Hepatocyte nuclear factor-4α reverses malignancy of hepatocellular carcinoma through regulating miR-134 in the DLK1-DIO3 region

Authors

  • Chuan Yin,

    1. Department of Gastroenterology, Changzheng Hospital, Second Military Medical University, Shanghai, China
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    • These authors contributed equally to this work.

  • Pei-Qin Wang,

    1. Department of Gastroenterology, Changzheng Hospital, Second Military Medical University, Shanghai, China
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    • These authors contributed equally to this work.

  • Wen-Ping Xu,

    1. Department of Gastroenterology, Changzheng Hospital, Second Military Medical University, Shanghai, China
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    • These authors contributed equally to this work.

  • Yuan Yang,

    1. Third Department of Hepatic Surgery, Eastern Hepatobiliary Surgery Hospital, Shanghai, China
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    • These authors contributed equally to this work.

  • Qing Zhang,

    1. Department of Gastroenterology, Changzheng Hospital, Second Military Medical University, Shanghai, China
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  • Bei-Fang Ning,

    1. Department of Gastroenterology, Changzheng Hospital, Second Military Medical University, Shanghai, China
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  • Ping-Ping Zhang,

    1. Department of Gastroenterology, Changzheng Hospital, Second Military Medical University, Shanghai, China
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  • Wei-Ping Zhou,

    1. Third Department of Hepatic Surgery, Eastern Hepatobiliary Surgery Hospital, Shanghai, China
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  • Wei-Fen Xie,

    1. Department of Gastroenterology, Changzheng Hospital, Second Military Medical University, Shanghai, China
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  • Wan-Sheng Chen,

    Corresponding author
    1. Department of Pharmacy, Changzheng Hospital, Second Military Medical University, Shanghai, China
    • Address reprint requests to: Xin Zhang, Department of Gastroenterology, Changzheng Hospital, Second Military Medical University, 415 Fengyang Road, Shanghai 200003, China. E-mail: zhang68@hotmail.com Fax: +86–21–8188–9624; or Wan-Sheng Chen, Department of Pharmacy, Changzheng Hospital, Second Military Medical University, 415 Fengyang Road, Shanghai 200003, China. E-mail: chenws126@126.com

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  • Xin Zhang

    Corresponding author
    1. Department of Gastroenterology, Changzheng Hospital, Second Military Medical University, Shanghai, China
    • Address reprint requests to: Xin Zhang, Department of Gastroenterology, Changzheng Hospital, Second Military Medical University, 415 Fengyang Road, Shanghai 200003, China. E-mail: zhang68@hotmail.com Fax: +86–21–8188–9624; or Wan-Sheng Chen, Department of Pharmacy, Changzheng Hospital, Second Military Medical University, 415 Fengyang Road, Shanghai 200003, China. E-mail: chenws126@126.com

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  • Potential conflict of interest: Nothing to report.

  • Supported by the National Natural Science Foundation of China (81071842, 81270033; Key Program 81230011; Creative Research Groups 30921006), the China National Key Projects for Infectious Disease (2013ZX10002007-007), the Shanghai Science and Technology Committee for Key Projects (11JC1416200 and 10431903600), and the Rising-Star Program (11QA1408800).

Abstract

Hepatocyte nuclear factor-4α (HNF4α) is a dominant transcriptional regulator of hepatocyte differentiation and hepatocellular carcinogenesis. There is striking suppression of hepatocellular carcinoma (HCC) by HNF4α, although the mechanisms by which HNF4α reverses HCC malignancy are largely unknown. Herein, we demonstrate that HNF4α administration to HCC cells resulted in elevated levels of 28 mature microRNAs (miRNAs) from the miR-379-656 cluster, which is located in the delta-like 1 homolog (DLK1) -iodothyronine deiodinase 3 (DIO3) locus on human chromosome 14q32. Consistent with the reduction of HNF4α, these miRNAs were down-regulated in human HCC tissue. HNF4α regulated the transcription of the miR-379-656 cluster by directly binding to its response element in the DLK1-DIO3 region. Interestingly, several miRNAs in this cluster inhibited proliferation and metastasis of HCC cells in vitro. As a representative miRNA in this cluster, miR-134 exerted a dramatically suppressive effect on HCC malignancy by down-regulating the oncoprotein, KRAS. Moreover, miR-134 markedly diminished HCC tumorigenicity and displayed a significant antitumor effect in vivo. In addition, inhibition of endogenous miR-134 partially reversed the suppressive effects of HNF4α on KRAS expression and HCC malignancy. Furthermore, a positive correlation between HNF4α and miR-134 levels was observed during hepatocarcinogenesis in rats, and decreases in miR-134 levels were significantly associated with the aggressive behavior of human HCCs. Conclusion: Our data highlight the importance of the miR-379-656 cluster in the inhibitory effect of HNF4α on HCC, and suggest that regulation of the HNF4α-miRNA cascade may have beneficial effects in the treatment of HCC. (Hepatology 2013; 58:1964–1976)

Abbreviations
CCK8

Cell Counting Kit-8

DEN

diethylinitrosamine

DIO3

iodothyronine deiodinase 3

DLK1

delta-like 1 homolog

DMRs

differentially methylated regions

HCC

hepatocellular carcinoma

HNF4α

hepatocyte nuclear factor-4α

MEGs

maternally expressed noncoding genes

Mirg

miRNA-containing gene

miRNA

microRNA

MREs

miRNA-responsive elements

Hepatocyte nuclear factor-4α (HNF4α), a member of the nuclear hormone receptor superfamily, is essential for the differentiation of the hepatic lineage and for maintaining the function of mature hepatocytes.[1-3] Loss of HNF4α expression is a critical event in the progression of hepatocellular carcinoma (HCC) and is inversely associated with HCC differentiation status.[4, 5] A previous study from this laboratory demonstrated that up-regulating HNF4α could reverse the malignant phenotypes of HCC by inducing redifferentiation of HCC cells to hepatocytes.[6] We also demonstrated that HNF4α administration could attenuate liver fibrosis and block hepatocarcinogenesis in rats.[7, 8] Interestingly, a recent study by others reported that transient inhibition of HNF4α could initiate hepatocellular oncogenesis through a microRNA (miRNA)-inflammatory feedback circuit.[9]

The imprinted delta-like 1 homolog (DLK1)-iodothyronine deiodinase 3 (DIO3) region on human chromosome 14q32 contains more than 60 miRNAs that are transcribed from only the maternal chromosome.[10] These miRNAs are organized into two clusters: one is between Meg3 and Meg8; the other (miR-379-656 cluster) is between Meg8 and miRNA containing gene (Mirg).[11, 12] A loss of the imprinted DLK1-DIO3 region results in developmental abnormalities and fetal lethality.[13] The degree of activation of this region is positively correlated with the pluripotency level of stem cells.[14, 15] The DLK1-DIO3 region is also a cancer susceptibility locus and dysregulation of the miRNAs in this region has been found in some human tumors.[16-18] Down-regulation of these miRNAs is regarded as a common molecular event in carcinogenesis, especially in many kinds of epithelial malignancy.[19-22] However, several studies have reported increased expression of these miRNAs in acute promyelocytic leukemia (APL), endometrial carcinosarcomas, and invasive cervical cancer.[23-25] A report by Luk et al.[26] indicated that the DLK1-DIO3 miRNA cluster is up-regulated in a cohort of HCC patients with poor survival due to a change in imprinting status of the locus.

In the present report, we demonstrate that HNF4α can induce transcription of the miR-379-656 cluster and that several miRNAs of this cluster exhibit inhibitory effects on HCC cells in vitro. We also show that miR-134, an miRNA in the miR-379-656 cluster, contributes to HNF4α-induced malignancy reversion by targeting KRAS.

Materials and Methods

Adenovirus Vectors

The recombinant AdHNF4α adenoviruses, expressing HNF4α or the AdGFP control, were constructed in our laboratory as described.[6] The human miR-134 gene was polymerase chain reaction (PCR)-amplified from Hep3B genomic DNA and cloned into the transfer plasmid, Adtrack-CMV. Homologous recombination with the AdEasy vector system and production of adenovirus AdmiR-134 were performed as described.[6]

Total RNA (Plus miRNA) Isolation and Real-Time Polymerase Chain Reaction (RT-PCR)

Total RNA plus miRNA from cultured cells or tissue was isolated using the miRVana miRNA isolation kit (Ambion), reverse transcribed, and then subjected to SYBR Green-based RT-PCR analysis. Expression levels of miRNAs were assessed as described with minor modification.[27] At least three independent experiments were carried out for each experimental condition. Sequences of primers are listed in Supporting Table 1.

miRNA Microarray and cDNA Microarray Analysis

For analysis of miRNA expression patterns, RNA samples from AdHNF4α or AdGFP-treated Hep3B cells were hybridized on a human miRNA microarray (G4470A, Agilent Technologies). Data were extracted using Feature Extraction Software v. 9.3 and analyzed using GeneSpring software (Agilent).

For cDNA microarray analysis, total RNA samples were profiled on a custom Affymetrix array by purification of poly(A)+ mRNA, generation of cDNA and labeled cRNA, and hybridization on a GeneChip Human Genome U133 Plus 2.0 Array (90047, Affymetrix, Santa Clara, CA). The microarray was scanned with an Affymetrix GeneChip Scanner 3000 and analyzed with GeneChip Operating Software.

Chromatin Immunoprecipitation (ChIP) Assay

Hep3B cells were cross-linked and processed according to the Millipore ChIP Assay Kit protocol. A mouse antihuman HNF4α monoclonal antibody (R&D Systems) or control IgG (Santa Cruz Biotechnology) was used for immunoprecipitation. Ten microliters of sonicated but preimmunoprecipitated DNA from each sample were used as input controls. RT-PCR analysis was carried out for HNF4α binding sites in the miR-379-656 cluster. At least three independent experiments were performed. The putative binding sites of HNF4α in the DLK1-DIO3 region were analyzed by JASPAR, a high-quality transcription factor binding profile database.[28] Sequences of the putative binding sites of HNF4α in the miR-379-656 cluster and the primers for ChIP-PCR are shown in Supporting Table 2. The HNF4α-RE in the promoter of the confirmed HNF4α target gene, NINJ1, was used as a positive control.[29]

Plasmid Construction and Luciferase Reporter Assay

To test HNF4α binding sites in the miR-379-656 cluster, the HNF4α-RE-LUC plasmid was generated by inserting a PCR-derived 533 bp fragment containing the HNF4α response element (RE) from the DLK1-DIO3 region into pGL3-Promoter (E1761, Promega). Hep3B cells preinfected with adenovirus for 24 hours were cotransfected with HNF4α-RE-LUC vectors together with the control pRL-SV40 vector (E2261, Promega). Luciferase activity was measured using the Dual-Glo Luciferase Assay System (E2920, Promega) 48 hours posttransfection. To detect miRNA-responsive elements (MREs) for miR-134 in the human KRAS gene, the KRAS 3′ untranslated region (UTR) containing the predicted miR-134 binding sites was cloned into psiCHECK2 (C8021, Promega). HCC cells were cotransfected with psiCHECK-KRAS-3′ UTR or control vector and synthetic miR-134 or as-miR-134, and the luciferase activity was measured. Mutant constructs were generated using PCR-directed mutagenesis with paired primers containing the mutant sequences. All constructs were verified by DNA sequencing. The primers for constructs are listed in Supporting Table 1. At least three independent transfection experiments were carried out for each condition.

Western Blot Analysis

Proteins were extracted using RIPA buffer (P0013B, Beyotime, Suzhou, China) supplemented with protease inhibitor cocktail (Merck), separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), and transferred to nitrocellulose membrane (HAHY00010, Millipore). Western blotting was performed using SuperSignal Western Blot Enhancer (Thermo Scientific) according to the manufacturer's instructions. Mouse anti-human KRAS monoclonal antibody or goat anti-human HNF4α antibody (Santa Cruz Biotechnology) were used as the primary antibody, and IRDyeTM800DX-conjugated anti-mouse or anti-goat immunoglobulins (LI-COR) were used as the secondary antibody. Detection was performed using the Odyssey Infrared Imaging System (LI-COR).

Cell Proliferation, Colony Formation, and Soft Agar Colony Formation Assays

For proliferation assays, HCC cells were transfected or infected for 6 hours and then plated into 96-well plates. Cell Counting Kit-8 (Dojinodo, Tokyo, Japan) was used to examine cell proliferation according to the manufacturer's instructions. For plate colony formation assays, Hep3B cells transfected or infected for 6 hours were seeded on 60-mm dishes. For soft agar colony formation assays, YY-8103 cells or MHCC-LM3 cells transfected or infected for 6 hours were resuspended in medium containing 0.5% low melting point agarose and seeded onto plates containing medium with 1% solidified agarose. After 2 to 3 weeks, colonies on plates or in soft agar were stained with 0.1% crystal violet, photographed, and counted. At least three independent experiments were performed for each condition.

In Vitro Migration and Invasion Assay

In vitro migration and invasion assays were performed by placing cells into the upper chamber of a transwell (BD Bioscience) without or with Matrigel, under serum-free conditions. Medium supplemented with 10% fetal calf serum (FCS) and 50 μg/mL fibronectin (BD Biosciences) was used as a chemoattractant in the lower chamber. After incubation for 24 or 48 hours, cells remaining on the upper chamber were removed with a cotton swab, while cells adhering to the lower membrane were stained with 0.1% crystal violet and photographed with an inverted microscope (Zeiss). The area of positive staining was measured using image analysis software (Image-Pro Plus 6.0, Media Cybernetics). Migration and invasion were calculated as the positive area percentages. At least three independent experiments were performed for each condition.

Animal Models

Male BALB/c nude mice (age 5∼6 weeks) or Wistar rats were purchased from the Shanghai Experimental Animal Center of the Chinese Academy of Sciences, Shanghai, China, and housed in a pathogen-free facility in the Experimental Animal Centre of the Second Military Medical University. All procedures were performed in accordance with the guidelines of the Committee on Animals of the Chinese Academy of Sciences.

To detect the effect of miR-134 on the tumorigenicity of HCC cells, 2 × 106 MHCC-LM3 or YY-8103 cells infected with AdmiR-134 or control virus AdGFP were injected subcutaneously into both flanks of each mouse (10 mice per group). Tumor formation was estimated as described.[6] To investigate the antitumor effect of AdmiR-134, a subcutaneously injected HCC model was established by injecting 2 × 106 MHCC-LM3 cells into BALB/c nude mice. Ten days after cell implantation, mice with comparable tumor size were randomly divided into two groups and given intratumoral injections of AdmiR-134 or AdGFP (2 × 109 pfu) twice a week (8 mice for each group). Tumor volume was serially calculated. Mice were euthanized and tumors were removed for further analysis 27 days after cell implantation.

The diethylinitrosamine (DEN) (Sigma-Aldrich)-induced HCC rat model was established as described.[8] The rat livers (which may contain tumor) were used for RNA extraction and RT-PCR.

Human Tissues

All human liver tissue samples were obtained from HCC patients receiving surgical resection at the Eastern Hepatobiliary Surgery Hospital (Shanghai, China). Written informed consent was obtained from all patients. All human experiments were approved by the Ethics Committee of the Second Military Medical University (Shanghai, China).

Statistical Analysis

All data are presented as the mean ± standard deviation. Data analyses were performed with Prism5 (GraphPad software, La Jolla, CA). For experiments involving three or more groups, data were evaluated using a one-way analysis of variance (ANOVA). For experiments involving only two groups, data were analyzed with the Student unpaired t test. The Kaplan-Meier method was used to calculate survival, and significance was determined by log-rank test. The Mann-Whitney U test was used for comparison of tumor weight and volume. Statistical significance was set at *P < 0.05, **P < 0.01, ***P < 0.001. P < 0.05 was considered statistically significant.

Additional details are described in the Supporting Material.

Results

HNF4α Induces Expression of the miR-379-656 Cluster in the DLK1-DIO3 Region

To determine the miRNAs regulated by HNF4α in Hep3B cells, we conducted microarray analyses to obtain miRNA expression profiles in AdHNF4α or AdGFP-treated Hep3B cells. Interestingly, HNF4α overexpression elevated a subset of miRNAs from the miR-379-656 cluster, which is in the DLK1-DIO3 region on chromosome 14q32 (Fig. 1A). RT-PCR was then used to verify the effect of HNF4α on the miR-379-656 cluster. Of the 53 miRNAs[30] in this cluster, 28 were induced in Hep3B cells treated with AdHNF4α, 14 of which were shown to be up-regulated by microarray analysis (Fig. 1B). The effect of HNF4α on the up-regulation of these miRNAs was confirmed in YY-8103 cells (Supporting Table 3). We then detected the expression of HNF4α and these miRNAs in 20 pairs of human HCC and their surrounding noncancerous liver tissues (Fig. 1C). Reduction of HNF4α was observed in 19 HCC samples, in which most of the 28 miRNAs, identified above, were also down-regulated (Supporting Table 4). In one sample with enhanced expression of HNF4α, the levels of 22 miRNAs in the miR-379-656 cluster were also increased (Supporting Table 5).

Figure 1.

HNF4α up-regulates the expression of the miR-379-656 cluster in the DLK1-DIO3 region. (A) A heat map diagram of the relative abundance of the hsa-miR-379-656 cluster by miRNA microarray analysis of Hep3B cells infected with AdHNF4α or AdGFP for 48 hours and 72 hours. (B) RT-PCR analysis of mature miRNA expression of the miR-379-656 cluster in Hep3B cells infected with AdHNF4α for 72 hours. Twenty-eight miRNAs were up-regulated by HNF4α overexpression (AdHNF4α/AdGFP >1.5). Data are presented as the mean ± SD of three independent experiments. (C) The levels of HNF4α and HNF4α-elevated miRNAs in the cluster in 20 paired HCC samples detected by RT-PCR. The results show the fold expression in HCC cancerous tissue (T) versus associated noncancerous tissue (N).

HNF4α Regulates the Transcription of the miR-379-656 Cluster

To investigate the mechanism by which HNF4α regulates the miR-379-656 cluster, we examined the levels of miRNA primary precursors in Hep3B cells treated with AdHNF4α or siHNF4α. The results showed that all the primary transcripts of the 53 miRNAs in the miR-379-656 cluster were increased by HNF4α overexpression and decreased by HNF4α knockdown (Fig. 2A,B), suggesting that HNF4α modulates the transcription of the cluster. We then used JASPAR[28] to analyze the HNF4α-REs in the region from 2 kb upstream of miR-379 to miR-656. Twenty-two putative HNF4α-REs were identified when the profile score threshold was set at 80% (Supporting Table 2). ChIP assays confirmed the binding of HNF4α to an HNF4α-RE between miR-329-2 and miR-494 (Fig. 2C,D). Luciferase assays showed that ectopic expression of HNF4α increased the activity of the HNF4α-RE in this cluster, which was impaired by mutation of the HNF4α-RE (Fig. 2E). Taken together, these data indicate that HNF4α activates the transcription of the miR-379-656 cluster by direct binding to a specific responsive element in this region.

Figure 2.

HNF4α activates the transcriptional activity of the miR-379-656 cluster by binding to the DLK1-DIO3 region. (A,B) The levels of primary precursors from the miR-379-656 cluster in Hep3B cells infected by AdHNF4α (A) or transfected by siHNF4α (B) were detected by RT-PCR. The results show the fold gene expression of the AdHNF4α-infected Hep3B group versus the AdGFP control group (A) and of the siHNF4α-transfected group versus the scramble control group (B). The dashed horizontal lines indicate the threshold for 1-fold increment (A) or decrement (B). Data are presented as the mean ± SD of three independent experiments, P < 0.05. (C) Schematic representation of the miR-379-656 cluster in the DLK1-DIO3 region, the validated response element of HNF4α (HNF4α-RE), and the mutation sequence of the HNF4α-RE. (D) ChIP assays revealed the binding of HNF4α to the miR-379-656 cluster region. RT-PCR (right) and semiquantitative PCR (left) were performed to examine the DNA fragments immunoprecipitated by HNF4α antibody with the primers for HNF4α-RE. DNA sample from immunoprecipitation with normal rabbit IgG (IgG) was used as control. The human NINJ1 promoter containing a consensus HNF4α-RE was used as a positive control. (E) Luciferase activity in HCC cells of a reporter plasmid harboring the HNF4α-RE from the miR-379-656 cluster. Mutation of HNF4α-RE abolished the transcriptional activity of HNF4α. Each value represents the mean ± SD of triplicate experiments.

miR-134 Suppresses Malignant Properties of HCC In Vitro

To evaluate the effect of the miRNAs in the miR-379-656 cluster on HCC cells, the 28 HNF4α-elevated miRNAs were transfected into Hep3B and YY-8103 cells. Proliferation assays showed that 14 of the 28 miRNAs repressed the growth of Hep3B cells by more than 30% (Supporting Fig. 1A). More significant suppression on proliferation was observed in YY-8103 cells transfected with miR-544, miR-134, or miR-541 (Supporting Fig. 1B). In addition, miR-381, miR-382, and miR-134 exerted marked inhibition on migration and invasion of YY-8103 cells (Supporting Fig. 1C). These data indicate that this cluster may play an important role in the antitumor effect of HNF4α. Because miR-134 displayed a profound effect on both proliferation and metastasis, we further examined the functional role of this cluster in HCC using miR-134 as a representative miRNA. miR-134 overexpression arrested cell growth and suppressed clonogenic survival of HCC cells (Fig. 3A,B; Supporting Fig. 2A,B). In contrast, inhibition of endogenous miR-134 by as-miR-134 promoted HCC cell growth and colony formation (Fig. 3C,D; Supporting Fig. 2C). In accordance with previous reports,[18, 31] transfection of miR-134 into YY-8103 cells decreased the G0/G1 population by 35% (P < 0.01) and increased the G2/M population by 117% (P < 0.01) (Supporting Fig. 2D). Moreover, overexpression of miR-134 decreased cell migration and invasion, whereas as-miR-134 treatment exacerbated the metastatic potential of HCC cells (Fig. 3E,F).

Figure 3.

miR-134 represses the malignant properties of HCC cells in vitro. (A) Cell proliferation of Hep3B cells transfected with miR-134 or scramble miRNA (Scr) was measured using the Cell Counting Kit-8 (CCK8). (B) Soft agar colony formation assays were performed with YY-8103 and MHCC-LM3 cells transfected with miR-134 or scramble miRNA (Scr). Colonies were counted 2 to 3 weeks after cells were plated in soft agar. (C) Proliferation assay for Hep3B cells transfected with as-miR-134 or scramble as-miRNA (Scr). (D) The clonogenic survival of HCC cells was enhanced by as-miR-134. (E,F) Cell migration and invasion were assessed by transwell assays of HCC cells transfected with miR-134 for 72 hours (E) or as-miR-134 for 48 hours (F). Each value represents the mean ± SD of triplicate experiments.

KRAS Is a Downstream Effector of miR-134

To identify the potential target of miR-134, we searched the Target Scan and Pictar databases and found that the 3′ UTR of the proto-oncogene, KRAS, contains four putative binding sites for miR-134 (Fig. 4A). Additionally, a complementary DNA (cDNA) microarray analysis demonstrated that HNF4α reexpression reduced the expression of KRAS in Hep3B cells (Supporting Table 6), which was validated by RT-PCR and western blot analysis (Supporting Fig. 3A,B). Interestingly, overexpression of miR-134 in cultured HCC cells significantly decreased both mRNA and protein levels of KRAS (Fig. 4B; Supporting Fig. 4A). In contrast, as-miR-134 increased KRAS expression (Fig. 4C; Supporting Fig. 4B). Luciferase assays revealed that miR-134 diminished the activity of the KRAS 3′ UTR in HCC cells (Supporting Fig. 4C). Mutation of motif 1 in the putative MREs of the KRAS 3′ UTR abrogated its response to miR-134 (Fig. 4A,D). These data suggest that KRAS is a direct target of miR-134. To evaluate whether the antitumor effect of miR-134 in vitro could be reversed by restoration of KRAS levels, YY-8103 cells were cotransfected with miR-134 and a plasmid expressing KRAS without its 3′ UTR. The result showed that overexpression of nontargetable KRAS reversed the suppressive effects of miR-134 on malignant properties of HCC cells, including proliferation, migration, and invasion (Fig. 4E,F).

Figure 4.

miR-134 represses KRAS expression in HCC cells. (A) Schematic representation of the predicted target regions of miR-134 in the 3′ UTR of KRAS and the mutated sequences of these target regions. (B) The mRNA (left) and protein (right) levels of KRAS in YY-8103 cells transfected with miR-134 or scramble miRNA (Scr). The mRNA level of KRAS was detected by RT-PCR 24 hours after transfection. The protein level of KRAS was analyzed by western blotting 24 hours and 48 hours after transfection. (C) RT-PCR (left) and western blot analysis (right) of KRAS expression in YY-8103 cells transfected with as-miR-134 or scramble as-miRNA (Scr). (D) In YY-8103 cells, miR-134 overexpression decreased the luciferase activity of the reporter plasmid carrying the KRAS 3′ UTR. Mutation of motif 1 in the putative target region of the KRAS 3′ UTR abrogated its response to miR-134. All values are relative to those of the scramble control (Scr). Vector: psiCHECK2; WT: psiCHECK2 containing wild-type KRAS 3′ UTR. (E) The proliferation of YY-8103 cells cotransfected with the indicated miRNA and plasmids. (F) The migration (left) and invasion (right) of YY-8103 cells cotransfected with the indicated miRNA and plasmids for 72 hours. Each value represents the mean ± SD of triplicate experiments.

miR-134 Suppresses Tumorigenicity of HCC Cells in Mice

To validate the effect of miR-134 on HCC in vivo, MHCC-LM3 cells infected with adenovirus expressing miR-134 (AdmiR-134) or control adenovirus (AdGFP) were subcutaneously injected into the flanks of BALB/c nude mice. In the AdGFP group, tumor nodules were detected in 8/10 mice on day 10 and in all subjects on day 19. In contrast, tumor nodules in the AdmiR-134 group were detected in only 1/10 mice on day 10 and in 6/10 mice on day 25 (Fig. 5A). Moreover, xenografts were also significantly smaller in the AdmiR-134 group compared with the control group at every timepoint (Fig. 5B; Supporting Fig. 5A). Similar results were also obtained for YY-8103 cells (Supporting Fig. 5B,C).

Figure 5.

miR-134 overexpression suppresses tumorigenicity of HCC cells in vivo. (A) The HCC-free survival of mice injected with AdmiR-134 or Ad-GFP-infected MHCC-LM3 cells was analyzed by the Kaplan-Meier method and compared using the log-rank test (n = 10). (B) The tumor size in mice injected with AdmiR-134 or AdGFP-infected MHCC-LM3 cells was estimated by serial calibration (n = 10). (C,D) Intratumoral injection of AdmiR-134 repressed HCC xenograft growth. A subcutaneous liver tumor model was established based on implantation of MHCC-LM3 cells. The HCC xenografts were then injected with AdmiR-134 or AdGFP. Tumor size was estimated by serial calculation (C) and tumor weight was measured at the endpoint (D) (n = 8). (E) RT-PCR analysis of expression levels of miR-134 and KRAS in the MHCC-LM3 tumor nodules injected with AdmiR-134 or Ad-GFP. (F) Representative images of KRAS and Ki67 immunohistochemistry of serial sections from MHCC-LM3 tumor nodules injected with AdmiR-134 or Ad-GFP . Magnification, ×200 and 400; Data in (B,C) are shown as mean ± SD. Horizontal lines in (D,E) indicate median values.

We then observed the antitumor effect of miR-134 on HCC xenografts established by subcutaneous injection of MHCC-LM3 cells into BALB/c nude mice. Intratumoral injection of AdmiR-134 significantly reduced the growth and the weight of tumor nodules compared with injection of AdGFP (Fig. 5C,D; Supporting Fig. 5D). RT-PCR revealed that miR-134 expression was significantly elevated in AdmiR-134-treated tumors, with a concomitant decrease of KRAS (Fig. 5E). Immunohistochemical staining of tumors showed that treatment with AdmiR-134 resulted in remarkable down-regulation of KRAS levels, accompanied by reduced Ki67 staining (Fig. 5F).

HNF4α Suppresses the Tumorigenic Capacity of HCC Partially Through Up-Regulation of miR-134

To determine the role of miR-134 in the HNF4α reversion of malignant phenotypes, as-miR-134 was transfected into HCC cells overexpressing HNF4α. As expected, as-miR-134 reversed the HNF4α-induced reduction in proliferation in Hep3B cells (Fig. 6A). Additionally, the suppressive effect of HNF4α on colony formation was partially reversed by as-miR-134 in Hep3B and YY-8103 cells (Fig. 6B; Supporting Fig. 6A). as-miR-134 also abrogated the inhibitory effects of HNF4α on migration and invasion (Fig. 6C,D). In addition, the repression of KRAS by ectopic HNF4α was also restored by as-miR-134 at the mRNA and protein levels (Fig. 6E,F; Supporting Fig. 6B,C). Consistently, luciferase assays also showed that inhibition of miR-134 reversed the effect of HNF4α on the KRAS 3′ UTR (Supporting Fig. 6D).

Figure 6.

as-miR-134 reverses the suppressive effects of HNF4α on HCC cells. (A) Hep3B cells infected with AdHNF4α or AdGFP and transfected with as-miR-134 or scramble control (Scr). Cell proliferation was detected on the 5th day after transfection. (B-D) Soft agar colony formation (B), migration (C), and invasion (D) of YY-8103 cells infected with AdHNF4α or AdGFP and transfected with as-miR-134 or scramble control (Scr). (E,F) Human KRAS expression was analyzed by RT-PCR (E) and western blot analysis (F) in Hep3B cells treated with the indicated virus and as-miRNA. Each value represents the mean ± SD of triplicate experiments. Left: the original data; right: the relative ratio of AdHNF4α versus AdGFP.

Low miR-134 Levels Are Correlated With Reduced HNF4α Levels in Hepatocarcinogenesis and Are Associated With Aggressive Behavior of Human HCC

To further determine the correlation of HNF4α and miR-134 in HCC, we examined their expression profiles in the DEN-induced HCC rat model (n = 4 at each indicated timepoint). In agreement with our previous study,[8] HNF4α expression decreased gradually after DEN administration. Interestingly, the transcript level of miR-134 (pri-miR134) was also suppressed in the process of hepatocarcinogenesis (Fig. 7A). A striking positive correlation between HNF4α and pri-miR-134 levels was observed in DEN-treated rat liver (Fig. 7B). We then analyzed the association of HNF4α and pri-miR-134 expression in human HCC samples (n = 71). As compared with their surrounding noncancerous tissues, 63% (45/71) and 70% (50/71) of the HCC tissues showed lower levels of HNF4α and pri-miR-134, respectively (Fig. 7C). Reduced pri-miR-134 expression was more frequent in HCCs with lower HNF4α levels relative to those with intermediate and high HNF4α levels (89% versus 38%; Fig. 7D). The correlation was particularly apparent in HCC subjects with alpha-fetoprotein (AFP) levels over 1,000 μg/L (r = 0.6241, P = 0.0003, n = 29; Fig. 7E).

Figure 7.

Suppressed miR-134 expression is correlated with reduced HNF4α levels in hepatocarcinogenesis. (A) The relative RNA levels of HNF4α and primary miR-134 (pri-miR-134) in a DEN-induced rat HCC model (n = 4 at indicated timepoints; all values are relative to the normal control). (B) The positive correlation between HNF4α and pri-miR-134 expression levels in the DEN-induced rat HCC model (r = 0.7429, P = 0.0002, n = 20). (C) The levels of HNF4α mRNA and pri-miR-134 in human HCC samples (n = 71) detected by RT-PCR. The results are presented as fold gene expression of HCC cancerous tissue (T) versus noncancerous adjacent tissue (N). (D) Reduced pri-miR-134 expression was more frequent in HCC samples with low HNF4α levels compared with samples with intermediate or high HNF4α levels. Low level: T/N < 1, P < 0.05; high level: T/N > 1, P < 0.05; intermediate: T/N = 1 or P > 0.05 (E) The positive correlation between the expression levels of HNF4α and pri-miR-134 in HCC subjects with high AFP levels (>1,000 μg/L). (r = 0.6241, P = 0.0003, n = 29).

The clinicopathological significance of pri-miR-134 levels in the above 71 patients with HCC was further analyzed. The median value of pri-miR-134 levels in HCC tissues was chosen as the cutoff point; 49.3% of HCCs (35/71) had low-level expression of pri-miR-134, and 50.7% of HCCs (36/71) had high-level expression of pri-miR-134 (Table 1). The low-level expression of pri-miR-134 in HCCs was associated with more aggressive pathological features, including liver cirrhosis (P = 0.0127), high levels of AFP (P = 0.0142), large tumor size (P = 0.0271), advanced tumor stage (P = 0.0051), presence of tumor microsatellites (P = 0.0434), and absence of tumor encapsulation (P = 0.0013) (Table 1).

Table 1. Clinicopathologic Correlation of miR-134 Down-Regulation in Human HCCs
 Pri-miR-134 
 All Cases Low Expression High Expression 
Variables(n = 71)(n = 35)(n = 36)P Valuea
  1. a

    χ2 test.

  2. b

    Mean age.

  3. HBsAg, hepatitis B surface antigen; AFP, α-fetoprotein.

Age (years)
≤ 51.7b3415 (44.1)19 (55.9)0.4028
> 51.73720 (54.1)17 (45.9) 
Sex
Male6233 (53.2)29 (46.8)0.0821
Female92 (22.2)7 (77.8) 
HBsAg
Absent199 (47.4)10 (52.6)0.8443
Present5226 (50.0)26 (50.0) 
Liver cirrhosis
Yes3221 (65.6)11 (34.4)0.0127
No3914 (35.9)25 (64.1) 
AFP (ng/ml)   
≤ 2091 (11.1)8 (88.9)0.0142
> 206234 (54.8)28 (45.2) 
Tumor size (cm)
≤ 581 (12.5)7 (87.5)0.0271
> 56334 (54.0)29 (46.0) 
Tumor multiplicity
Single5627 (48.2)29 (51.8)0.7247
Multiple158 (53.3)7 (46.7) 
Tumor staging (TNM)
I176 (35.3)11 (64.7)0.0051
II226 (27.3)16 (72.7) 
III3021 (70.0)9 (30.0) 
IV22 (100.0)0 (0.0) 
Venous invasion
Absent3014 (46.7)16 (53.3)0.7047
Present4121 (51.2)20 (48.8) 
Tumor microsatellite
Absent3513 (37.1)22 (62.9)0.0434
Present3622 (61.1)14 (38.9) 
Tumor encapsulation
Absent3122 (71.0)9 (29.0)0.0013
Present4013 (32.5)27 (67.5) 

Discussion

HNF4α is a transcription factor that plays a key role in hepatocyte differentiation and in the maintenance of hepatic function. It is well established that the miRNAs at the DLK1-DIO3 imprinting locus are critical for the differentiation of stem cells and for the development of the mouse embryo.[14, 15, 32] The DLK1-DIO3 miRNA cluster is up-regulated in c-MET mouse liver tumors and in a subgroup of HCC patients[26]; however, the expression status and function of this miRNA cluster in human HCC are largely unknown. In the current study, we demonstrated that the HNF4α-regulated miR-379-656 cluster in the DLK1-DIO3 region is suppressed in the majority of HCC tumor tissues. Experiments in cultured HCC cells confirmed a suppressive effect of the miR-379-656 cluster on malignant phenotypes.

The DLK1-DIO3 imprinted locus contains three paternally expressed protein-coding genes and several maternally expressed noncoding RNA genes (MEGs).[33] Differentially methylated regions (DMRs) are key control elements regulating the expression of imprinted regions and in the DLK1-DIO3 locus include an intergenic DMR (Ig-DMR) located between Dlk1 and Meg3 and a DMR spanning the Meg3 promoter (Meg3-DMR). Maternal deletion of Meg3 and a small portion of its promoter abolishes expression from all the MEGs in the region[32]; therefore, it is believed that all Megs are transcribed from the Meg3 promoter as one long transcript. However, Fiore et al.[34] reported that the transcription factor, myocyte enhancing factor 2 (Mef2), could activate the transcription of the miR-379-656 cluster through direct binding to the upstream of the cluster. In this study, we found that ectopic expression of HNF4α did not elevate expression of all MEGs in this region (data not shown), but transactivated the expression of the miR-379-656 cluster. These data suggest that these MEGs are regulated by gene-specific elements, in addition to Meg3 control of the one giant polycistronic RNA.

miR-134 was first identified as a brain-specific microRNA, and is implicated in the control of neuronal microstructure.[35] Silencing miR-134 results in neuroprotection and prolongs seizure-suppressive actions in mice.[36] miR-134 also regulates the differentiation of mouse embryonic stem cells.[37] Overexpression of miR-134 induces cell cycle arrest in human pituitary tumor cells.[18] In the present study, we found that the level of miR-134 transcription in the liver gradually reduced during the development of HCC in a chemically induced HCC rat model. A reduction of miR-134 levels was also observed in the majority of human HCC tumor samples, and was associated with aggressive phenotypes of the disease. More interestingly, malignant phenotypes of HCC cells could be manipulated by changing miR-134 expression, both in vitro and in vivo. Together, these results suggest that miR-134 plays a crucial role in the carcinogenesis and progression of HCC and prompt the exploration of antitumor effects of other miRNAs in this cluster.

Previous studies have revealed that miR-134 can target Nanog, Sox2, c-Myc, nuclear receptor liver receptor homolog 1, and Oct4.[17, 37, 38] These genes are important for the proliferation and fate-determining properties of stem/progenitor cells and are also involved in hepatocarcinogenesis. The proto-oncogene KRAS is a central regulator of intracellular signal transduction pathways in malignant transformation, including PI3K-AKT, vascular endothelial growth factor, Wnt-β-catenin, and nuclear factor kappa B (NF-κB) pathways. It has been reported that KRAS is not frequently mutated and only one of 35 tumors had up-regulation of KRAS in human HCC.[39] However, KRAS was found to be up-regulated in most of HCC samples in this study (Supporting Fig. 7). Evidence suggests that KRAS is a bona fide target of several miRNAs (including let-7, miR-30C, miR-143, and miR-96) that can inhibit cancer cell proliferation and metastasis.[40-43] This study demonstrates that KRAS is a direct target of miR-134. More important, nontargetable KRAS reversed the miR-134-mediated suppression of HCC malignancy, suggesting that inhibition of the oncoprotein KRAS contributes to the suppressive effect of miR-134 on HCC.

Recently, Hatziapostolou et al.[9] reported that HNF4α can modulate inflammatory signaling to prevent and suppress hepatocellular carcinogenesis through up-regulation of miR-124. We identified here a positive correlation between miR-134 and HNF4α in HCC pathogenesis. We also show that miR-134 acts as an important functional effector of HNF4α for KRAS suppression and reversion of HCC malignancy. These findings suggest regulating the HNF4α-miRNA cascade could be developed as a strategy for the treatment of HCC.

In summary, we have identified a novel mechanism by which HNF4α reverses HCC malignancy through up-regulation of an miRNA cluster in the DLK1-DIO3 region, particularly miR-134. Further investigations of the other miRNAs in this cluster are merited.

Ancillary

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