Role of microRNA-199a-5p and discoidin domain receptor 1 in human hepatocellular carcinoma invasion
© Shen et al. 2010
Received: 13 January 2010
Accepted: 27 August 2010
Published: 27 August 2010
Micro-ribonucleic acid (miRNA)-199a-5p has been reported to be decreased in hepatocellular carcinoma (HCC) compared to normal tissue. Discoidin domain receptor-1 (DDR1) tyrosine kinase, involved in cell invasion-related signaling pathway, was predicted to be a potential target of miR-199a-5p by the use of miRNA target prediction algorithms. The aim of this study was to investigate the role of miR-199a-5p and DDR1 in HCC invasion.
Mature miR-199a-5p and DDR1 expression were evaluated in tumor and adjacent non-tumor liver tissues from 23 patients with HCC undergoing liver resection and five hepatoma cell lines by the use of real-time quantitative RT-PCR (qRT-PCR) analysis. The effect of aberrant miR-199a-5p expression on cell invasion was assessed in vitro using HepG2 and SNU-182 hepatoma cell lines. Luciferase reporter assay was employed to validate DDR1 as a putative miR-199a-5p target gene. Regulation of DDR1 expression by miR-199a-5p was assessed by the use qRT-PCR and western blotting analysis.
A significant down-regulation of miR-199a-5p was observed in 65.2% of HCC tissues and in four of five cell lines. In contrast, DDR1 expression was significantly increased in 52.2% of HCC samples and in two of five cell lines. Increased DDR1 expression in HCC was associated with advanced tumor stage. DDR1 was shown to be a direct target of miR-199a-5p by luciferase reporter assay. Transfection of miR-199a-5p inhibited invasion of HepG2 but not SNU-182 hepatoma cells.
Decreased expression of miR-199a-5p contributes to increased cell invasion by functional deregulation of DDR1 activity in HCC. However, the effect of miR-199a-5p on DDR1 varies among individuals and hepatoma cell lines. These findings may have significant translational relevance for development of new targeted therapies as well as prognostic prediction for patients with HCC.
- AJCC/UICC :
American Joint Committee on Cancer and International Union Against Cancer
- AUROC :
area under receiver operating characteristics
- β-gal :
- CI :
- DDR1 :
discoidin domain receptor-1
- GAPDH :
- HCC :
- HMBS :
- HPRT1 :
hypoxanthine phosphoribosyl-transferase 1
- MGB :
minor grove binder
- miRNA :
- miR-199a-5p :
microRNA excised from the 5' arm of microRNA-199a precursor
- NT :
- qRT-PCR :
quantitative real-time reverse transcription polymerase chain reaction
- RIN :
RNA integrity number
- RNU44 :
small nucleolar RNA C/D box 44
- SD :
- SDHA :
succinate dehydrogenase complex, subunit A
- SE :
- siRNA :
short interfering RNA
- UTR :
- WST :
water soluble tetrazolium
Hepatocellular carcinoma (HCC) is the fifth most common malignancy worldwide and has an increasing incidence in western countries . Although the risk factors for HCC are well characterized, the molecular pathogenesis of this particular tumor type is not well understood . Micro-ribonucleic acids (miRNAs) represent an abundant class of endogenous small RNA molecules of 20-25 nucleotides in length  capable of mediating a vast gene regulatory network . MiRNAs can regulate gene expression by direct cleavage of targeted messenger-RNAs (mRNAs) or by inhibiting translation through complementarity to targeted mRNAs at the 3'untranslated regions (UTRs) . Computational analysis indicates that the total number of miRNAs may be greater than 1% of the protein coding genes in the human genome . To date, 721 human miRNAs are annotated in the miRBase release 14.0 database . Genes targeted by miRNAs control multiple biological processes in health and disease , including cancer development . Accumulating evidence suggests that some miRNAs may function as oncogenes or tumor suppressors . Recently, miRNA expression patterns have been investigated in HCC [11–16]. Although decreased expression of miR-199a-5p has been frequently demonstrated in HCC [11, 12, 15], functional analysis and translational relevance of this phenomenon has not been defined. The discoidin domain receptor (DDR) belongs to a novel class of receptor tyrosine kinases with a characteristic discoidin homology domain, stalk region, transmembrane region, juxtamembrane region, and kinase domain . The DDR family consists of two members, DDR1 and DDR2, which can be alternatively spliced into five DDR1 isoforms (DDR1a-e) . Over-expression of DDR1 was detected in several human cancers including breast , ovary , and lung . The precise mechanism(s) by which these receptors may contribute to oncogenesis are not yet known. Targeted deletion of DDR1 in mice results in severe defects in placental implantation and mammary gland development , suggesting a potential role in cell migration and extracellular matrix degradation. Over-expression of DDR1 has been shown to increase the migration and invasion of hepatoma cells in vitro , implicating a causal role of DDR1 in promoting tumor progression. DDR1 is predicted to be a potential target of miR-199a-5p using publicly available PicTar (4-way), TargetScanS, and miRanda algorithms . Thus, we postulated that aberrantly expressed miR-199a-5p may contribute to invasion by modulation of DDR1 expression in HCC patients.
Patients, tissues, cell lines, and cultures
Demographic data of patients (n = 23)
Value ± SD1 (%)
63.6 ± 15.5
Underlying liver disease
Alcoholic liver disease
Nonalcoholic fatty liver disease
Multiple tumoral nodules
Tumor size - diameter of the biggest nodule (cm)
8.6 ± 4.3
Tumor grade 2
Generation of firefly luciferase constructs
Standard molecular biology techniques were used for generation of all constructs. For generation of a reporter vector bearing a human DDR1 fragment with putative miR-199a-5p binding sites, target sequences were cloned in the pMIR-REPORT Luciferase vector (Ambion). The pMIR-REPORT™ miRNA Expression Reporter Vector System consists of an experimental vector with a firefly luciferase reporter gene under the control of a cytomegalovirus promoter/termination system and an associated beta-galactosidase (β-gal) reporter gene control plasmid. The 3'UTR of the luciferase gene contains a multiple cloning site for insertion of predicted miRNA binding targets. By cloning a predicted miRNA target sequence into pMIR-REPORT, the luciferase reporter is subjected to regulation that mimics the miRNA target. The pMIR-REPORT β-gal reporter plasmid is used for transfection normalization. A human DDR1 3'UTR 457-bp fragment bearing all 4 putative binding sites for miR-199a-5p, which are identical among all the DDR1 splice variants, was generated by RT-PCR from total RNA extracted from HepG2 cells. Primers (SpeI and HindIII restriction sites are underlined) used to amplify this fragment were 5'-ACTAGTTTCCTTCCTAGAAGCCCCTGT-3' (forward primer) and 5'-AAGCTTCCCCAAT CCCAATATTTACTCC-3' (reverse primer) (Eurofins MWG Operon, Ebersberg, Germany). The PCR product was purified on agarose gel, isolated and first inserted into the pGEM-T easy vector (Promega) following the manufacturer's instructions. The pGEM-T plasmids were digested with the appropriate restriction enzymes and electrophoresed in agarose gel. The isolated insert was then excised from the gel, purified and subsequently subcloned in the HindIII/SpeI site of the pMIR-REPORT Luciferase vector. Plasmid constructs were verified for correctness by DNA sequencing using ABI PRISM® 3130 Genetic Analyzer (Applied Biosystems).
Pre-miR™ miRNA precursors of miR-199a-5p and non-targeting control miRNA precursors (Pre-miR™ miRNA Precursor Molecules-Negative Control #1) were purchased from Ambion, Inc. (Austin, TX, USA). Short interfering RNA (siRNA) against DDR1 mRNA (DDR1-siRNA) and a negative control siRNA were obtained from Qiagen (Hilden, Germany). Transfections of miRNA, siRNA as well as cotransfections of miRNA precursors and reporter vectors were performed using Lipofectamine 2000 (Invitrogen Corporation, Carlsbad, CA, USA). Conditions for HepG2 and SNU-182 cells were optimized to yield transfection efficiencies of 78% and 67%, respectively, with a cell viability > 80%. GAPDH knockdown and cell viability were both assessed by the KDalert™glyceraldehyde-3-phosphate dehydrogenase (GAPDH) assay kit (Ambion) for small RNA transfection.
Primers and probes for miR-199a-5p, DDR1 and reference genes.
(6-FAM) TTCAGTTGAGGAACAGGT (MGB*)
(6-FAM) ACTGAACATGAAGGTCTT (MGB*)
(SYBR Green assay)
Actinomycin D treatment for determination of mRNA stability
HepG2 cells were plated in 24-well plates and transfected with 10 nM of miR-199a-5p, miR-control or 100 nM si-DDR1 as a positive control. After transfection for 6 hours, cells were incubated with or without 4 μg/ml of actinomycin D (Sigma-Aldrich, Chemie GmbH, Steinheim, Germany) for additional 36 hours. Total RNA was extracted from cells and DDR1 mRNA was quantified by qRT-PCR described above. The expression levels of DDR1 are presented as values normalized against 106 copies of β-actin transcripts.
Luciferase reporter assay
The pMIR-DDR1-3'-untranslated region (UTR) luciferase vector, containing the putative binding site for miR-199a-5p in the multiple cloning site within the 3' UTR of the luciferase gene in the pMIR-REPORT™ miRNA Expression Reporter Vector (Ambion) was constructed according to the manufacturer's instructions. HepG2 cells were plated at 2 × 105 cells/well in triplicate in 12-well plates. The pMIR-DDR1-3'-UTR construct (200 ng) together with β-gal expression vector pMIR-REPORT β-gal (200 ng) (Ambion) was cotransfected with Pre-miR™ miRNA Precursor Molecules or negative control miRNA precursors (Ambion). Luciferase assays and β-gal enzyme assays were performed 24 hours after transfection according to the manufacturer's protocol (Promega Corporation, Madison, WI, USA). Firefly luciferase activity was normalized to β-gal expression for each sample.
Matrigel matrix invasion assay
Cell invasion assays were conducted using BD BioCoat Matrigel Invasion chambers (BD Biosciences Clontech, Heidelberg, Germany). Briefly, 48 hours after transfection 4 × 104 HepG2 cells or 2 × 104 SNU-182 cells were seeded into the top chamber with a Matrigel coated filter and 750 μl Dulbecco's Modified Eagle Medium containing 5% fetal bovine serum was used as a chemoattractant. Simultaneously, 100 μl of the cell suspension was seeded into 96-well plate in five replicates for cell number normalization using WST-1 assay. Inserts were incubated at 37°C with 5% CO2 for 22 hours. After incubation, cells that were still on the upper side of the filters were mechanically removed. Cells that migrated to the lower side were fixed with 100% methanol and stained with 1% toluidine blue (Sigma-Aldrich) in 1% borax (Sigma-Aldrich). Cells were counted in five fields for triplicate membranes at 10× magnification using an inverted optical microscope (Nikon ECLIPSE TS100, Nikon, Japan). The WST-1 assay was also performed and the invasion index was normalized to cell numbers.
Cell proliferation assay
Cell proliferation was assessed using the water-soluble tetrazolium-1 (WST-1) assay following the manufacturer's protocol (Roche Applied Science). Cells were plated at the density of 8,000/well in 96-well plates (BD Biosciences, Rockville, MD), transfected as described above, and incubated at 37 °C. Cell proliferation was assessed after 72 hours in 5 replicates.
Transfected cells were washed once with ice-cold PBS and lyzed in 1 × blue loading buffer (Cell Signaling Technology, Danvers, MA, USA) supplemented with a protease inhibitor cocktail (Roche Diagnostics). Protein samples were subjected to 10% of SDS-PAGE and blotted with primary antibody selectively recognizing DDR1 (C-20, Santa Cruz Biotechnology). To determine the amounts of loaded proteins, blots were probed with GAPDH (Cell Signaling Technology). Protein bands were visualized using ECL Plus Western blotting detection reagents (Amersham Biosciences, Buckinghamshire, UK) after incubation with appropriate HPR-conjugated secondary antibodies (Jackson Immuno Research Laboratories, West Grove, PA, USA), followed by exposure to Kodak Bio-Max films (Carestream Health, Paris, France).
Data are expressed as mean ± SD unless otherwise indicated. Categorical data are described as frequency of the subjects with a specific characteristic. Chi-square test or Fisher's exact test was used for comparing categorical data and Student's t-test, Mann-Whitney-U-test, one-way ANOVA or Kruskal-Wallis test, when appropriate, was used for comparing continuous variables. Spearman's rank test correlation coefficient was used to measure the degree of association between two quantitative variables. Agreement of quantitative variables was evaluated by the correlation coefficient (r). To identify potential predictors of AJCC/UICC stage III-IV, univariate and multivariate analyses were performed. A forward stepwise selection method was used to select variables for the multivariate regression model. Two-tailed p-values less than 0.05 were considered statistically significant. Statistical analysis was performed using SPSS software version 12.0 (SPSS Inc., Chicago, IL, USA).
Patient demographic data
A total of 23 patients were enrolled in the study and their characteristics are shown in Table 1. The mean age of the patients was 63.6 ± 15.5 years and 69.6% were male. There was no difference between cirrhotic and non-cirrhotic patients regarding number and size of tumors, AJCC/UICC stage, recurrence, or intrahepatic metastasis.
Decreased miR-199a-5p expression in human HCC tissues and cell lines
Expression of DDR1 in human HCC tissues and cell lines
Results of univariate analysis for factors associated with advanced HCC stage at diagnosis
AJCC/UICC stage I-II
AJCC/UICC stage III-IV
69.4 ± 8.0
60 ± 18.1
Multiple tumor nodules
Diameter of the biggest nodule (cm)
6.7 ± 4.4
9.8 ± 4.0
Poorly differentiated HCC (G4)
Mir-199a expression (median value)
DDR1 expression (median value)
MiR-199a-5p differentially regulates the expression of DDR1 in HepG2 and SNU-182 cells
DDR1 mRNA is predicted to be a potential target of miR-199a-5p by TargetScanS
predicted consequential pairing of target region (top)
and miRNA (bottom)
Position 1165-1185 of DDR1 3' UTR
| | | | | | |
Position 1199-1219 of DDR1 3' UTR
| | | | | |
Position 1260-1280 of DDR1 3' UTR
| | | | | | |
Position 1383-1403 of DDR1 3' UTR
| | | | | | |
MiR-199a-5p differentially modulates invasion of hepatoma cell lines in vitro
MiR-199a-5p does not modulate proliferation of HepG2 and SNU-182 cells in vitro
To characterize the effect of miR-199a-5p on hepatoma proliferation, we performed overexpression studies using the miR-199a-5p specific precursor. Proliferation of HepG2 and SNU-182 cells was neither altered by precursor miR-199a-5p (p = 0.486 and p = 0.073, respectively) nor by DDR1-siRNA (p = 0.980 and p = 0.141, respectively) (Figure 6B). These studies indicate that DDR1 is rather involved in the process of tumor cell invasion than in tumor growth.
Decreased miR-199a-5p expression in HCC has been repeatedly reported, but its functional relevance has not been elucidated to date [11, 12, 15]. A pivotal role for miRNAs in the process of malignant transformation has been suggested in the literature [9, 31]. However, the precise molecular mechanisms by which miRNAs modulate tumor cell biology are largely unknown. MiRNAs from animals were first reported to repress translation without affecting mRNA levels . More recent evidence indicated that miRNAs and siRNAs can control post-transcriptional gene expression by directing the endonuclease cleavage of target mRNA, which is referred to as "slicer" activity . Endonucleolytic cleavage is generally favored by perfect base-pairing between miRNA and mRNA. Some mismatches, however, can be tolerated and still allow endonucleolytic cleavage to occur . The majority of animal miRNAs are only partially complementary to their targets . Several reports have shown that animal miRNAs can also induce significant degradation of target mRNAs despite imperfect mRNA-miRNA base-pairing [36, 37], referred to as "slicer"-independent decay. This phenomenon emphasizes mRNA degradation as an important aspect of miRNA-mediated repression of gene expression. There is also some evidence that "slicer"-independent mRNA decay induced by miRNAs might occur through promotion of mRNA decapping and 5'-3'-degradation . The contribution of translational repression or mRNA degradation to gene silencing appears to differ for each miRNA:target pair and is likely to depend on the particular set of proteins bound to the 3'UTR of the mRNA . MiR-199a-5p, which is partially complementary to the 3' UTR of DDR1 mRNA, induced significant degradation of DDR1 mRNA in hepatoma cells in our study. Thus, DDR1 has been experimentally validated as a target gene of miR-199a-5p. MiR-199b-5p, which has a very similar nucleotide sequence, is also predicted to target DDR1. However, miR-199b-5p was only detectable in four out of 24 tissue samples in a miRNA microarray assay (data not shown) and it has never been reported to be de-regulated in HCC in literature. Therefore, the regulatory function of miR-199b-5p was not further assessed in this study.
DDR1 is a tyrosine kinase receptor for collagen  and its activation can cause tumor invasion which appears to be mediated by matrix metalloproteinases (MMP)2 and MMP9 [19, 23]. Indeed, we found a positive correlation between the expression of DDR1 and MMP2 in our patient cohort (data not shown), further supporting the important role of DDR1 for tumor invasion. Consistent with recently published findings  and our results indicating a critical role for DDR1 in HCC progression, we found a significant correlation between the expression of DDR1 and AJCC/UICC tumor stage. Analyzing predictive factors for advanced tumor stage at the time of HCC diagnosis, we found a positive correlation between AJCC/UICC stage III-IV and poor tumor differentiation, presence of macrovascular invasion, and high DDR1 expression. In addition, multivariate logistic regression analysis identified high DDR1 expression as the single independent factor associated with advanced tumor stage, and, hence, poor prognosis. In line with this clinicopathological observation, we found that DDR1 gene silencing by transfection of miR-199a-5p into HepG2 cells significantly decreased tumor cell invasion in vitro. However, our studies also indicate that DDR1 is rather involved in the process of tumor invasion than in tumor growth. These results demonstrated that miR-199a-5p is capable to modulate tumor cell invasion at least in part by targeting DDR1. Although in our study DDR1 has been validated as a target gene of miR-199a-5p, no significant correlation between miR-199a-5p and DDR1 mRNA expression was found in tumor samples from our patient cohort. In addition, SNU-182 hepatoma cells exhibited increased levels of expression of both miR-199a-5p and DDR1 mRNA. Transfection of miR-199a-5p did not induce a change in DDR1 mRNA expression, but significantly down-regulated DDR1 protein in SNU-182 cells. However, no significant inhibitory effect on tumor invasion was noted. Considering the preexisting high expression of miR-199a-5p in SNU-182 cells, our results might hint at a certain independence of DDR1 to miR-199a-5p-mediated gene regulation and function in these cells. Our data hint at a more complex regulation network between DDR1 and miR-199a-5p in HCC. One reason might be that HCC is a very heterogeneous tumor entity and distinct cellular components might interfere with the effect of miR-199a-5p on DDR1 . For instance, by the use of miRNA target prediction algorithms other 23 miRNAs were also predicted to target DDR1 . In addition, DDR1 has been demonstrated to be a direct transcriptional target of the p53 tumor suppressor gene  and, therefore, the p53 status in tumor cells may also affect the expression of DDR1. Finally, recent findings reveal a more diverse role for small RNA molecules in the regulation of gene expression than previously recognized. For instance, miRNAs can act also as translation activators under specific cellular conditions . In addition, double strand RNAs can activate rather than repress gene expression by targeting non-coding regulatory regions in gene promoters . To date, the only reported stimulatory effect of miRNA on RNA expression is represented by the interaction between miR-122 and replication of hepatitis C virus RNA in hepatocytes . MiR-199a-5p expression has also been shown to be diversely deregulated in other cancer types. For instance, miR-199a-5p was also found to be down-regulated in ovarian cancer  and oral squamous cell carcinoma , but up-regulated in cervical carcinoma  and bronchial squamous cell carcinoma . Moreover, increased expression of miR-199a-5p has been considered a signature for high metastatic risk and a poor prognosis in uveal melanoma .
Thus, the complexity of the regulation of mRNA by miRNA encountered in our and other studies indicates that the effect of miRNA on its target gene is cell type and environment dependent. However, our study demonstrates a previously uncharacterized biological function of miR-199a-5p such as the ability to inhibit tumor invasion through targeting DDR1.
Less than half of patients with HCC are eligible for potential curative treatment including liver resection and transplantation because of advanced tumor stage at time of diagnosis. The combination of clinical and biological predictors may increase diagnostic accuracy of tumor staging, thus permitting optimized therapeutic management of HCC patients. Although DDR1 expression was shown to be the only predictive factor for advanced HCC, our study was clearly limited by the small sample size which may tend to overestimate the prognostic value of DDR1 expression. Thus, prospective studies that seek and independently validate the prognostic utility of DDR1 expression for patients with HCC in a larger and carefully selected cohort should be conducted. Patient survival after surgical treatment is hampered by frequent tumor recurrence and systemic chemotherapy is largely ineffective . In recent years, kinase inhibitors have become an attractive target class for drug development , and it was shown recently that systemic application of a multikinase inhibitor improves survival of patients with HCC . Further investigation of therapeutic strategies targeting the miR-199a-5p-DDR1 signaling network is therefore warranted. In conclusion, identification of the miR-199a-5p:DDR1 target pair and its crucial role in tumor cell invasion highlight the translational relevance for both prognostic prediction and targeted molecular therapy for patients with HCC.
The authors thank Dr. Brigitte Pützer for kindly providing the HuH-7 cell line and critical reading the manuscript, Dr. Guido Marquitan and Dr. Ruth Broering for helpful suggestions, and Anne Achterfeld for technical assistance. This study was funded in part by 107-05710/IFORES program (V.R.C.) and 107-05470/IFORES program (S.B.) of the University of Essen.
This article is published under license to BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.