A Novel GSK-3 beta–C/EBP alpha–miR-122–InsulinLike Growth Factor 1 Receptor Regulatory Circuitry in Human Hepatocellular Carcinoma Chunxian Zeng,1 Ruizhi Wang,1 Daochuan Li,2 Xue-Jia Lin,1 Qing-Kun Wei,1 Yunfei Yuan,3 Qing Wang,2 Wen Chen,2 and Shi-Mei Zhuang1 miR-122 is a highly abundant, hepatocyte-specific microRNA. The biomedical significance and regulatory mechanisms of miR-122 remain obscure. We explored the role of miR-122 in tumorigenesis in the context of gene regulatory network. The miR-122 promoter and its transactivator were identified by way of luciferase reporter system, electrophoretic mobility shift, and chromatin immunoprecipitation assays. The miR-122 regulatory circuitry and its implication in hepatocarcinogenesis were identified using livers of different development stages, human hepatocellular carcinoma (HCC) tissues and cell lines, and aflatoxin B1 (AFB1)-transformed cells. We characterized the 25.3 to 24.8 kb region upstream of miR122 precursor as miR-122 promoter. Further investigation revealed that deletion of predicted CCAAT/enhancer-binding protein alpha (C/EBPa) binding sites C/EBPa knockdown significantly reduced miR-122 promoter activity and endogenous miR-122 expression; and C/ EBPa directly interacted with the miR-122 promoter in vitro and in vivo. These data suggest that C/EBPa is a transactivator for miR-122 transcription. We further demonstrated that miR-122 suppressed insulin-like growth factor 1 receptor (IGF-1R) translation and sustained glycogen synthase kinase-3 beta (GSK-3b) activity. The activated GSK-3b not only repressed cell proliferation, but also activated C/EBPa, which maintained miR-122 levels and thereby enforced IGF-1R suppression. Interestingly, down-regulation of miR-122 and C/EBPa, and up-regulation of IGF-1R were frequently observed in HCC tissues, and decreased miR-122 levels were associated with worse survival of HCC patients. Moreover, AFB1 exposure resulted in decreased activity in GSK-3b, C/EBPa, and miR-122 and increased levels of IGF-1R, whereas restoration of miR-122 suppressed the tumorigenicity of HCC and AFB1-transformed cells. Conclusion: We have identified a novel GSK-3b–C/EBPa–miR-122–IGF-1R regulatory circuitry whose dysfunction may contribute to the development of HCC. Our findings provide new insight into miR-122’s function and the mechanisms of hepatocarcinogenesis. (HEPATOLOGY 2010;52:1702-1712)
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icroRNAs (miRNAs) repress the expression of protein-coding genes through binding to the 30 untranslated region (UTR) of target messenger RNAs (mRNAs).1 An understanding of the roles of miRNA in tumorigenesis is emerging. Aberrant expression of miRNAs has been observed in both benign and malignant tumors. miRNA expression sig-
natures can reflect the developmental lineage of human tumors and the clinical behaviors of cancers. Deregulation of miRNAs may confer cells with malignant phenotypes, including uncontrolled cell proliferation, resistance to apoptosis, and the capability of invasion and metastasis.2-4 However, most of the published studies that explore the implication of miRNA in
Abbreviations: AFB1, aflatoxin B1; C/EBPa, CCAAT/enhancer-binding protein alpha; ChIP, chromatin immunoprecipitation; EGFP, enhanced green fluorescent protein; EMSA, electrophoretic mobility shift assay; GSK-3b, glycogen synthase kinase-3 beta; HCC, hepatocellular carcinoma; IGF-1R, insulin-like growth factor 1 receptor; IgG, immunoglobulin G; miRNA, microRNA; mRNA, messenger RNA; NC, negative control; pre–miR-122, miR-122 precursor; qPCR, quantitative real-time polymerase chain reaction; RT-PCR, reverse-transcription polymerase chain reaction; siRNA, small interfering RNA; TSS, transcription start site; UTR, untranslated region. From the 1Key Laboratory of Gene Engineering of the Ministry of Education, State Key Laboratory of Biocontrol, School of Life Sciences; the 2Department of Toxicology, School of Public Health; and the 3Department of Hepatobiliary Oncology, Cancer Center, Sun Yat-sen University, Guangzhou, People’s Republic of China. Received May 20, 2010; accepted July 19, 2010. Supported by grants from the Ministry of Science and Technology of China (2005CB724600, 2010CB912803), the National Natural Science Foundation of China (30925036, 30630055, and 30925029), and the Ministry of Health of China (2008ZX10002-019). 1702
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tumorigenesis have focused on identifying individual targets of miRNA rather than elucidating miRNA function in the context of gene regulatory network. As in other solid tumors, multiple genetic and epigenetic changes in protein-coding genes have been described in hepatocellular carcinoma (HCC).5 However, the evidence accumulated so far cannot explain the full complexity of HCC. Recent studies suggest that dysfunction of miRNAs also contributes to HCC development.6-10 miR-122 is a hepatocyte-specific miRNA that facilitates the replication of hepatitis C virus11 and regulates the metabolism of lipids12 and the expression of hepatic circadian genes.13 Recently, few targets of miR122, including cyclin G1, serum response factor, insulinlike growth factor 1 receptor (IGF-1R), a disintegrin and metalloprotease family-10 and -17, have been experimentally validated.14-16 Frequent miR-122 downregulation is observed in HCC tissues.7,14-16 To date, neither the mechanisms underlying miR-122 deregulation nor the regulatory networks of miR-122 have been investigated. In an attempt to explore the role of miR122 in tumorigenesis in the context of gene regulatory network, we identify the promoter region of miR-122, reveal that CCAAT/enhancer-binding protein alpha (C/ EBPa) directly transactivates miR-122 transcription, and demonstrate that decreased C/EBPa activity represents an important mechanism responsible for miR-122 deregulation in HCC. We further disclose a novel glycogen synthase kinase-3 beta (GSK-3b)-C/EBPa-miR-122IGF-1R regulatory circuitry whose dysfunction is implicated in HCC development.
Patients and Methods Databases used for bioinformatic analysis and details about reagents and experimental procedures can be found in the Supporting Information Materials and Methods. Patients and Human Specimens. Normal liver tissues were obtained from patients undergoing resection of hepatic hemangiomas, and paired HCC and adjacent nontumor liver tissues were obtained from patients undergoing HCC resection at the Cancer Center at Sun Yat-sen University. All tissues were obtained from the Cancer Center’s Bank of Tumor Resources. Both tumor and nontumor tissues were his-
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tologically confirmed. No local or systemic treatment had been conducted before surgical procedure. After surgical resection, no other anticancer therapy was administered before relapse. Patient characteristics are provided in Supporting Information Table 1. Informed consent was obtained from each patient, and the study was approved by the Institute Research Ethics Committee at the Sun Yat-sen University Cancer Center. Cell Lines. The cell lines used were HEK293T, the HCC cell lines HepG2 and Huh-7, the immortalized human fetal liver cell line L02 and its aflatoxin B1 (AFB1)-transformed subline L02-T,17 and the miR-122 stably expressing cell line HepG2-miR-122 and its control line HepG2-Mock. Animal Studies. All mouse experiments were approved by the Institutional Animal Care and Use Committee at School of Life Sciences at Sun Yat-sen University. Experimental procedures were performed in accordance with the Guide for the Care and Use of Laboratory Animals (NIH Publication No. 80-23, revised 1996) and were performed according to the institutional ethical guidelines for animal experiments. BALB/c nude mice were employed for tumorigenicity analysis. Livers from fetus, postnatal, and adult C57BL/6 mice were collected. Oligonucleotides and Plasmids. We used the following miRNA and small interfering RNA (siRNA) oligonucleotides (Genepharma, Shanghai, China): miR-122 mimics; miR-122 harboring a mutated seed region; siIGF-1R, siC/EBPa, and siGSK-3b that targeted human IGF-1R (3192-3212 nt, GeneBank accession no. NM_000875.3), C/EBPa (819-839 nt, GeneBank accession no. NM_004364.2), and GSK-3b (1816-1836 nt, GeneBank accession no. NM_001146 156.1) transcripts, respectively; and a negative control (NC) RNA duplex for both miRNA and small interfering RNA (siRNA). The sequence-specific miR-122 inhibitor (anti–miR122) and its control (anti–miR-NC) were obtained from Dharmacon (Lafayette, CO). All oligonucleotide sequences are listed in Supporting Information Table 2. We used the following plasmids: firefly luciferase reporter plasmids for verifying miR-122–targeted 30 -UTR and the miR-122 promoter region; pSi-shC/EBPa that expressed siRNA targeting 222240 nt of C/EBPa (GeneBank accession no. NM_004364.2); pc3-gab-C/ EBPa, pc3-gab-C/EBPa-T222A/T226A, and p3XFLAG-
Address reprint requests to: Shi-Mei Zhuang, School of Life Sciences, Sun Yat-sen University, Xin Gang Xi Road #135, Guangzhou 510275, P. R. China. E-mail:
[email protected] or
[email protected]; fax: (86)-20-84112169. C 2010 by the American Association for the Study of Liver Diseases. Copyright V View this article online at wileyonlinelibrary.com. DOI 10.1002/hep.23875 Potential conflict of interest: Nothing to report. Additional Supporting Information may be found in the online version of this article.
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GSK-3b-S9A that expressed wild-type C/EBPa, T222A/ T226A mutant C/EBPa, and S9A mutant GSK-3b, respectively; and retrovirus vector (pRetroX–miR-122) for expressing the miR-122 precursor (pre–miR-122). The pSi-shC/EBPa, pc3-gab-C/EBPa, and pc3-gab-C/ EBPa-T222A/T226A vectors also expressed enhanced green fluorescent protein (EGFP). Cell Transfection and Infection. RNA oligonucleotides were transfected using Lipofectamine RNAiMAX. A final concentration of 50 nM duplex or 200 nM miRNA inhibitor was used unless indicated. RNA transfection efficiency is approximately 70%-80%10 and the overexpression of miRNA mimic persists for at least 4 days.8 Lipofectamine 2000 was used for transfection of plasmid alone or together with RNA oligonucleotides; GenJet In Vitro DNA Transfection Reagent (SignaGen Laboratories, MD) was used for transfection of Huh-7 with pSi-shC/EBPa or pSi-Vector. For infection, target cells were infected 3 to 4 times with retroviral supernatant supplemented with 4 lg/mL polybrene (Millipore, Billerica, MA). Luciferase Reporter Assay. Luciferase reporter assays were applied to characterize the miR-122 promoter and miR-122–targeted 30 -UTR. pRL-TK or pRL-CMV, both of which express Renilla luciferase, was cotransfected to correct the differences in both transfection and harvest efficiencies. The activity of the firefly luciferase reporter, which carried miR-122 promoter or miR-122-targeted 30 -UTR, was normalized to the Renilla luciferase activity. Rapid Amplification of Complementary DNA Ends. The miR-122 primary transcript from normal liver tissue was characterized using the 50 -Full RACE Kit (TaKaRa, Japan). Total RNA was treated with calf intestinal phosphatase and tobacco acid pyrophosphatase, followed by ligation to an RNA adaptor and gene-specific primed reverse-transcription polymerase chain reaction (RT-PCR) to amplify the 50 -end of the transcript. Electrophoretic Mobility Shift Assay. Electrophoretic mobility shift assay (EMSA) was conducted using a Gel Shift Assay System (Promega, Madison, WI). The probes, corresponding to the predicted C/EBPa binding sequences on the miR-122 promoter (Supporting Information Table 2), were end-labeled with c-32P adenosine triphosphate, then incubated with Huh-7 nuclear extract. For competition assay, nuclear extract was preincubated with 100-fold molar excess of unlabeled oligonucleotides prior to adding labeled probe. For antibody-supershift assay, nuclear extract was preincubated with anti-C/EBPa antibody or normal immunoglobulin G (IgG) before adding to the binding reaction.
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Chromatin Immunoprecipitation Assay. The formaldehyde cross-linked chromatin complexes were immunoprecipitated using anti-C/EBPa or normal IgG (as a negative control), then collected with Protein A/G PLUS-Agarose. The DNA–protein cross-link was reversed by heating, after which the DNA was subjected to PCR. Analysis of Cell Growth. Bromodeoxyuridine incorporation and anchorage-independent growth assays were used to examine the capability of cells to proliferate and to grow in soft agar. Analysis of Gene Expression. Northern blotting, semiquantitative RT-PCR, and quantitative real-time RT-PCR (qPCR) were performed to evaluate RNA levels. Immunoblotting and immunohistochemical staining were used to detect protein level. Bioinformatic Tools and Statistical Analysis. Data are presented as the mean 6 SEM of at least three independent experiments. Analyses on the differences between groups were conducted using GraphPad Prism version 4.0 (GraphPad Software, Inc., San Diego, CA). Unless indicated, the differences between groups were analyzed using a Student t test when only two groups or assessed using one-way analysis of variance when more than two groups were compared. Two-factor analysis was performed using two-way analysis of variance with a posttest for subsequent comparisons of individual factors. Overall survival was calculated from the date of tumor resection to the time of death. KaplanMeier survival curves and Cox proportional hazard regression analysis, which were applied to identify prognostic factors, were performed using SPSS version 13.0 (SPSS Inc., Chicago, IL). All statistical tests were two-sided. P values
