MicroRNA-1 and microRNA-499 downregulate the expression of the ets 1 proto-oncogene in HepG 2 cells

MicroRNAs may function to promote or suppress tumor development, depending on the cellular context. The important role of microRNAs in regulating molecular pathways underlying tumorigenesis has been emphasized in hepatocellular carcinoma (HCC). MicroRNAs regulate gene expression via post-transcriptional mechanisms by inhibiting translation or by degrading mRNA. In this study, we show that microRNA-1 (miR-1) and microRNA-499 (miR-499) are capable of repressing the expression of the ets1 protooncogene, which plays a fundamental role in the extracellular matrix (ECM) degradation, a process required for tumor cell invasion and migration. We used luciferase reporter assays to demonstrate that miR-1 and miR-499 target the 3' untranslated region (UTR) of ets1. Overexpression of miR-1 and miR-499 in HepG2 cells led to downregulation of ets1 mRNA and protein as assessed by quantitative reverse transcription PCR and western blot analysis. Furthermore, overexpression of miR-1 and miR-499 inhibited the invasion and migration of HepG2 cells in matrigel invasion and transwell migration assays, respectively. These results suggest that miR-1 and miR-499 may play an important role in the pathogenesis of HCC by regulating ets1.


Introduction
Hepatocellular carcinoma (HCC) is the sixth most common malignancy and the third most common cause of cancer-related deaths worldwide, claiming over one million lives annually (1).The highest incidence rates are reported in East Asia (2).The prognosis of patients with HCC is poor, with a 5-year survival rate after diagnosis of ~10% (3).Ets1 is the founding member of the Ets family of transcription factors and has been shown to promote invasive behavior in multiple cell types (4)(5)(6)(7)(8)(9)(10).The regulation of matrix metalloproteases MMP-1, MMP-3, MMP-9 and urokinase type plasminogen activator (uPA) expression have been ascribed to Ets1 (4)(5)(6)(7)(8)(9)(10).Expression of Ets1 is also associated with poor prognosis in patients with tumors including breast cancer, ovarian tumor, and hepatocellular carcinoma.
Various molecular alterations occur in pre-neoplastic nodules and escalate in HCC, including dysregulation of well-known molecular pathways in carcinogenesis (11)(12)(13)(14).The important role of microRNAs (miRNAs) in regulating these pathways has been emphasized.MiRNAs are small (18-24 nucleotides), evolutionarily conserved, endogenous, single-stranded, non-coding RNA molecules, that negatively modulate gene expression in animals and plants.Mature miRNAs operate via sequence-specific interactions with the 3' untranslated region (UTR) of cognate mRNA targets, causing suppression of translation and mRNA decay (15,16).A large body of evidence suggests that the multigene regulatory capacity of miRNAs is dysregulated and exploited in cancer.Indeed, miRNA loci are often targeted by genetic and epigenetic defects, and miRNA signatures facilitating tumor classification and the prediction of clinical outcome have been reported (17,18).A global reduction of miRNA abundance appears to be a general trait of human cancers, playing a causal role in the transformed phenotype (19)(20)(21).Aberrant expression of miRNA has also been linked to a variety of cancers, including HCC (22)(23)(24)(25).
In the current study, we show that the ets1 proto-oncogene, which is highly expressed in HCC (26), is targeted by miR-1 and miR-499.MiR-1 and miR-499 specifically inhibit the expression of Ets1.Overexpression of miR-1 and miR-499 in the HepG2 HCC cell line inhibited cellular invasion and migration.Taken together, these results suggest that Est1 is negatively regulated by miR-1 and miR-499 in HepG2 cells, which may contribute to the invasive and migratory potential of hepatocellular carcinoma.

Materials and methods
Cell Culture.HepG2 and HEK 293 cell lines (American Type Culture Culture Collection, Manassas, VA, USA) were
Transwell cell invasion and migration assays.For invasion assays, transfected HepG2 cells were serum-starved for 18 h in DMEM containing 0.1% (v/v) FBS.Cells were trypsinized and resuspended in the same medium and 2x10 5 cells were added to the upper chamber of each well (6.5 mm in diameter, 8 µm pore size; Corning, Inc., Corning, NY, USA) coated with 30 mg/cm 2 matrigel extracellular matrix (ECM) gel (Sigma-Aldrich, St. Louis, MO, USA).Medium containing 0.1% (v/v) FBS, supplemented with hepatocyte growth factor (HGF) (20 ng/ml) (ProSpec-Tany TechnoGene, Ltd., East Brunswick, NJ, USA) was placed in the lower compartment of the chamber.After incubation for 24 h at 37˚C, cells on the upper membrane surface were removed by carefully wiping with a cotton swab, and the filters were fixed by treatment with 95% (v/v) ethanol for 30 min.Cells were stained with 0.2% (w/v) crystal violet solution for 30 min.Cells adhering to the undersurface of the filter were counted (five high-power fields/chamber) using an inverted microscope.Migration assays were performed as described above, excluding the use of matrigel and with an incubation time of 12 h.

Statistical analysis.
All values are reported as the means ± standard deviation.Differences were assessed by two-tailed Student's t-test of Excel software.P<0.05 was considered statistically significant.

Results
Interaction of miR-1 or miR-499 with the 3'UTR of Ets1 mRNA.In order to identify miRNAs regulating Ets1, we used TargetScan (www.targetscan.org),an online software program, to predict miRNAs targeting 3'UTR of the ets1 mRNA.This analysis revealed that the 3'UTR of ets1 contains putative sites for >20 miRNAs.We tested the ability of a subset of these miRNAs (miR-1, miR-139, miR-181a, miR-200b, miR-221, miR-365, miR-429 and miR-499) to target the ets1 3'UTR using luciferase reporter assays.Our analysis showed that only miR-1 and miR-499 induced an obvious decrease in relative luciferase activity (Fig. 1A).Further investigation showed that the putative target sites for miR-1 and miR-499 are conserved in mammalian species (Fig. 1B).The target sites for miR-499 and miR-1 locate in the front and rear fragment of 3'UTR of ets1 mRNA, respectively.To investigate this potential interaction experimentally, the 3'UTR of ets1 mRNA was divided into two fragments, Ets1-3'UTR-1 and Ets1-3'UTR-2, and sub-cloned into a modified pGL3 control plasmid (pGL3m), as  previously described (27).We then tested the ability of miR-1 or miR-499 to inhibit luciferase activity of pGL3m-Ets1-3'UTR-1 or pGL3m-Ets1-3'UTR-2 following co-transfection into HEK 293 cells.Our analysis demonstrated that both miR-1 and miR-499 induced a significant decrease in relative luciferase activity (~40%) compared with vector transfected cells (Fig. 1D).To test the specificity of this interaction, miR-1 and miR-499 overexpression constructs were co-transfected with ets1 luciferase reporter constructs containing substitutions disrupting the miRNA target sites (Fig. 1C).We did not observe any decrease in relative luciferase activity in miRNA transfected cells compared with vector control (Fig. 1D).
miR-1 and miR-499 downregulate Ets1 expression.To investigate whether miR-1 or miR-499 affect endogenous Ets1 protein expression, we transfected miR-1 and miR-499 duplexes into HepG2 cells and analyzed Ets1 expression after 48 h by western blot analysis.As a positive control, cells were also transfected with Ets1 siRNA.We found that miR-1 and miR-499 dramatically reduced the expression of Ets1 protein compared to negative control (Fig. 2A).qRT-PCR analysis of ets1 mRNA expression 48 h after transfection with miR-1 and miR-499 duplexes, revealed that the level of Ets1 mRNA was also significantly reduced compared to negative control (Fig. 2B).There were no significant differences on Ets1 protein and mRNA levels between overexpression of individual miRNAs or co-transfection of both miR-1 and miR-499.
miR-1 and miR-499 negatively regulate cell invasion and migration in vitro.HGF (hepatocyte growth factor), a cytokine also known as scatter factor (SF), can significantly promote the invasion of hepatoma carcinoma cells (28).Moreover, Ets1 has been shown to play a key role in the acquisition of invasive behavior by inducing the expression of MMP-1, MMP-3, MMP-9 and uPA (4).Given that miR-1 and miR-499 are capable of affecting Ets1 expression, we examined the effect of overexpressing these miRNAs on HGF-induced invasiveness of HepG2 cells using the matrigel invasion assay system.As shown in Fig. 3A, miR-1 and miR-499 significantly reduced HGF-induced invasion of HepG2 cells.Next, we examined the effect of miR-1 and miR-499 on HGF-induced migration of HepG2 cells using transwell migration assays.Similar to the invasion assays, miR-1 and miR-499 also inhibited the migration behavior of HepG2 cells (Fig. 3C).To eliminate the possibility of off-target effects, we transfected cells with Ets1 siRNA.In a similar manner to miRNA overexpression, we found that knockdown of Ets1 inhibited the invasion and migration induced by HGF (Fig. 3A and C).

Discussion
Growing evidence indicates that Ets1 plays a key role in the invasive behavior of many mammalian tumors.In this study, we demonstrate that miR-1 and miR-499 negatively regulate the ets1 proto-oncogene at the post-transcriptional level, via conserved sites within the 3'UTR.Furthermore, overexpression of miR-1 or miR-499 inhibited the invasion and migration of HepG2 cells in vitro, emphasizing the essential role of these two miRNAs in hepatic oncogenesis and tumor behavior.
In general, miRNAs may function as both tumor suppressors and oncogenes in tumors.The correlation between the expression of specific miRNAs and cancer has been widely observed.It has also been shown that a global reduction of miRNA abundance may be a general trait of human cancers (19)(20)(21).This suggests that miRNAs may have a crucial function in cancer progression (29).
Overexpression of Ets1 is highly associated with many types of cancer.Ets1 expression is generally higher in invasive tumors than in benign tumors (6,9), and is indicative of poor prognosis (7,9,26,30).The expression of Ets1 is also correlated with histological differentiation of HCC (26).This suggests that Ets1 is higher in poorly differentiated HCC and may yield relative biological information to HCC.
There is growing evidence to show that Ets1 may also be involved in the regulation of c-Met, the receptor for HGF/SF, which induces migration (35).Furthermore, c-Met may also activate Ets1, as HGF/SF has been shown to stimulate Ets1 activity through the Ras/Raf/MEK1/ERK1/2 pathway in MDCK cells (36).
In our study, we found that miR-1 and miR-499 inhibit HGF-induced invasion and migration in HCC HepG2 cells, by repressing the expression of the ets1 proto-oncogene.These results indicate that miR-1 and miR-499 may represent candidates for anticancer therapy.In conclusion, miR-1 and miR-499 inhibit Ets1 expression by binding to the 3'UTR of the ets1 mRNA, thereby reducing HGF-induced cell invasion and migration.

Figure 2 .
Figure 2. miR-1 and miR-499 regulate Ets1 expression at the post-transcriptional level.(A) Ets1 protein was measured in HepG2 cells by western blot analysis 48 h after transfection with synthetic miR-1 and miR-499 duplexes, or Ets1 siRNA.β-actin was used as an internal loading control.(B) The effect of miR-1 and miR-499 on ets1 mRNA expression was analyzed by qRT-PCR.Ets1 levels were calculated using GAPDH as an internal control.Results represent the mean of triplicate qRT-PCR assays ± standard deviation ( * P<0.05).

Figure 3 .
Figure 3. miR-1 and miR-499 suppress invasion and migration of HepG2 cells by inhibiting Ets1 expression.(A) Cell invasion was assayed using matrigel invasion chambers.HepG2 cells were transfected with miR-1 and miR-499 duplexes or Ets1 siRNA and plated 48 h after transfection in 24-well transwell plates.Mock-transfected cells (no miRNA or siRNA) or cells transfected with negative control siRNA were used as controls.Cells were added to the upper chamber of each well.After 24 h of incubation, cells that invaded through the pores to the under surface of the membrane were fixed, stained and counted.Five random microscopic fields were counted for each treatment.(B) Cell numbers represent the average count of five random microscopic fields.Each bar represents the mean ± standard deviation of the counts from a single representative experiment ( *** P<0.001).(C) miR-1 and miR-499 blocked cell migration as assayed in transwell chambers, following incubation for 12 h and using HGF (20 ng/ml) as a chemoattractant.Cells that migrated through the filter were fixed and stained with crystal violet.Representative images of the lower surface of membrane are shown.(D) Results represent the average of three experiments ± standard deviation from a representative experiment ( *** P<0.001).

Figure 4 .
Figure 4.A schematic representation showing how miR-1 and miR-499 modulate the invasive and migratory behaviors of HepG2 cells via modulation of Ets1.