Article first published online: 7 JAN 2013
Copyright © 2012 American Association for the Study of Liver Diseases
Volume 57, Issue 1, pages 162–170, January 2013
How to Cite
Takata, A., Otsuka, M., Yoshikawa, T., Kishikawa, T., Hikiba, Y., Obi, S., Goto, T., Kang, Y. J., Maeda, S., Yoshida, H., Omata, M., Asahara, H. and Koike, K. (2013), MicroRNA-140 acts as a liver tumor suppressor by controlling NF-κB activity by directly targeting DNA methyltransferase 1 (Dnmt1) expression. Hepatology, 57: 162–170. doi: 10.1002/hep.26011
Potential conflict of interest: Nothing to report.
Supported by Grants-in-Aid from the Ministry of Education, Culture, Sports, Science and Technology, Japan (#22390058, #23590960, and #20390204) (M. O., T. G., and K. K.); Health Sciences Research Grants from the Ministry of Health, Labor and Welfare of Japan (Research on Hepatitis) (to K. K.); National Institutes of Health Grant R01AI088229 (to Y. J. K.); the Miyakawa Memorial Research Foundation (to A. T.); and grants from the Sagawa Foundation for Promotion of Cancer Research, the Astellas Foundation for Research on Metabolic Disorders, and the Cell Science Research Foundation (to M. O.).
- Issue published online: 7 JAN 2013
- Article first published online: 7 JAN 2013
- Accepted manuscript online: 17 AUG 2012 12:34AM EST
- Manuscript Accepted: 18 JUL 2012
- Manuscript Received: 30 MAR 2012
MicroRNAs (miRNAs) are small RNAs that regulate the expression of specific target genes. While deregulated miRNA expression levels have been detected in many tumors, whether miRNA functional impairment is also involved in carcinogenesis remains unknown. We investigated whether deregulation of miRNA machinery components and subsequent functional impairment of miRNAs are involved in hepatocarcinogenesis. Among miRNA-containing ribonucleoprotein complex components, reduced expression of DDX20 was frequently observed in human hepatocellular carcinomas, in which enhanced nuclear factor-κB (NF-κB) activity is believed to be closely linked to carcinogenesis. Because DDX20 normally suppresses NF-κB activity by preferentially regulating the function of the NF-κB-suppressing miRNA-140, we hypothesized that impairment of miRNA-140 function may be involved in hepatocarcinogenesis. DNA methyltransferase 1 (Dnmt1) was identified as a direct target of miRNA-140, and increased Dnmt1 expression in DDX20-deficient cells hypermethylated the promoters of metallothionein genes, resulting in decreased metallothionein expression leading to enhanced NF-κB activity. MiRNA-140-knockout mice were prone to hepatocarcinogenesis and had a phenotype similar to that of DDX20 deficiency, suggesting that miRNA-140 plays a central role in DDX20 deficiency-related pathogenesis. Conclusion: These results indicate that miRNA-140 acts as a liver tumor suppressor, and that impairment of miRNA-140 function due to a deficiency of DDX20, a miRNA machinery component, could lead to hepatocarcinogenesis. (HEPATOLOGY 2013)
Hepatocellular carcinoma (HCC) is the third most common cause of cancer-related mortality worldwide.1 Although multiple major risk factors have been identified, such as infection with hepatitis viruses B or C, the molecular mechanisms underlying HCC development remain poorly understood, hindering the development of novel therapeutic approaches. Therefore, a better understanding of the molecular pathways involved in hepatocarcinogenesis is critical for the development of new therapeutic options.
Nuclear factor-κB (NF-κB) is one of the best-characterized intracellular signaling pathways. Its activation is a common feature of human HCC.2-4 It acts as an inhibitor of apoptosis and as a tumor promoter4, 5 and is associated with the acquisition of a transformed phenotype during hepatocarcinogenesis.6 In fact, studies using patient samples suggest that NF-κB activation in the liver leads to the development of HCC.7 Although there are conflicting reports,8 activation of the NF-κB pathway in the liver is crucial for the initiation and promotion of HCC.4
MicroRNAs (miRNAs) are small RNA molecules that regulate the expression of target genes and are involved in various biological functions.9-12 Although specific miRNAs can function as either suppressors or oncogenes in tumor development, a general reduction in miRNA expression is commonly observed in human cancers.13-22 In this context, it can be hypothesized that deregulation of the machinery components involved in miRNA function may be related to the functional impairment of miRNAs and the pathogenesis of carcinogenesis.
In this study, we show that the expression of DDX20, an miRNA-containing ribonucleoprotein (miRNP) component, is frequently decreased in human HCC. Because DDX20 is required for both the preferential loading of miRNA-140 into the RNA-induced silencing complex and its function,23 we hypothesized that DDX20 deficiency would lead to hepatocarcinogenesis via impaired miRNA-140 function. MiRNA-140 knockout mice were indeed more prone to hepatocarcinogenesis, and we identified a possible molecular pathway from DDX20 deficiency to liver cancer.
Materials and Methods
Mouse and Liver Tumor Induction.
MiRNA-140−/− mice have been described.24 Recombinant murine tumor necrosis factor-α (TNF-α) (25 μg/kg; Wako, Osaka, Japan) was injected into the tail vein, and the mice were sacrificed 1 hour later. To induce liver tumors, 15-day-old mice received an intraperitoneal injection of diethylnitrosamine (DEN) (25 mg/kg body weight), and were sacrificed 32 weeks later. All animal experiments were performed in compliance with the regulations of the Animal Use Committee of the University of Tokyo and the Institute for Adult Disease, Asahi Life Foundation.
Detailed Materials and Methods.
The detailed experimental procedures of clinical samples, cells, plasmids, reporter assays, reverse-transcription polymerase chain reaction (RT-PCR) analysis, antibodies, western blotting, cell assays, immunohistochemistry, microarray analysis, methylation analysis, and electrophoretic mobility-shift assay are described in the Supporting Information.
Statistically significant differences between groups were determined using a Wilcoxon rank-sum test. A Wilcoxon signed-rank test was used for statistical comparisons of protein expression levels between HCC and surrounding noncancerous tissues.
DDX20 Expression Is Frequently Decreased in HCC.
The expression levels of proteins reported to be miRNP components (Dicer, Ago2, TRBP2, DDX20 [also known as Gemin3], and Gemin4)26 were initially determined via immunohistochemistry in HCC and noncancerous background liver tissues from 10 patients. DDX20 expression was lower in HCC tissue compared with the surrounding noncancerous tissue in 8 of 10 cases, whereas expression of the other genes was unchanged (Table 1 and Supporting Fig. 1). Therefore, and because DDX20 was recently identified as a possible liver tumor suppressor in mice,27 we determined its role as a human HCC suppressor.
|Gene ID||Gene Symbol||Decreased||Increased||No Change|
DDX20 protein expression was lower in several HCC cell lines, such as Huh7 and Hep3B (Fig. 1A), compared with normal hepatocytes. DDX20 protein levels were also lower in human HCC needle biopsy specimens than in surrounding noncancerous liver tissue (Fig. 1B). Immunohistochemical analysis confirmed that DDX20 expression was frequently lower in HCC than in surrounding noncancerous liver tissue (Fig. 1C,D). Specifically, 47 of 70 cases examined showed reduced DDX20 protein expression in HCC versus background noncancerous liver tissue (Fig. 1D and Supporting Table 1). These results indicate that the expression of DDX20, an miRNP component, is frequently reduced in human HCC, and suggest that this reduced DDX20 expression might be involved in the pathogenesis of a subset of HCC cases.
NF-κB Activity Is Enhanced by DDX20 Deficiency.
Because DDX20 knockout mice are embryonic-lethal,28 DDX20 has been suggested to have important biological roles. DDX20, a DEAD-box protein,29 was originally found to interact with survival motor neuron protein.30 Later, it was identified as a major component of miRNPs,31 which may mediate miRNA function. As we have reported, DDX20 is preferentially involved in miRNA-140-3p function,23 acting as a suppressor of NF-κB activity in the liver.32 DDX20-knockdown PLC/PRF/5 cells exhibit enhanced NF-κB activity23 (Fig. 2A). Whereas the proliferation rates of DDX20-knockdown cells and control cells were comparable (Fig. 2B), apoptotic cell death after stimulation with TNF-related apoptosis-inducing ligand (TRAIL), which induces both cell apoptosis and NF-κB activation,33 was significantly reduced in DDX20-knockdown cells (Fig. 2C). Similar results were obtained using DDX20-knockdown HepG2 cells (Supporting Fig. 2A-D). Conversely, NF-κB activity was reduced, but cell proliferation remained unchanged, in Hep3B cells stably overexpressing DDX20 (Fig. 2D,E). Sensitivity to TRAIL-induced apoptosis was restored in these cells (Fig. 2F). Similar results were also obtained using Huh7 cells (Supporting Fig. 2E-H). These data confirm a previous report that DDX20 deficiency enhances NF-κB activity and the downstream events of this pathway.
Metallothionein Expression Is Decreased by DDX20 Deficiency.
Next, to investigate the biological consequences of DDX20 deficiency, we examined the changes in transcript levels in DDX20-knockdown cells using microarrays (GEO accession number: GSE28088). The expression of genes driven by NF-κB that are related to carcinogenesis, such as FASLG, IRAK1, CARD9, and Galectin-1, were enhanced significantly in DDX20-knockdown cells, as expected (Table 2). To determine the mechanism underlying the enhanced NF-κB activation in DDX20-deficient cells, we searched for candidate genes and noticed that the expression levels of a group of metallothioneins (MTs), such as MT1E, MT1F, MT1G, MT1M, MT1X, and MT2A, were all significantly decreased when DDX20 was deficient (Table 3). The decreased expression of MTs in DDX20-knockdown HepG2 and PLC/PRF/5 cells was confirmed via quantitative RT-PCR (Fig. 3a and Supporting Fig. 3). Expression of MT-3, which was not altered in the microarray analysis, was similarly unaltered in quantitative RT-PCR analysis. Notably, it was already known that MTs are frequently silenced in human primary liver cancers.34-36 In addition, MT knockout mice have enhanced NF-κB activity, likely due to reactive oxygen species, and these mice are more prone to hepatocarcinogenesis.37 These results suggest that DDX20 deficiency enhances NF-κB activity by decreasing the expression of MTs, which could facilitate the development of liver cancer.
|RefSeq ID||Symbol||Description||Ratio||Representative Gene Function|
|NM_000639||FASLG||Fas ligand||3.5||NF-κB target, apoptosis|
|NM_052813||C9orf151||CARD9||2.5||NF-κB cascade, NF-κB target|
|NM_014959||CARD8||Tumor up-regulated CARD-containing antagonist of CASP9 (TUCAN)||2.2||NF-κB target|
|NM_131917||FAF1||FAS-associated factor 1 (hFAF1)||1.9||Cytoplasmic sequestering of NF-κB, NF-κB target|
|NM_020644||TMEM9B||Transmembrane protein 9B precursor||1.9||Positive regulation of NF-κB transcription factor activity|
|NM_017544||NKRF||ITBA4 protein||1.9||Negative regulation of transcription|
|NM_006247||PPP5C||Protein phosphatase T||1.8||Positive regulation of NF-κB cascade|
|NM_001569||IRAK1||IRAK-1||1.7||Positive regulation of NF-κB transcription factor activity|
|NM_177951||PPM1A||Protein phosphatase 1A||1.7||Positive regulation of NF-κB cascade|
|NM_018098||ECT2||Epithelial cell-transforming sequence 2 oncogene||1.6||Positive regulation of NF-κB cascade|
|NM_002305||LGALS1||Galectin-1 (putative MAPK-activating protein MP12)||1.6||Positive regulation of NF-κB cascade|
|NM_015093||TAB2||TAK1-binding protein 2||1.6||Positive regulation of NF-κB cascade|
|NM_004180||TANK||TRAF-interacting protein||1.5||NF-κB cascade|
|NM_014976||PDCD11||Programmed cell death protein 11||1.5||rRNA processing|
|NM_015336||ZDHHC17||Putative NF-κB–activating protein 205||1.5||Positive regulation of NF-κB cascade|
|NM_002503||NFKBIB||IKB-β||1.5||Cytoplasmic sequestering of NF-κB|
|NM_138330||ZNF675||Zinc finger protein 675||1.5||Negative regulation of NF-κB transcription factor activity|
|MTL5||Metallothionein-like 5 (Tesmin)||1.12|
MiRNA-140 Directly Targets Dnmt1.
Because MT expression is regulated principally by CpG island methylation in their promoter regions,38, 39 we examined the quantitative methylation status of MT promoters in DDX20-knockdown cells. The CpG islands of the MT1E, MT1G, MT1M, MT1X, and MT2A promoters, and the CpG shores of the MT1F promoters, were significantly more highly methylated under DDX20-deficient conditions, as determined by the comprehensive Illumina Quantitative Methylation BeadChip method (Table 4, Supporting Table 2, and GSE 37633). A crucial step in DNA methylation involves DNA methyltransferase (Dnmt), which catalyzes the methylation of CpG dinucleotides in genomic DNA.40 The methylation status of MT promoters is mediated specifically by Dnmt1.41 Because Dnmt1 contains a predicted miRNA-140-3p target site in its 3′ UTR, with a perfect match to its seed sequences (Fig. 3B), and because the effects of miRNA-140-3p activity were impaired in DDX20-knockdown cells,23 it was hypothesized that whereas miRNA-140 normally targets and suppresses Dnmt1 protein expression, miRNA-140-3p dysfunction due to DDX20 deficiency results in enhanced Dnmt1 expression, leading to hypermethylation of MT promoters. Consistent with this hypothesis, Dnmt1 expression was increased significantly in DDX20-knockdown cells (Fig. 3C). miRNA-140 precursor overexpression suppressed activity of the Dnmt1 3′ UTR reporter construct, the effect of which was lost when two mutations were introduced into its seed sequences (Fig. 3D). MiRNA-140 precursor overexpression suppressed Dnmt1 protein expression (Fig. 3E). These results indicate that miRNA-140 directly targets Dnmt1 and suppresses its expression in the normal state. Consistently, decreased DDX20, increased Dnmt1, and decreased MT expression were detected together in human clinical HCC samples, as determined via immunohistochemistry (Fig. 3F). By contrast, miRNA-140 precursor-overexpressing Huh7 cells showed increased expression of MTs and reduced NF-κB activity in vitro (Supporting Fig. 4A,B). Moreover, the increase in the number of spheres formed from PLC/PRF/5 cells due to DDX20 knockdown was antagonized by treatment with an NF-κB inhibitor or a demethylating agent (Supporting Fig. 5). Taken together, these results suggest that the up-regulated Dnmt1 protein expression caused by functional impairment of miRNA-140-3p due to DDX20 deficiency results in decreased expression of MTs via enhanced methylation at the CpG sites in their promoters. This may lead to enhanced NF-κB activity and cellular transformation at least in vitro.
|Symbol||CpG Island Methylation Ratio||Target ID|
MiRNA-140 Is a Liver Tumor Suppressor.
To further examine the biological consequences of functional impairment of miRNA-140 due to DDX20 deficiency, we determined the phenotypes of miRNA-140 knockout (miRNA-140−/−) mice (Fig. 4A). Similar to the in vitro DDX20 knockdown results, Dnmt1 expression was increased and MT levels decreased in the liver tissue of these mice (Fig. 4B). NF-κB–DNA binding activity was enhanced in the livers of miRNA-140−/− mice after tail-vein injection of TNF-α, a crucial cytokine that induces NF-κB activity and hepatocarcinogenesis (Fig. 4C). As was found in MT knockout mice, phosphorylation of p65 at serine 276, which is critical for p65 activation, was significantly increased in the livers of miRNA-140−/− mice after DEN exposure, which induces NF-κB activation and liver tumors37 (Fig. 4D). Notably, the size and number of liver tumors that developed 8 months after DEN exposure were markedly elevated in miRNA-140−/− mice compared with control mice (Fig. 4E,F). These results indicate that miRNA-140−/− mice are indeed more prone to liver cancer development and suggest that miRNA-140 acts as a liver tumor suppressor, probably by suppressing NF-κB activity, although we cannot completely exclude other molecular mechanisms. Nonetheless, these results also suggest that the impairment of miRNA-140 function due to DDX20 deficiency may lead to hepatocarcinogenesis in humans, as we have observed in miRNA-140−/− mice (Supporting Figs. 6 and 7).
Here, we report that miRNA-140−/− mice have increased NF-κB activity and are more prone to HCC development. In addition, we show that DDX20, an miRNP component, is frequently decreased in human HCC tissues. Because DDX20 deficiency preferentially causes impaired miRNA-140 function,23 the functional impairment of miRNA-140 may result in phenotypes similar to those of miRNA-140−/− mice and may lead to hepatocarcinogenesis. In support of the hypothesis that DDX20 dysfunction is involved in hepatocarcinogenesis, DDX20 is located at 1p21.1-p13.2, a frequently deleted chromosomal region in human HCC,27 and DDX20 was recently identified as a possible liver tumor suppressor in a functional screen in mice.27 Although the possibility that intracellular signaling pathways other than miRNA-140 may also be involved in the biological consequences of DDX20 deficiency cannot be denied, we believe that functional impairment of miRNA-140 plays a major role in the phenotypes induced by DDX20 deficiency, based on the phenotypic similarities.
Changes in miRNA expression levels have been reported in various tumors.7, 12, 42 However, in this study, we found that reduced expression of an miRNA machinery component might lead to carcinogenesis, at least in part, through functional impairment of miRNAs. Recent studies have shown that components of the RNA interference machinery are associated with the outcome of ovarian cancer patients,43 and that single-nucleotide polymorphisms in miRNA machinery genes can be used as diagnostic risk markers.44, 45 Therefore, the impairment of miRNA function caused by deregulated miRNA machinery components may also be involved in carcinogenesis.
Our study identified Dnmt1 as a critical target of miRNA-140. The decreased MT expression due to the CpG promoter methylation induced by Dnmt1 resulted in enhanced NF-κB activity. This finding was consistent with the results obtained using MT gene knockout mice, in which enhanced NF-κB activation promoted hepatocarcinogenesis.37 The decrease in MT expression that results from increased Dnmt1 expression caused by functional impairment of miRNA-140, together with increased NF-κB activation and hepatocarcinogenesis in MT knockout mice,37 supports the concept that the DDX20/miRNA-140/Dnmt1/MT/NF-κB pathway may play a crucial role in hepatocarcinogenesis. However, we cannot fully exclude the possibility that other intracellular signaling pathways are also involved in the induction of hepatocarcinogenesis by miRNA-140 or DDX20 deficiency, because the precise role of NF-κB in hepatocarcinogenesis has not been clearly defined,8 although constitutive activation of NF-κB signaling has been frequently detected in human HCCs.46 The mechanisms by which DDX20 expression is initially decreased and the reason its locus is frequently deleted in HCC remain to be elucidated. However, because DDX20 expression is also regulated by methylation of its CpG promoter,47 once this pathway is deregulated, decreased DDX20 expression could be maintained by a positive feedback mechanism, even without deletion of its locus.27
In conclusion, this study shows that miRNA-140 acts as a liver tumor suppressor. We show that DDX20, an miRNP component, is frequently decreased in human HCC, which may induce hepatocarcinogenesis via impairment of miRNA-140 function. These results suggest the importance of investigations of not only aberrant miRNA expression levels,12, 14, 17, 48 but also deregulation of miRNP components,22 with subsequent impairment of miRNA function as molecular pathways and possible therapeutic targets for carcinogenesis and other diseases.
- 41Epigenetic regulation of metallothionein-i gene expression: differential regulation of methylated and unmethylated promoters by DNA methyltransferases and methyl CpG binding proteins. J Cell Biochem 2006; 97: 1300-1316., , , , , .
Additional Supporting Information may be found in the online version of this article.
|HEP_26011_sm_SuppFig1.tif||1281K||Supporting Information Figure 1.|
|HEP_26011_sm_SuppFig2.tif||1508K||Supporting Information Figure 2.|
|HEP_26011_sm_SuppFig3.tif||1157K||Supporting Information Figure 3.|
|HEP_26011_sm_SuppFig4.tif||1174K||Supporting Information Figure 4.|
|HEP_26011_sm_SuppFig5.tif||1181K||Supporting Information Figure 5.|
|HEP_26011_sm_SuppFig6.tif||3161K||Supporting Information Figure 6.|
|HEP_26011_sm_SuppFig7.tif||1250K||Supporting Information Figure 7.|
|HEP_26011_sm_SuppTab1.doc||140K||Supporting Information Table 1.|
|HEP_26011_sm_SuppTab2.doc||133K||Supporting Information Table 2.|
Please note: Wiley Blackwell is not responsible for the content or functionality of any supporting information supplied by the authors. Any queries (other than missing content) should be directed to the corresponding author for the article.