-122 expression. Data are presented in a matrix format in which rows and columns represent genes and HCC samples, respectively. Differentially expressed genes are grouped in two clusters, A and B. HCC samples cluster in two groups, C1 and C2, which greatly differ in terms of miR-122 level (C1: 2.7±0.4; C2: 0.6±0.4; mean±s.e.m; Po0.001). Genes included in cluster A and cluster B are up- and downregulated in cluster C1 HCC, respectively. As expected, cluster B includes several predicted miR-122 target genes (*). (b) Gene set enrichment analysis using the genes embedded in the clusters A and B as signatures and the gene expression profiles derived from the experimental inhibition of miR-122 in mice (Krutzfeldt et al., 2005). Cluster A and B genes are significantly enriched in the profiles corresponding to control mice (enrichment score (ES) ¼ 0.578, Po0.001), and the mice treated with antagomir-122 (ES ¼ 0.658, Po0.001), respectively.

Table 2 Ingenuity pathway analysis of genes correlated with miR-122 HNF3A, HNF3B, HNF4A, HNF4G and HNF6 expression in hepatocellular carcinoma (Supplementary Figure 5). Gene network analysis not Positive correlation Negative correlation only confirmed activation of the HNF4A signature in HCC-derived cell lines expressing miR-122 but also Disease and disorders revealed strong interactions with other liver-enriched 6 9 Hepatic system disease (Po10À ) Cancer (Po10À ) transcription factors (Figure 4b). Molecular and cellular functions In this context, HNF4A was directly evaluated as a Lipid metabolism (Po10À9) Cell cycle (Po10À5) function of miR-122 expression. As shown in Figures Amino-acid metabolism Cell assembly and org. (Po10À5) 4c–e, HNF4A expression was directly correlated with (Po10À3) the expression of miR-122, both on the mRNA and Physiological system development and functions protein levels. Furthermore, in the high miR-122 Tumor morphology (Po10À3) Immune response (Po10À3) expressing cell lines, HNF4A was particularly enriched Embryonic development Connective tissue development in the nuclear fraction compared with the cytoplasmic (Po10À3) (Po10À3) fraction, reflecting its greater transcriptional activity À3 Tumor morphology (Po10 ) (Figure 4e). The latter is consistent with a central role of Top canonical pathways HNF4 revealed by the network analysis in the miR-122 Fatty acid metabolism Actin cystosk. Signaling overexpressing HCC cell lines (Figure 4b). Despite a (Po10À10) (Po10À4) strong positive correlation, the exact relationship Xenobiotic metabolism Hypoxia signaling (Po10À3) À5 between miR-122 and HNF4A appears to be highly (Po10 ) 0 5 complex. The lack of miR-122 seed region in the 3 UTR Coagulation system (Po10À ) FXR/RXR activation (Po10À4) of HNF4A transcript makes HNF4A an unlikely direct target of miR-122. Accordingly, the experimental Top Tox functions: hepatotoxicity modulation of miR-122 expression did not affect 6 Liver cholestasis (Po10À ) Hepatocellular carcinoma HNF4A mRNA and protein levels (data not show). It (P 10À3) o is also dubious whether HNF4A directly drives the expression of miR-122, as the silencing of HNF4A by small interfering RNA did not significantly change the expression of miR-122 (Figure 5). Conversely, the istic of fully differentiated hepatocytes (Supplementary knockdown of HNF1A, HNF3A or HNF3B was able Table 2). These included genes encoding apolipoproteins to reduce miR-122 expression, implying that mir-122 (APO-A1), coagulation factors (F2) and acute phase may be under the transcriptional control of these proteins (AHSG). More importantly, miR-122 showed a transcription factors (Figure 5). These results provide strong positive correlation with the expression level of new insight into the transcriptional regulation of the numerous liver-enriched transcription factors essential miR-122 gene and implicate miR-122 as a marker of for hepatocytic differentiation, such as HNF1A, hepatocyte differentiation.

Oncogene miR-122 repression is a marker of tumor progression in HCC C Coulouarn et al 3531

Figure 4 High level of miR-122 in hepatocellular carcinoma (HCC) defines a specific gene expression profile linked to HNF4A. (a, b) Ingenuity pathway analysis applied to the genes whose expression was positively correlated with the expression of miR-122 in human primary HCC (a) and HCC-derived cell lines (b). Liver-enriched transcription factors, HNF4A in particular, were identified as the central regulators in the gene signature associated with a high miR-122 expression. (c–e) In HCC-derived cell lines, the loss of miR-122 is associated with a loss of HNF4A. (c) The evaluation of HNF4A mRNA level in HCC-derived cell lines indicated a positive correlation between HNF4A and miR-122. (d) The assessment of HNF4A expression in protein extracts isolated from the cell lines that show high versus low miR-122 further indicates a positive correlation between miR-122 and HNF4A expression. High miR-122 expressing cell lines, such as PLC/PRF/5, Huh-1, Hep40 or Huh-6, show considerably higher HNF4A protein levels compared with SNU-182, SNU-387 and SNU-398 lines, which show low miR-122levels. (e) Analysis of HNF4A expression in cytoplasmic (C) and nuclear (N) compartments indicates a nuclear enrichment of HNF4A in the high miR-122 expressing cell lines.

Loss of miR-122 coincides with acquisition of invasive expression during tumor progression and the potential phenotype relevance of miR-122 restoration for HCC therapy. Lastly, we analysed the genes that were negatively correlated with miR-122 (cluster B in Figure 3a). Expression profiling indicated that the loss of miR-122 expression was associated with a higher expression of Discussion numerous genes related to metastasis, including genes involved in cell motility (RAC1, RHOA), angiogenesis Accumulating evidence suggests a causal role for (VEGF), hypoxia (HIF1A) and epithelial–mesenchymal miRNAs in human cancer (Esquela-Kerscher and Slack, transition (vimentin) (Supplementary Table 1, Supple- 2006). In this study, we have evaluated the clinical and mentary Figure 6). It is noted that RHOA and RAC1 functional relevance of liver-specific miR-122 for human were two central molecules in a network associated with HCC. We show that the repression of miR-122 cell motility (Figure 6a). Therefore, we investigated the correlates with clinically relevant parameters, such as impact of miR-122 loss on cell motility. As shown in etiology, tumor size and differentiation grade. More Figure 6b, the experimental inhibition of miR-122 in importantly, miR-122 repression is specific for liver Huh-1 cells resulted in an increase of migration and tumors, which are characterized by poor prognosis, and invasion. Furthermore, by validating the importance of coincides with suppression of hepatic phenotype as well miR-122 in the control of cell motility, the ectopic as gain of metastatic properties. expression of miR-122 was able to reduce the migration Our findings show a link between the expression levels and invasion of SK-Hep1 cells (Figure 6c). These data of miR-122 and HBV or HCV infection. By analysing emphasize the critical role of the loss of miR-122 the expression of miR-122 in a cohort of HCC with

Oncogene miR-122 repression is a marker of tumor progression in HCC C Coulouarn et al 3532 different etiology, we have established that the down- of HCV replication has been found to be closely regulation of miR-122 occurs mainly in the HCC arising correlated with the miR-122 expression levels (Jopling in the HBV-infected livers. In the HCC developed with et al., 2005; Chang et al., 2008), and further studies have HCV infection, miR-122 expression was either main- implicated miR-122 in HCV translation, suggesting the tained or increased, in agreement with recent observa- involvement of miR-122 in HCV tropism to the liver tions (Varnholt et al., 2008). Interestingly, the efﬁciency (Henke et al., 2008). Therefore, we speculate that HCV not only requires the host-expressed miR-122 for its replication, but also it may either directly induce miR-122 expression or indirectly inhibit miR-122 downregulation. P<0.05 P<0.01 P<0.05 P<0.01 Similar to the transcription factors, tissue speciﬁcity 1.2 of miRNA expression may imply a role in differentia- 1 tion. Owing to its high abundance in the liver, it has 0.8

0.6

0.4 mRNA level 0.2

1.2 P<0.05 P<0.01 P<0.05 ns

0.8 * * * 0.6

0.4 miR-122 level 0.2

0 HNF1AHNF3A HNF3B HNF4A

NC siRNA Figure 5 The silencing of HNF1A, HNF3A and HNF3B results in a decrease of miR-122 level. The high miR-122 expressing PLC/PRF/5 cell line was transfected with 40 nM of specific small interfering RNA (siRNA) directed against HNF-1A, -3A, -3B and -4A (siRNA, black column) versus negative control (NC, white column). Forty-eight hours after the transfection, the efficiency of the silencing was confirmed by the decreased level of the respective mRNA (upper panel). As shown in the lower panel, the silencing of HNF-1A, -3A and -3B significantly reduces the level of miR-122.

Figure 6 MicroRNA-122 (miR-122) affects the propensity of cancer cells to migrate and invade. (a) Ingenuity pathway analysis was performed on the genes upregulated in hepatocellular carcinoma samples showing a low miR-122 expression (cluster B in Figure 3). This procedure revealed a network associated with cell motility and identiﬁed RAC1 and RHOA as two central regulators. (b) Inhibition of miR-122 results in the acquisition of migrating and invasive properties. The effect of miR-122 inhibition on cellular migration and invasion was assessed in Huh-1 by transfecting 20 nM of either anti-miR-122 (black column) or negative control (NC-Anti-miR, white column). Invasive and migrating activity was measured by using the BD BioCoat Matrigel Invasion Chambers. Data indicate that the loss of miR-122 increases the migrating and invasive properties of Huh-1 cells. (c) Conversely, in the miR-122 negative SK-Hep1 cell line, the restoration of miR-122 by transfecting miR-122 precursor molecules results in a decreased migration and invasion (Pre-miR-122, black column) when compared with cells transfected with negative control (NC-Pre-miR, white column).

Oncogene miR-122 repression is a marker of tumor progression in HCC C Coulouarn et al 3533 been suggested that miR-122 may maintain the hepatic Supporting these data, our results show that HCC differentiation status by repressing the genes that are cell lines known to exhibit a more invasive pheno- normally not expressed by hepatocytes (Girard et al., type also displayed a decreased miR-122 expression 2008). The temporal expression pattern of miR-122 (Coulouarn et al., 2008). In addition, a significant during liver development is consistent with its involve- enrichment of genes related to metastasis (RHOA, ment in liver differentiation (Chang et al., 2004). In VEGF, HIF1A, Vimentin) was observed both in HCC support of this notion, recent work showed that the and cell lines lacking miR-122 expression (Supplemen- promotion of endodermal differentiation of human tary Table 1). Accordingly, we provide direct evidence embryonic stem cells by NaButyrate was associated that the modulation of miR-122 greatly influenced the with an increase of miR-122 expression and a parallel propensity of cancer cells to migrate and invade. Indeed, activation of several hepatocyte-specific genes (Tzur the loss of miR-122 resulted in the acquisition of et al., 2008). According to our findings in human HCC migrating and invasive properties and the restoration and HCC-derived cell lines, the expression of miR-122 of miR-122 was able to reverse this phenotype. In an was tightly correlated with the expression of numerous attempt to identify the underlying molecular mechan- genes characteristic of the differentiated hepatocytes. isms responsible for the impact of miR-122 on cell In agreement with the current view of miR-122 as a motility, we used miRNA target prediction algorithms regulator of lipid and cholesterol metabolism, the on genes negatively correlated with miR-122. Interest- expression of abundant apolipoprotein genes was found ingly, an evolutionary conserved binding site for to be correlated with miR-122 levels (Krutzfeldt et al., miR-122 was detected in the 30 UTR of RHOA mRNA 2005; Esau et al., 2006; Elmen et al., 2008). The specific (Supplementary Figure 8a), and the analysis of downregulation of miR-122 in poorly differentiated the secondary structure of RHOA 30 UTR indicated tumors as well as in tumors characterized by the that the predicted binding site coincided with a hairpin- hepatoblast gene signature supported miR-122 as an loop structure, making this sequence probably access- important determinant of hepatic differentiation. More- ible for an interaction with miR-122 (Supplementary over, in two independent data sets, we identified Figure 8b). RHOA is frequently overexpressed in HCC HNF4A as the central regulatory transcription factor and several groups correlated the increase of RHOA in the network of genes that were differentially expression with tumor progression, cell differentiation expressed as a function of miR-122 expression. HNF4A and metastasis (reviewed by Grise et al., 2009). More- is a liver-enriched transcription factor controlling the over, RHOA overexpression in liver has been directly differentiation-dependent gene expression in developing correlated with the acquisition of an aggressive pheno- and adult livers (Odom et al., 2004). Although we type, both in vitro and in vivo (Yoshioka et al., 1995; initially speculated that HNF4A was the missing link Xue et al., 2008). In addition, gene reporter experiments between the differentiation status and the transcription suggested a direct functional interaction between miR- of miR-122 gene, the additional gene-silencing experi- 122 and RHOA 30 UTR (Supplementary Figure 8c). ments suggested a more important role for other Nevertheless, the experimental inhibition or induction of liver-enriched transcription factors, such as HNF1A, miR-122 failed to show a consistent modulation of HNF3A and HNF3B. The promoter region of miR-122 RHOA on a protein level. Also, the acquisition of gene has not been yet identified but a binding site for invasive properties observed on miR-122 inhibition HNF3B has been located 5 kb upstream of miR-122 (Figure 6b) appeared to be independent of RHOA, as (Supplementary Figure 7). Although further experi- the silencing of RHOA did not significantly abrogate ments are needed to address the functionality of this this response. The absence of RHOA modulation on a binding site, our data provide the first evidence for the protein level could be due to an enhanced stability of the tissue specificity of miR-122 expression. protein. Alternatively, RHOA transcript may be tightly Using a comparative genomic approach based on regulated by positive and negative signals. For example, the experimentally well defined gene expression signa- the increase in the oncogenic miR-155, which was tures, we have identified earlier several clinically relevant recently reported to target and reduce the level of subtypes of HCC with significant differences in the RHOA (Kong et al., 2008), may counteract the miR-122 biological properties and clinical outcome (Lee et al., effect. These observations raise important questions 2004a, b, 2006; Kaposi-Novak et al., 2006; Coulouarn regarding the regulation of transcript stability by et al., 2008). Importantly, we showed here that the miRNAs and suggest that the integration of activating repression of miR-122 was characteristic of HCC, which and inhibitory signals driven by different miRNAs may displayed a hepatoblast, c-Met or late transforming define the level of a particular transcript. Interestingly, growth factor-b signature. These HCC subtypes were while writing this paper, Tsai et al. (2009) published characterized by increased invasiveness, tumor recur- that miR-122 regulates intrahepatic metastasis by rence and decreased patient survival whereby raising a controlling the disintegrin and metalloprotease possibility that the loss of miR-122 may predispose to ADAM17. Together, these data not only emphasize the acquisition of invasive phenotype. Consistent with the critical role of miR-122 in migration and invasion this, miR-122 was recently described as a metastasis- but also suggest that miR-122 contribution to tissue related miRNA in HCC (Budhu et al., 2008), and remodeling is broader than a direct effect on actin repression of miR-122 was reported in highly metastatic cytoskeleton organization through a RHOA-dependent human breast cancer cell lines (Tavazoie et al., 2008). mechanism.

Oncogene miR-122 repression is a marker of tumor progression in HCC C Coulouarn et al 3534 In conclusion, our work sheds new light on the clinical DNA microarray analysis and functional implications of miR-122 expression in liver The gene expression data used in this study (GEO accession cancer. We identified miR-122 as a new diagnostic and numbers: Human microarray platform, GPL1528; human prognostic miRNA marker for HCC progression. Given HCC microarray data, GSE1898) were generated using a that the modulation of miR-122 expression became a genome-wide set of long-mer human oligonucleotides obtained from Illumina (San Diego, CA, USA) and printed reality in animal models (Czech, 2006; Elmen et al., 2008), at the Advanced Technology Center (National Cancer our study provides a rationale for the classification and Institute). Target preparation and hybridization on micro- development of novel therapy for human HCC. arrays were described earlier (Lee and Thorgeirsson, 2002). Differentially expressed genes were identified by a univariate two-sample t-test with a random variance model. Permutation Materials and methods P-values for significant genes (Po0.01) were computed on the basis of 10 000 random permutations as described earlier Human tissue samples (Coulouarn et al., 2006). Expression profiles were clustered by A total of 64 HCC tissues and 28 matched nontumor the Cluster 3.0 program (http//rana.lbl.gov/EisenSoftware. surrounding liver tissues were obtained from patients under- html) using an unsupervised hierarchical procedure and going partial hepatectomy as treatment for HCC. Tissue visualized with the TreeView program (http//rana.lbl.gov/ banking was approved by the Institutional Review Board of EisenSoftware.html) (Coulouarn et al., 2006). the US National Cancer Institute. The determination of proliferation, apoptosis and ubiquitination index was described earlier (Lee et al., 2004b). Total RNAs were isolated Gene set enrichment analysis and pathway analysis using the CsCl density gradient centrifugation method Gene set enrichment analysis was performed using the web- (Coulouarn et al., 2006). based tool developed by Broad Institute (www.broad.mit.edu/ gsea/). Enrichment scores were determined after 1000 permutations. The Ingenuity pathway analysis tool was used to Cell lines examine the functional associations between differentially HCC-derived cell lines were obtained from American Type expressed genes and to generate the highest significant gene Culture Collection (ATCC, Rockville, MD, USA) and networks (www.ingenuity.com). Biologically relevant networks RIKEN Cell Bank (Ibaraki, Japan) and maintained in were identified using the scoring system provided by Ingenuity Dulbecco’s modified Eagle’s medium/F12 medium supplemen- (Mountain View, CA, USA). ted with 2 mM glutamine and 10% fetal bovine serum, as described (Lee and Thorgeirsson, 2002). Total RNAs were extracted from cells at 80% confluence using the RNeasy mini miRNA target prediction kit (Qiagen, Valencia, CA, USA). Potential miR-122 target among genes negatively correlated with miR-122 expression was determined using the publicly available miRanda (www.microrna.org) and TargetScan Cell transfection (www.targetscan.org) algorithms. The secondary structure Transfections were carried out using siPORT NeoFX accord- of potential target transcripts was explored using the ing to the manufacturer’s instructions (Ambion, Austin, TX, web-based RNA analyser tool (www.wb2x01.biozentrum. USA). RNA and protein analysis were conducted after 48 h uni-wuerzburg.de). incubation in normal culture condition. miRIDIAN miRNA Hairpin Inhibitors and Mimics from Dharmacon (Waltham, MA, USA) were used for the specific inhibition and over- RHOA 30 UTR reporter analysis expression of miR-122. The amount of small RNA was The human sequence of the RHOA 30 UTR was amplified adjusted to a final concentration of 20 nM. The efficiency and by PCR using Phusion high-fidelity DNA polymerase specificity of the transfections were evaluated by confocal (New England Biolabs, Ipswich, MA, USA) and cloned into a microscopy and quantitative reverse transcription–PCR (Sup- SacI- and HindIII-cut pMIR-Report luciferase vector plementary Figure 9). Silencer select small interfering RNAs (Ambion). A mutant construct was generated by introducing along with the negative control no. 1 (Ambion) were used mutations in the putative miR-122-binding site. Mutations for the specific knockdown of HNF1A (s13870), HNF3A were introduced by stand overlap PCR using mutagenic (s6689), HNF3B (s6692), HNF4A (s6698), FOXO1 (s5259) primers. All constructs were sequenced to confirm that the and RHOA (s760). desired mutations had been obtained. Firefly luciferase constructs containing the wildtype or the mutant variant of RHOA 30 UTR were co-transfected with a b-galactosidase Real-time reverse transcription–PCR of miR-122 control vector in the presence of 20 nM miR-122 inhibitor or Expression of miR-122 was measured using the mirVana negative control. Transfections were performed with 0.1 mg quantitative reverse transcription–PCR miRNA Detection Kit firefly luciferase constructs and 0.01 mg b-galactosidase nor- and SYBR Green I (Ambion). Total RNA was reverse malization vector using Lipofectamine 2000 (Invitrogen, transcribed at 37 1C for 30 min with an ArrayScript Enzyme Carlsbad, CA, USA). Luciferase and b-galactosidase activities mix including miR-122 specific RT primers according to the were assessed 48 h after transfection using a dual-light manufacturer’s instructions. Quantitative PCR analysis was chemiluminescent reporter gene assay system (Applied Biosys- performed with an IQ-5 real-time PCR detection system (Bio- tems, Foster City, CA, USA). Rad Laboratories, Hercules, CA, USA) in a 96-well reaction plate. Melting analysis of the PCR products was conducted to validate the amplification of the specific product. Quantitative Invasion and migration assays ÀDDCt values were determined using the 2 method, where the DCt The effect of miR-122 on invasion and migration was value of a given RNA sample was calculated by subtracting the evaluated by using the BD BioCoat Matrigel Invasion Ct value of miR-122 from the Ct value of 5S small RNA used Chambers (San Jose, CA, USA) as described (Coulouarn for normalization. et al., 2008).

Oncogene miR-122 repression is a marker of tumor progression in HCC C Coulouarn et al 3535 Immunoblotting Abbreviations Protein expression analysis was as described earlier (Cou- louarn et al., 2006). Protein extraction was performed using HCC, hepatocellular carcinoma; miRNA, microRNA. M-PER and NE-PER reagents (Pierce, Rockford, IL, USA). Twenty micrograms of protein extract were subjected to SDS– polyacrylamide gel electrophoresis and transferred to poly- Conflict of interest vinylidene difluoride membranes. Membranes were probed with a goat polyclonal HNF4A antibody and a rabbit The authors declare no conflict of interest. polyclonal RHOA antibody (Santa Cruz Biotechnology, Santa Cruz, CA, USA). Detection was performed by ECL Acknowledgements (Amersham Biosciences, Piscataway, NJ, USA) after incubation of an horseradish peroxidase-conjugated secondary anti- This project was supported in part by the Intramural Research body (Santa Cruz Biotechnology). Program of the Center for Cancer Research.

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Supplementary Information accompanies the paper on the Oncogene website (http://www.nature.com/onc)

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