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Introduction

Hepatocellular carcinoma (HCC) is one of the most common and aggressive malignant tumors worldwide and the second leading cause of cancer-related mortality (1). Approximately 10 million HCC patients die every year in China. To date, surgical resection and liver transplantation are the only curative treatment options (2). However, tumor metastasis remains the leading cause of death in HCC patients after resection (3). The proliferation and migration of HCC cells are markedly increased by various growth factors and cytokines such as vascular endothelial growth factor (VEGF) (4), tumor necrosis factor-α (TNF-α) (5) and granulocyte colony-stimulating factor (G-CSF) (6).

VEGF is one of the most potent stimulants of progression in several tumor types, mainly because it modulates its target transcription factors through multiple signaling pathways (7,8). Indeed, expression of signaling molecules in the VEGF pathway is elevated in several cardiovascular disorders including acute myocardial infarction, coronary artery disease and atherosclerosis (9–11). However, the exact mechanism(s) involved in the process by which VEGF modulates hepatoma cell proliferation and migration remain unclear. Thus, identification of novel molecular mechanisms, particularly novel inhibitors, in the VEGF-dependent hepatoma cell proliferation and migration process has enormous therapeutic potential.

MicroRNAs (miRNAs) are RNA fragments typically composed of only 20–22 nucleotides. They regulate target genes variously at the RNA and/or protein levels and as a result control downstream cellular processes including proliferation, differentiation, and survival (12). Many lines of evidence have implicated miRNAs in modulating hepatoma cell function by targeting the transcriptional factors or signaling molecules involved in tumor cell proliferation and migration such as miR-25, miR-520e and miR-21-3p (2,13,14). Deregulation of miRNAs expression has also been reported in numerous human cancer types, the miRNAs functioning as tumor suppressors in these cases (15). Recently, miR-199a-5p was reported to be an antagonist of tumor cell proliferation. Shi et al revealed that overexpression of miR-199a-5p in porcine preadipocytes significantly promoted cell proliferation while attenuating lipid deposition in subsequent adipocytes (16). Dai et al also demonstrated repression of sustained endoplasmic reticulum stress and hepatocyte apoptosis by endogenous miR-199a-5p by blocking the IRE1α-related pathway (17). Underexpression of miR-199a-5p contributes to the rise of cell invasion in HCC via the functional deregulation of DDR1 activity (18). Hsu et al also showed that upregulation of miR-199a-5p suppresses cell proliferation, motility, and angiogenesis of these ectopic stem cells by targeting the 3′-untranslated region of VEGFA (19).

Here, we found by miRNA microarray expression analysis that miR-199a-5p, which is abundantly expressed in normal human liver cells, was significantly downregulated in human hepatoma cells. On this basis, we hypothesized that miR-199a-5p regulates tumorigenesis through some unknown pathway. We then found that miR-199a-5p inhibits human hepatoma cell proliferation and migration through targeting the NOR1 gene, which was previously cloned in our laboratory in the Third Xiang Ya Hospital of Central South University (Hunan, China) and identified as a novel tumor suppressor gene (20).

Materials and methods

Cell source and culture

The human normal liver (THLE-3) and human hepatoma (HepG2) cell lines were obtained from ATCC. Both cell lines were cultured in RPMI-1640 (Gibco, USA) supplemented with 10% fetal bovine serum (FBS) (Gibco), 10 mg/ml streptomycin and 10,000 U/ml penicillin in a humidified atmosphere of 5% CO2 at 37°C. The sequence of miR-199a-5p is ccc agt gtt cag act acc tgt tc, and the anti-sequence is ggg tca caa gtc tga tgg aca ag.

Microarray analysis

Total RNAs obtained from cells were subjected to the Mammalian miRNA Array Service V4.0 (CapitalBio Corp., Beijing, China) and analyzed.

PCR and quantitative RT-PCR analysis

Total RNAs were extracted using TRIzol reagent (Invitrogen, Carlsbad, CA, USA) following the manufacturer's instructions. PCR was performed for miR-199a-5p (F_Pr: 5′-TCCAGCTGGGCCCAGTGTTCAGACTAC-3′ ; R_Pr: 5′-GTGTCGTGGAGTCGGCAATTC-3′), human NOR1 (F_Pr: 5′-TGTTAGGCTAGCGATTGAGTTATTTGCTTACAC-3′ and R_Pr: 5′-CTGCACGAATTCGGTTAAGTATGGCCCGATCTA-3′). SYBR® Green assays (Invitrogen) was used for quantitative RT-PCR. All samples were run in triplicate in 96-well reaction plates using the Applied BioSystems 7300 Sequence Detection system (Perkin-Elmer Applied Biosystems, Foster City, CA, USA). The reactions started at 95°C for 5 min followed by 38 cycles of 95°C for 15 sec, 60°C for 30 sec and 72°C for 30 sec. All experiments were repeated three times.

Western blotting

The anti-NOR1 rabbit monoclonal antibody (Epitomics, CA, USA) diluted at 1:1,000, and the anti-GAPDH rat monoclonal antibody (Beyotime, China) at 1:1,000, were used for western blotting. Band intensities were quantified by grayscale and analyzed.

Cell proliferation and migration

Cell proliferation was measured by the MTT assay (MTT cell proliferation assay kit, Invitrogen™). HepG2 cells (2×104 per well) were seeded on cell culture inserts (8-µm pore size) (Millipore Cell, USA) with serum-free RPMI-1640 in triplicate. Those inserts were then put into a 24-well culture plate. Twelve hours later the medium was removed and the inserts were washed with PBS and stained after fixation. After rinsing with water, images were photographed in three random fields (×400). Cell migration was tested and quantified.

Luciferase reporter assay

The pMIR-NOR1-3′-(UTR) luciferase vector containing the putative binding site for miR-199a-5p in multiple cloning sites in the 3′-UTR of pMIR-REPORT™ microRNA Expression Reporter Vector (Ambion) was constructed according to the manufacturer's instructions. HepG2 cells were plated at 2×105 cells/well in 12-well plates in triplicate. The pMIR-NOR1-3′-UTR vector (200 ng) together with the β-gal expressing vector pMIR-REPORT β-gal (200 ng) (Ambion) were co-transfected with either Pre-miR™ miRNA precursor molecules or negative control miRNA precursors (Ambion). Luciferase assays and β-gal enzyme assays were performed 24 h after the transfection according to the manufacturer's protocol (Promega Corp., Madison, WI, USA). Firefly luciferase activity was normalized to β-gal expression for each sample.

Construction of lentiviral vector

The lentiviral miR-199a-5p overexpression system (LV-miR-199a-5p) was amplified by PCR. The two ends of miR-199a-5p were linked with restriction enzyme cutting sites. The PCR products of the target gene and pSMPUW-U6-Puro lentiviral expression vector (Cell Biolabs, Inc.) were respectively digested with BamHI and SalI. After purification by electrophoretic separation, the target fragments were induced into competent cells followed by incubation in Luria-Bertani broth containing ampicillin to select overnight. Single colonies were picked as putative products. The products of PCR amplification were identified by restriction endonuclease digestion and sequenced.

Lentiviral packaging and titer determination

The lentiviral vector and pRSV-Rev lentivirus package vector (Invitrogen) were co-transfected into HepG2 cells. Supernatants were collected by centrifugation 24 and 48 h after transfection, respectively, for concentration tests. The lentivirus titers at each time-point were analyzed by the hole-by-dilution titer assay.

Lentiviral transduction

HepG2 cells were infected with lentivirus (LV-miR-199a-5p) when they were 50–70% confluent. In our pre-experiment, the MOI (multiplicity of infection) value gradients were 0, 10, 50 and 100. Corresponding doses of LV-miR-199a-5p were, respectively, added. LV-GFP was transduced as control. The green fluorescent protein (GFP) expression levels were measured 72 h after transduction in order to determine the optimal MOI value, which was used in subsequent experiments.

Statistical analysis

All results are presented as means ± SEM of at least three independent experiments, unless otherwise indicated. Student's t-test was used to assess differences between two groups. P<0.05 was considered statistically significant.

Results

The expression levels of miRNAs in HepG2 and THLE-3

To identify miRNAs that contribute to the proliferation of liver cells, we performed microarray analysis of miRNA expression in a normal human liver cell line (THLE-3) and a human hepatoma cell line (HepG2). There were significant differences between the two cell lines in the expression of 149 miRNAs, 58 of which were upregulated and 91 downregulated. Consistent with previous reports, miR-let-7e was significantly downregulated in the cancer cells (21). miRNAs with significantly altered expression levels are listed in Table I; they include miR-199a-5p, which was significantly downregulated in the hepatoma cells.

Table I The expression levels of miRNAs in HepG2 and THLE-3 cells.

Table I

The expression levels of miRNAs in HepG2 and THLE-3 cells.

miRNA name	score(d)	Fold change
miR-199a-5p	−61.3854	0.16945
miR-18b	−54.7323	0.11342
miR-let-7e	−43.3242	0.12435
miR-193a	−36.3287	0.08793
miR-224	−28.6543	0.15276
miR-28	−25.9657	0.25738
miR-19a	−20.1985	0.09486
miR-434	−16.9874	0.28677
miR-122a	94.3249	28.43587
miR-422b	85.4535	25.45325
miR-520e	70.45278	23.97742
miR-134	63.32455	14.554325
miR-198	43.31445	8.43529
miR-202	28.45289	3.23455
miR-382	20.35185	2.55325
miR-520b	13.31457	1.98097

miR-199a-5p expression was attenuated in VEGF-induced HepG2 cells

VEGF contributes to some key stages of tumorigenesis, including the function of cancer stem cells and tumor initiation (22). To confirm the importance of miR-199a-5p in regulating tumorigenesis, we performed miRNA microarray analysis in HepG2 cells at 0, 3, 6, and 24 h after VEGF stimulation with an IC50 of 60 ng/ml (Fig. 1B).

Figure 1 miR-199a-5p expression is attenuated in VEGF-induced HepG2 cells. HepG2 cells, seeded in 6-well plates, were cultured with 0.5% FBS for 48 h before treatment with either VEGF (60 ng/ml) or RPMI-1640 as a negative control. (A) VEGF caused a dose-dependent decrease of miR-199a-5p expression in HepG2 cells in 24 h, which was tested by qRT-PCR (n=3) (P<0.05). (B) VEGF caused a time-dependent decrease of miR-199a expression in HepG2 cells, detected by qRT-PCR (n=3) (P<0.05).

The relative expression levels of miR-199a-5p were normalized to U6, the endogenous control. The results showed that VEGF stimulation on HepG2 cells caused a significant dose-dependent reduction in the expression levels of miR-199a (Fig. 1A). These findings revealed that miR-199a and VEGF expressions are negatively correlated.

Effects of micro-RNA199a-5p and VEGF on HepG2 proliferation

To examine the effects of microRNA199a-5p in HepG2 cells further, we examined whether its restoration would affect VEGF-induced cell proliferation. We constructed a lentiviral vector expressing miR-199a-5p (LV-miR-199a-5p) and showed that transductions with increasing multiplicity of infections (MOI) of LV-miR-199a-5p significantly elevated miR-199a-5p expression in HepG2 cells (Fig. 2A). Forced expression of miR-199a-5p by LV-miR-199a-5p strongly inhibited cell proliferation over the range 10-100 MOI (Fig. 2B). The effects of miR-199a-5p on HepG2 cell proliferation were further elucidated by measuring the expression of PCNA, a marker of tumor cell proliferation. VEGF significantly increased PCNA expression, but this was markedly attenuated in miR-199a-5p-overexpressing cells (Fig. 2C and D), while miR-199a-5p knock-down by anti-miR-199a-5p increased proliferation (Fig. 2E and F).

Figure 2 miR-199a-5p inhibits VEGF-induced HepG2 proliferation. (A) HepG2 cells were infected with LV-miR-199a-5p or LV-GFP at different MOI. LV-miR-199a-5p induced the overexpression of miR-199a-5p in a dose-dependent manner. (B) HepG2 cells were infected with LV-miR-199a-5p (0–100 MOI) or LV-GFP (0–100 MOI), treated with or without VEGF (60 ng/ml) for 24 h. LV-miR-199a-5p significantly decreased VEGF-induced cell proliferation, shown by the MTT proliferation assay (n=3) P<0.05. (C) HepG2 cells infected with LV-miR-199a-5p (100 MOI) or LV-GFP (100 MOI) were incubated with VEGF (60 ng/ml) for 24 h and analyzed for PCNA protein levels by western blotting. (D) PCNA protein levels in the western blots were quantified by grayscale and analyzed (n=3). (E) The inhibitory effect of anti-microRNA199a-5p on miR-199a-5p expression in HepG2 cells. (F) Anti-miR-199a-5p (80 nM) significantly restored HepG2 cell proliferation stimulated by VEGF (60 ng/ml), detected by the MTT proliferation assay (n=3).

Effects of micro-RNA199a and VEGF on hepatoma cell migration

Hepatoma cell migration is also thought to make an important contribution to the progression of HCC. To determine whether miR-199a-5p is also involved in this process, we performed cell migration experiments, and the results verified that miR-199a-5p suppresses tumorigenesis. miR-199a inhibited cell migration induced by VEGF overexpression (Fig. 3A and B). Moreover, consistent with our hypothesis, the inhibition of miR-199a expression by anti-miR-199a restored the capacity of HepG2 cells for migration (Fig. 3C and D). These results suggest that miR-199a modulates tumorigenesis by changing key factors in the tumor microenvironment.

Figure 3 miR-199a-5p inhibits VEGF-induced hepatoma cell migration. (A) HepG2 cells were seeded in triplicate on inserts and incubated for 12 h with or without either VEGF or LV-micro-RNA199a before staining. (B) The number of migrated cells was quantified (×400). Results were confirmed by triple independent experiments. (C) After infection with anti-miR-199a-5p or negative control oligos, HepG2 cells were seeded in triplicate on inserts and incubated for 12 h with or without VEGF; migrated cells were counted (×400). (D) The number of migrated cells was quantified (×400). Results from three independent experiments are reported.

NOR1 is a direct target of miR-199a-5p

Our previous study revealed NOR1 as a candidate tumor suppressor that inhibits the development and/or progression of tumors (23). In this study, when we searched the target scan database, we found that NOR1 is a potential target of miR-199a-5p. As shown in Fig. 4A, human NOR1 mRNA has a potential miR-199a-5p binding site in its 3′-UTR. To verify whether miR-199a-5p binds directly to the 3′-UTR sequence of NOR1 mRNA, down-regulating its expression, the 3′-UTR sequence containing the putative binding was cloned into an assay-ready luc-UTR reporter vector. The constructed vector was then co-transfected with either miR-199a-5p or control plasmid into HepG2 cells. As shown in Fig. 4B, the luciferase activity was inhibited in the miR-199a-5p-induced cells but not in the control group. To verify that NOR1 is a functional target gene of miR-199a-5p, we induced miR-199a-5p into HepG2 cells and found that NOR1 expression was decreased at both the mRNA and protein levels despite VEGF treatment (Fig. 4C–E). We therefore measured the proliferation and migration of HepG2 cells treated with NOR1 to test its role in the inhibition of tumorigenesis by miR-199a-5p. The results indicate that overexpression of NOR1 significantly inhibits the proliferation (Fig. 5A) and migration (Fig. 5B and C) of HepG2 cells.

Figure 4 NOR1 is a direct target of miR-199a-5p. (A) The sequences of wild-type and mutated NOR13′-UTR (NOR13′-UTR and NOR13′-UTR2, respectively) binding sites for miR-199a-5p are shown. (B) HepG2 cells were co-transfected by the luciferase reporter vectors carrying either NOR13′-UTR or NOR13′-UTR with miR-199a-5p, anti-miR-199a-5p, or negative microRNA. Luciferase was assayed 48 h after transfection. (C) miR-199a-5p or negative microRNAs were transfected into HepG2 cells for 24 h with or without VEGF (60 ng/ml) treatment. Then total RNAs were extracted and the NOR1 mRNA level was measured by qRT-PCR. (D) Stimulatory effects of miR-199a-5p overexpression on NOR1 expression after a 6-h VEGF treatment. (E) NOR1 protein levels were analyzed by grayscale.

Figure 5 miR-199a-5p inhibits cell proliferation and migration through targeting NOR1. (A) HepG2 cells were infected with either GFP, NOR1, miR-199a-5p or miR-199a-5p supplemented with NOR1, treated with or without VEGF (60 ng/ml). The cell proliferation levels were tested by the MTT method, n=3 (P<0.05). (B) HepG2 cells were infected as in (A) and transferred into cell inserts at 2×104 cells/insert, and then cell migration was measured after stimulation by VEGF for 24 h, n=3. (C) The number of migrating cells was quantified (×400). Results from three independent experiments are reported.

Discussion

miR-199a-5p has been shown to have a wide range of functions and can behave differently in different systems and diseases. For example, it is a major regulator of lung fibroblasts and livers in injured tissues (24,25). Rapid downregulation of miR-199a-5p is required for upregulation of HIF1α to protect cells exposed to hypoxia (26). Its behavior in tumors could be more complicated. In different contexts it can function either as an oncogene (27,28) or as a tumor suppressor (29,30). In our previous study, miR-199a-5p was confirmed as a tumor suppressor in hepatocellular carcinoma. However, details of the mechanism by which it regulates tumorigenesis remained unclear. In our present study, we demonstrated that it inhibits HepG2 cell proliferation and migration through targeting NOR1. We also found that miR-199a-5p expression in hepatocellular carcinoma was markedly lower than in normal liver cells because it was downregulated both time- and dose-dependently by VEGF stimulation, which induces the deterioration of hepatocellular carcinoma. Restoration of miR-199a-5p markedly attenuated VEGF-induced HepG2 cell proliferation and migration, and knock-down of miR-199a-5p by anti-miR-199a-5p had the opposite effect on tumorigenesis.

MicroRNAs are highly conserved small regulatory RNAs that antagonize the expression of target genes by hybridizing to specific binding sites in the 3′-untranslated regions (UTR) of many mRNAs (31). Upon microRNA-guided recruitment of a multi-protein complex, either the target mRNA is degraded directly or its translation is blocked, depending on the complementarity between the microRNA and its binding site (32). In our study, we confirmed that miR-199a-5p inhibited cell proliferation and migration via NOR1 by binding directly to the 3′-UTR of its mRNA. NOR1, the gene for which is located in a 120 kb region at 1p34.3, was first isolated from nasopharyngeal cells (NPCs) (23). Zeng et al showed that NOR1 mRNA expression was frequently downregulated in NPCs (33). This epigenetic silencing of NOR1 impaired the cellular protective response to environmental stresses by normal NPCs and promoted nasopharyngeal carcinogenesis (34). Our previous study confirmed NOR1 as a tumor suppressor, the expression of which was significantly downregulated in nasopharyngeal carcinoma, hepatocellular carcinoma and gastric cancer (20). Previously, regulation of NOR1 expression by microRNAs had not been demonstrated. In this study, we showed that miR-199a-5p binds directly to the 3′-UTR of human NOR1 mRNA and downregulates NOR1 expression at both the mRNA and protein levels with or without VEGF stimulation. Overexpression of NOR1 strongly inhibited the proliferation and migration of HepG2 cells, i.e., miR-199a-5p is a novel post-transcriptional regulator of NOR1.

In conclusion, this study identified miR-199a-5p as a potential regulator of human HepG2 cells that acts by targeting the 3′-UTR of NOR1 mRNA. miR-199a-5p expression was substantially downregulated in human hepatocellular carcinoma but not in normal liver cells. Re-establishment of its expression markedly inhibited both cell proliferation and migration in response to VEGF stimulation. Our study provides novel insight into the molecular mechanisms associated with tumorigenesis, and suggests a potential therapeutic target for human HCC treatment.

In conclusion, this study showed that miR-199a-5p antagonizes tumorigenesis owing to its inhibitory role in cell proliferation and migration. Through binding to the 3′-UTR of human NOR1 mRNA, miR-199a-5p enhances NOR1 expression, and then NOR1 inhibits cell proliferation and migration induced by VEGF.

Acknowledgments

This study was supported by National Natural Science Foundation of China (NSFC) (grant nos. 81573091, 30300383, 81072270 and 81101828), and Fundamental Research Funds for the Central Universities (no. 2011JQ030).

References