MicroRNA-34a Inhibits Uveal Melanoma Cell Proliferation and Migration through Downregulation of c-Met

Abstract

purpose. MicroRNAs (miRNAs) are endogenously expressed, noncoding, small RNAs that inhibit protein translation through binding to target mRNAs. Recent studies have demonstrated that miRNAs can regulate tumor cell proliferation and migration. MicroRNA-34a (miR-34a), a potential key effector of the p53 tumor-suppressor gene, was studied as a potential tumor suppressor in uveal melanoma.

methods. Northern blot analysis was performed to detect the expression level of miR-34a in uveal melanoma cells and melanocytes. Subsequently, melanoma cell proliferation and migration were examined by MTS cell proliferation and transwell migration assays, respectively. The target of miR-34a was predicted by bioinformatics and confirmed using a luciferase assay. In addition, expression of c-Met and cell cycle-related proteins was determined by Western blotting and immunofluorescence after the introduction of miR-34a.

results. miR-34a is actively expressed in melanocytes but not in uveal melanoma cells based on Northern blot analysis. Transfection of miR-34a into uveal melanoma cells led to a significant decrease in cell growth and migration. After identification of two putative miR-34a binding sites within the 3′ UTR of the human c-Met mRNA, miR-34a was shown to suppress luciferase activity using HEK293 cells with a luciferase reporter construct containing the binding sites. miR-34a was confirmed to downregulate the expression of c-Met protein by Western blotting and immunofluorescence. Furthermore, the introduction of miR-34a downregulated phosphorylated Akt and cell cycle-related proteins.

conclusions. These results demonstrate that miR-34a acts as a tumor suppressor in uveal melanoma cell proliferation and migration through the downregulation of c-Met.

Uveal melanoma is the most common primary intraocular malignancy in human adults, and the uvea is the second most common site for primary melanoma after the skin. ¹ Uveal melanoma eventually spreads to the liver in up to 50% of patients; most have subclinical evidence of metastasis at the time of diagnosis. ² Currently, no effective therapeutic regimen is available for patients with metastatic disease because of the lack of understanding of the biology of tumor growth and dissemination. ³ Treatment of uveal melanoma has been based largely on local regional control without directed therapy because of the lack of understanding of the molecular mechanisms behind uveal melanoma dissemination. ³ Recent investigations, however, are changing our understanding of its tumor biology and are helping to identify novel prognostic factors and targets for clinical therapy. 

MicroRNAs (miRNAs) are an abundant class of endogenously expressed, nonprotein-coding, short (20–25) nucleotide RNAs that regulate gene expression. ⁴ These small RNA molecules negatively regulate the translation and stability of target mRNAs through direct binding of complementary sequences at their 3′ untranslated regions (3′ UTRs). Computational analysis suggests that miRNAs may help regulate more than 30% of the protein-coding genes in diverse processes such as development, metabolism, cell proliferation, and differentiation. ^{5 6 7} Aberrant posttranscriptional regulation of mRNAs by miRNAs can lead to oncogenesis with increased cell proliferation, decreased apoptosis, and enhanced metastatic potential of affected cells. ^{8 9} Since its discovery in 1993 as a developmental modulator, ¹⁰ miRNA is increasingly found to be an important regulator of tumorigenesis. ¹¹ Multiple oncogenes and tumor-suppressor genes are linked to miRNA expression, including the Ras proto-oncogene, antiapoptotic gene BCL2, and the potent p53 tumor-suppressor gene. For example, let-7 acts as a tumor suppressor in the lungs because its downregulation in lung carcinoma is accompanied by an increase of the Ras protein. ¹² miR-15 and miR-16 induce apoptosis by targeting the mRNA of the antiapoptotic gene BCL2, ¹³ which is a key player in many types of human cancer, including leukemia, lymphoma, and carcinoma. Recently, microRNA-34a (miR-34a) was found to be a proapoptotic transcriptional target of the p53 tumor-suppressor gene effector network. ^{14 15 16 17 18 19 20 21 22} These studies suggest that miRNAs are involved in cancer formation through the regulation of cell growth and apoptosis. 

In this study, we investigated the function of miR-34a in uveal melanoma cells because p53 gene defects have been shown to lead to the development of melanoma. ²³ First, we showed that miR-34a was downregulated in uveal melanoma cells, and its introduction into these tumor cells through transfection led to the inhibition of growth and migration. We also computationally identified c-Met mRNA as a potential target of miR-34a and demonstrated that miR-34a decreases endogenous c-Met protein levels in uveal melanoma cells. Tumor cells that express high levels of c-Met are known to be more aggressive tumors and to have higher metastatic potential. ²⁴ Therefore, the inhibition of c-Met expression by miR-34a can slow the malignant progression of tumor cells. Furthermore, the introduction of miR-34a downregulates phosphorylated Akt and cell cycle-related proteins, an alternative pathway to suppress the metastatic potential of these tumors. In summary, these data suggest that miR-34a, a potent effector of the p53 transcriptional network, acts as a suppressor of uveal melanoma cell proliferation and migration. 

Materials and Methods

Cell Culture and Tumor Specimens

The human uveal melanoma cell lines M17, M21, M23, and SP6.5 (kind gift of Guy Pelletier, Research Center of Immunology, Quebec, Canada) were isolated from Caucasian patients with primary choroidal melanoma and grown in Dulbecco modified Eagle medium (DMEM; Invitrogen, Carlsbad, CA) supplemented with 10% fetal bovine serum (FBS; Hyclone, Logan, UT) and incubated at 37°C in a humidified incubator containing 5% CO₂, as described. ^{25 26} The human melanocyte cell line (D78) was isolated and cultured as previously described. ²⁷ HEK-293 cells, purchased from ATCC (Manassas, VA), were grown under the same conditions. Three human uveal melanoma specimens were obtained from patients treated at the Eye Hospital, Wenzhou Medical College (Wenzhou, China), with documented informed consent in each case. All studies and procedures involving human tissue were approved by the Wenzhou Medical College institutional review board. Patient samples involved in the study were used in accordance with the tenets of the Declaration of Helsinki. 

Northern Blot Analysis

Total RNA was extracted from cell lines or tissue samples with reagent (Trizol; Invitrogen), and integrity was confirmed with the use of spectrophotometry and formaldehyde/agarose gel electrophoresis. Ten micrograms of total RNA were dissolved in buffer (Gel Loading Buffer II; Ambion, Austin, TX), heated at 95°C for 3 minutes, loaded onto denaturing 15% Tris-borate-EDTA (TBE)-urea gels, separated on a 15% denaturing urea-PAGE gel for 1 hour, and transferred onto positively charged nylon membranes (GE Healthcare Life Sciences, Piscataway, NJ) followed by cross-linking through UV irradiation. The RNA blots were prehybridized at 68°C for 1 hour (Ultrahyb-Ultrasensitive Hybridization Buffer; Ambion) and subjected to hybridization with 3′-digoxigenin (DIG)-labeled locked nucleic acid (LNA) probe for miR-34a (100 ng/mL) overnight at 42°C. The LNA-modified oligonucleotide probe was obtained from Exiqon (Vedbaek, Denmark). One hundred picomoles of the probe were DIG-labeled (DIG Oligonucleotide 3′-End labeling kit; Roche, Mannheim, Germany). After hybridization, membranes were rinsed and then washed three times with a low-stringency buffer (2× SSC and 0.1% SDS). Detection was performed with a detection kit (DIG Luminescent Detection Kit; Roche, Penzberg, Germany) according to the manufacturer’s instructions. In brief, membranes were blocked in blocking buffer for 30 minutes and then incubated with alkaline phosphatase–conjugated anti–DIG antibody for 60 minutes, followed by washing three times in washing buffer. After equilibration in detection buffer, blots were incubated with chemiluminescent substrate CDP-star and exposed to Kodak film (Biomax MR; Eastman Kodak, Rochester, NY). DIG-labeled U6 RNA probe was used as an internal control. 

Cell Proliferation Assay

M23 and SP6.5 cells were plated at 3 × 10³ cells/well in 96-well plates (Costar, High Wycombe, UK) for each transfection. Transfections were performed using reagent (Lipofectamine 2000; Invitrogen). For each well, 50 nM miR-34a precursor molecule (Ambion) or a negative control precursor miRNA (Ambion) was transfected into cells. Pre-miR miRNA precursor molecules (Ambion) are small, chemically modified, double-stranded RNA molecules designed to mimic endogenous mature miRNAs once properly transfected and expressed by recipient cells. (For convenience, the miR-34a precursor is termed miR-34a after transfection throughout the article.) The negative control is a scrambled oligonucleotide that has been validated not to produce identifiable effects on known miRNA function (http://www.ambion.com/catalog/CatNum.php?17100). After 24-hour culture, cell proliferation was assessed using assay (CellTiter 96 Aqueous MTS (3-[4,5-dimethylthiazol-2-yl]-5-[3-carboxymethoxyphenyl]-2-[4-sulfophenyl]-2H-tetrazolium, inner salt); Promega, Madison, WI) according to the manufacturer’s instructions. Briefly, reagent (CellTiter 96 AQueous One Solution Reagent; Promega) was added to each well and incubated at 37°C for 3 hours. Cell proliferation was assessed by measuring the absorbance at 490 nm with a microtiter plate reader (Molecular Devices, Sunnyvale, CA). 

Transwell Migration Assays

M23 and SP6.5 cells were grown in DMEM containing 10% FBS to approximately 60% confluence and transfected with 50 nM miR-34a precursor molecule or a negative control. After 24 hours, the cells were harvested by trypsinization and washed once with D-Hanks solution (Invitrogen). To measure cell migration, 8-mm pore size-culture inserts (Transwell; Costar) were placed into the wells of 24-well culture plates, separating the upper and the lower chambers. In the lower chamber, 400 μL DMEM containing recombinant human HGF (20 ng/mL; R&D Systems, Minneapolis, MN) was added. Then 1 × 10⁵ cells were added to the upper chamber. After 24 hours of incubation at 37°C with 5% CO₂, the number of cells that had migrated through the pores was quantified by counting 10 independent visual fields under the microscope (Zeiss, Oberkochen, Germany) using a 20× objective, and cell morphology was observed by staining with hematoxylin and eosin. 

Luciferase Reporter Assays

The 3′UTR of human c-Met was amplified from human genomic DNA and cloned into pMIR-REPORT vector (Ambion) by directional cloning. The resultant plasmid was designated pLuc-MET 3′UTR. To generate the pLuc-MET 3′UTR-Mut construct, seed regions were mutated from CACUGCC to GUGACGG, removing all complementarity to nucleotides 1–7 of miR-34a (QuickchangeXL Mutagenesis Kit; Stratagene, La Jolla, CA). HEK-293 cells were cotransfected with 0.4 μg firefly luciferase reporter vector and 0.02 μg control vector containing Renilla luciferase, pRL-SV40 (Promega), using Lipofectamine 2000 (Invitrogen) in 24-well plates (Costar). Each transfection was carried out in four wells. For each well, 50 nM miR-34a precursor molecule (Ambion) or a negative control precursor miRNA (Ambion) was cotransfected with the reporter constructs (see 3 Fig. 4 ). Luciferase assays were performed 24 hours after transfection (Dual Luciferase Reporter Assay System; Promega). Firefly luciferase activity was normalized to Renilla luciferase activity. 

Immunofluorescence Staining

M23 and SP6.5 cells were transfected with miR-34a precursor or a negative control, then grown on coated glass coverslips for 24 hours. Cells were fixed in 4% paraformaldehyde solution for 15 minutes at room temperature and permeabilized with 0.1% Triton X-100 in Tris-buffered saline (TBS) for 3 minutes. Cells were blocked in TBS containing 5% bovine serum albumin (BSA) for 1 hour, followed by incubation with primary antibody anti–c-Met in the blocking solution for 1 hour. After washing with TBS, Cy3-conjugated secondary antibody in the blocking solution was added to the cells for 1 hour. Cells werewashed and then stained for 15 minutes with 4′-6-Diamidino-2-phenylindole (DAPI) to display the nuclei. Cells were mounted in a fluorescent mounting medium, and images were captured using a spinning disc confocal microscope (DSU; Olympus, Tokyo, Japan). 

Western Blot Analysis

M23 and SP6.5 cells (1 × 10⁵) were seeded and grown in DMEM with 10% FBS in six-well plates for 24 hours. After transfection, the cells were washed with cold phosphate-buffered saline (PBS) and subjected to lysis in a lysis buffer (50 mM Tris-Cl, 1 mM EDTA, 20 g/L sodium dodecyl sulfate [SDS], 5 mM dithiothreitol, 10 mM phenylmethylsulfonyl fluoride). Equal amounts of protein lysates (50 μg each) and rainbow molecular weight markers (Amersham Pharmacia Biotech, Amersham, UK) were separated by 10% SDS-PAGE, then electrotransferred to nitrocellulose membranes. Membranes were blocked with a buffer containing 5% nonfat milk in PBS with 0.05% Tween 20 for 2 hours and incubated overnight with antibody at 4°C. After a second wash with PBS containing 0.05% Tween 20, the membranes were incubated with peroxidase-conjugated secondary antibodies (Santa Cruz Biotechnology, Santa Cruz, CA) and developed with an enhanced chemiluminescence detection kit (Pierce, Rockford, IL). Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was used as a loading control. Antibodies for total ERK1/2, phosphorylated-ERK1/2 (threonine 202/tyrosine 204), total Akt, phosphorylated-Akt (serine 473, D9E), phosphorylated-Rb (serine 795), and phosphorylated-cdc2 (threonine 161) were from Cell Signaling Technology (Beverly, MA), and those for c-Met, E2F1, and E2F3 were from Santa Cruz Biotechnology. 

Statistical Analysis

All data were shown as the mean ± SEM. Differences between transfections with miR-34a and a negative control were analyzed using the Student’s t-test. Statistical significance was accepted at P < 0.05. 

Results

MicroRNA-34a Expression Was Downregulated in Uveal Melanoma Cells and Specimens

To determine whether miRNA was involved in the regulation of tumorigenesis of uveal melanoma cells, we first compared miR-34a expression in normal melanocytes and uveal melanoma cells. Northern blot analysis was performed using DIG-labeled LNA probe for miR-34a to detect its expression in uveal melanoma cell lines, including M17, M21, M23, and SP6.5, and the normal uveal melanocyte cell line D78. miR-34a was expressed in melanocytes (Fig. 1A) . In contrast, miR-34a expression was undetectable in all the uveal melanoma cell lines examined (Fig. 1A) . To determine the expression pattern of miR-34a in primary human uveal melanomas, total RNA from three human uveal melanoma specimens was analyzed by Northern blotting. Consistent with the results from uveal melanoma cell lines, miR-34a was not expressed at detectable levels in those three samples (Fig. 1B) . These results indicate that miR-34a expression is downregulated in human uveal melanoma. 

MicroRNA-34a Inhibited Proliferation of Uveal Melanoma Cells

Because miR-34a expression was undetectable in uveal melanoma cells, we first sought to determine whether the introduction of miR-34a had any biological effect on uveal melanoma cells. Two uveal melanoma cell lines, M23 and SP6.5, were transfected with the miR-34a precursor or a negative control. After transfection, the MTS assay was carried out to assess growth inhibition at days 1, 2, 3, 4, and 5. Although transfection with the negative control did not affect uveal melanoma cell proliferation and viability, miR-34a caused a dramatic inhibition of cell proliferation in M23 and SP6.5 uveal melanoma cells compared with that of control over a 5-day interval (Fig. 2) . A reduction in cell number was detected in each cell line by day 2 after transfection, and the differences between cells transfected with the miR-34a precursor and cells transfected with a negative control were statistically significant starting from day 3. A significant decrease in cell number persisted through day 5 (35% ± 5.2% decrease for M23 cells and 30% ± 4.5% for SP6.5 cells; P < 0.01; Fig. 2 ). Thus, these results suggest that miR-34a inhibits cell proliferation and plays an important role in regulating uveal melanoma cell growth. 

MicroRNA-34a Inhibited Migration of Uveal Melanoma Cells

We next assessed the effect of miR-34a on uveal melanoma cell migration, a prerequisite for malignant transformation and metastasis, using a transwell migration assay. After transfection of M23 and SP6.5 cells with either a miR-34a precursor or a control precursor, vertical migration studies were performed. Cells were seeded on culture inserts and the ability of cells to migrate to the underside of the inserts was determined in the presence of hepatocyte growth factor (HGF). As shown in Figure 3 , HGF-induced migration was significantly decreased when comparing miR-34a–transfected cells with negative control transfected cells (186 ± 15 vs. 425 ± 34 in M23 cells, 134 ± 8 vs. 224 ± 17 in SP6.5 cells; n = 3 each; P < 0.01). Therefore, the introduction of miR-34a resulted in reduced cell motility in response to HGF. 

c-Met Is a Target of miR-34a

Having demonstrated a functional role for miR-34a in uveal melanoma cells, we explored the cellular mechanisms underlying miR-34a–mediated cell proliferation and migration. TargetScan (http://www.targetscan.org) was conducted for miR-34a target prediction. As a result, two potential binding sites of miR-34a were predicted in the 3′ UTR of the c-Met mRNA (Fig. 4A) . Alignment between the predicted miR-34a target sites and miR-34a, the conserved 7-bp “seed” sequence for miR-34a:mRNA pairing is shown (Fig. 4A) . To test the specific regulation of c-Met through the two predicted binding sites, we amplified the c-Met 3′ UTR sequence and inserted it downstream of the firefly luciferase coding region of the pMIR-REPORT vector (Fig. 4B) . Mutants with the putative binding sites were prepared as described (see Materials and Methods). As expected, luciferase activity of the wild-type pLuc-MET 3′ UTR construct was significantly inhibited after the introduction of miR-34a into HEK293 cells, similar to those described by others, ¹⁷ but not by the negative control (Fig. 4C) . Mutations of the two c-Met 3′ UTR-binding sites completely abolished the ability of miR-34a to regulate luciferase expression (Fig. 4C) . These results demonstrated that c-Met is a potential target of miR-34a. 

Introduction of miR-34a Downregulated c-Met Expression in Uveal Melanoma Cells

To confirm that miR-34a was indeed responsible for the downregulation of c-Met in uveal melanoma cells, M23 and SP6.5 cells were transfected with the miR-34a precursor molecule or a negative control. Northern blot analysis was performed to detect the expression of miR-34a. As expected, miR-34a was present only when the cells were transfected with miR-34a (Fig. 5A) . Western blot analysis showed that though c-Met expression was not affected by transfection with a negative control compared with untransfected cells, c-Met expression was dramatically reduced when M23 and SP6.5 cells were transfected with miR-34a (Fig. 5B) . To further confirm the downregulation of c-Met by miR-34a, c-Met expression was also examined by immunostaining. As shown in Figure 5C , the expression of c-Met was decreased in cells transfected with miR-34a. 

Introduction of miR-34a Downregulated Activation of Akt and Cell Cycle-Related Proteins

c-Met has been shown to activate diverse intracellular signaling pathways. ²⁸ To examine the intracellular proteins affected by miR-34a in uveal melanoma cells, we next determined the expression patterns of ERK1/2 and Akt after the downregulation of c-Met by miR-34a. M23 and SP6.5 cells were transfected with either the miR-34a precursor or a negative control. Cells were solubilized and subjected to Western blot analysis with phosphorylation-specific antibodies to Akt and ERK1/2, molecules involved in the two major signaling pathways previously shown to be stimulated by HGF. ^{24 29} As shown in Figure 6A , the downregulation of c-Met by miR-34a led to significant reduction of phosphorylated-Akt in M23 and SP6.5 cells but had no effect on ERK1/2 phosphorylation. Total Akt and total ERK1/2 were not affected when comparing miR-34a transfection to negative control transfection (Fig. 6A) . 

In addition to its effects on phosphorylated-Akt, ectopic miR-34a delivery downregulated the phosphorylation of cell cycle regulatory proteins such as the retinoblastoma protein (Rb) and cdc2 in M23 and SP6.5 cells (Fig. 6B) . We also examined members of the E2F transcriptional factor family after transfection with miR-34a into uveal melanoma cells. Whereas E2F1 or E2F2 was not changed, the introduction of miR-34a decreased E2F3 protein expression in M23 and SP6.5 cells (Fig. 6Band data not shown). Taken together, these results demonstrated that the induction of miR-34a suppressed c-Met expression and cell cycle-related genes, thus inhibiting cell proliferation and migration. 

Discussion

Emerging evidence suggests that miRNA may have a regulatory role in the pathogenesis of cancer development in humans through the suppression of genes involved in cell proliferation, differentiation, and demise. Flanked by the insight that global suppression of miRNA leads to increased tumorigenesis and cellular transformation, studies to identify cancer-related miRNAs have unveiled a list of potential candidates important for human tumors. ^{8 9 30 31} Analysis of the p53 gene products identified miR-34a as a direct transcriptional target and an important component of the tumor-suppressor network. miR-34a helps to regulate cell cycle progression, DNA repair, and apoptosis after its elaboration within the cellular machinery. ^{14 17} Previous studies have demonstrated that miR-34a inhibits the proliferation of colorectal, pancreatic, and neuroblastoma cell lines through posttranscriptional silencing of messenger RNAs. ^{16 20} Thus far, little is known about the role of miR-34a in uveal melanoma. 

Although p53 has been identified as a potential source of miR-34a, downstream effectors of miR-34a remain a mystery. The MET oncogene is a cell surface receptor tyrosine kinase upregulated in a variety of tumor cells similar in scope to p53 mutants. ^{28 32} c-Met activation, through aberrant HGF paracrine stimulation, can contribute to tumor growth, invasiveness, and metastasis. ^{32 33 34 35} Overall, MET mutations occur at a far lower frequency than expected given the types of aggressive tumors seen with p53 mutants. However, the expression pattern of MET mutations is seemingly similar to the ones with miR-34a deficiency. Therefore, we examined the c-Met expression pathway of uveal melanoma cell lines after transfection with miR-34a. 

In the present study, we demonstrated that miR-34a is an important component during uveal melanoma development, with its effects linked to the c-Met signaling pathway; miR-34a, expressed in melanocytes, is downregulated in uveal melanoma cells and tumor specimens (Fig. 1) . Interestingly, a recent miRNA analysis has revealed that miR-34a is also downregulated in cutaneous melanoma cell lines and primary tumor specimens. ³¹ Transfection of miR-34a into uveal melanoma cells restored its inhibitory activity and led to significant growth retardation (Fig. 2) . Tumor cell migration, a cornerstone capability necessary for tumor metastasis, has been shown to be affected by the presence of miRNAs. For example, a recent study on miR-10b demonstrated that it enhanced tumor invasion and metastasis in breast cancer through the expression of RHOC, ³⁶ an important player in metastasis found in various types of carcinoma. Here, we were able to illustrate for the first time that miR-34a can regulate uveal melanoma cell migration through its target gene c-Met. A series of transwell experiments outlined in Results illustrated that uveal melanoma cell migration can be inhibited with the restoration of miRNA-34a activity (Fig. 3) . By targeting c-Met in an HGF-dependent fashion, miR-34a inhibited uveal melanoma cell migration dramatically. 

In addition to its regulation of c-Met activity, miR-34a downregulated phosphorylated Akt and cell cycle-related proteins, including Rb, cdc2, and E2F3 (Fig. 6) . We have shown previously that PI3K/Akt signaling pathway was involved in HGF-induced migration of uveal melanoma cells through activation of the HGF transmembrane receptor tyrosine kinase c-Met. ³³ Because c-Met levels are directly affected by miR-34a, its downstream effects are similarly altered in melanoma cells. Activated cdc2 kinase, which plays a central role in G₂-M phase transition through the phosphorylation of the threonine 161 residue, ³⁷ was significantly reduced after the introduction of miR-34a in uveal melanoma cells (Fig. 6B) . The retinoblastoma (Rb) gene was also affected by miR-34a (Fig. 6B) , which was the first tumor-suppressor gene identified in humans and one of the most important genes in cell cycle regulation and tumorigenesis. ³⁸ The major action of Rb as a tumor suppressor is to control the G₁-S transition in proliferating cells. ³⁸ miR-34a, which downregulates Rb, in turn, downregulates the transcriptional activity of its downstream targets. ³⁹ E2F3, a member of the E2F family, is a positive regulator of the cell cycle progression based on knockout studies. ⁴⁰ Among all known members of the E2F family, E2F3 has previously been shown to be the strongest candidate for the miR-34a target. ^{16 20} E2F3 downregulation based on Western blot analysis (Fig. 6B) , along with Rb expression, supports the notion that miR-34a can also inhibit cell proliferation through cell cycle protein regulation. 

In summary, we demonstrated that miR-34a modulates several signaling pathways involved in cell proliferation and migration. miR-34a downregulates c-Met, which in turn inhibits cell proliferation and migration through the Akt signaling pathway in an HGF-dependent fashion. miR-34a also affects cell proliferation through the cell cycle proteins Rb, cdc2, and E2F3. Specifically, we were able to show suppressed cellular expression of miR-34a in melanoma cell lines and tumor specimens (Fig. 1) . Restoration of this miRNA leads to the inhibition of tumor cell growth and migration (Figs. 2 3) . Thus, these findings suggest that miR-34a may play an important role in regulating the development of uveal melanoma. Our studies will hopefully have important clinical consequences in the treatment of uveal melanoma. 

 Supported in part by National Natural Science Foundation of China Grant 30772385 and Zhejiang Provincial Natural Science Foundation of China Grant Y2080853.

 Submitted for publication August 6, 2008; revised October 3, 2008; accepted January 26, 2009.

 Disclosure: D. Yan, None; X. Zhou, None; X. Chen, None; D.-N. Hu, None; X.D. Dong, None; J. Wang, None; F. Lu, None; L. Tu, None; J. Qu, None

 The publication costs of this article were defrayed in part by page charge payment. This article must therefore be marked “advertisement” in accordance with 18 U.S.C. §1734 solely to indicate this fact.

 

Figure 1.

 

Downregulation of miR-34a expression in human uveal melanoma cell lines and human melanoma specimens. (A) Northern blot analysis was performed to detect the expression of miR-34a in uveal melanoma cell lines, including M17, M21, M23, and SP6.5, and in the melanocyte cell line D78. miR-34a was expressed in melanocytes but not in melanoma cells at the detectable level. U6 snRNA was used as an internal control. (B) miR-34a was not expressed at the detectable level in three human uveal melanoma specimens. U6 snRNA was used as an internal control. U6, U6 snRNA.

Figure 2.

 

miR-34a inhibited the proliferation of uveal melanoma cells. MTS cell proliferation assay was carried out on days 1 to 5, as indicated, after lipofectamine transfection of uveal melanoma cells M23 and SP6.5 with either the miR-34a precursor (miR-34a, 50 nM) or a negative control (NC) scrambled oligonucleotide. The NC does not encode for any known miRNA. Cell populations transfected with miR-34a had significantly fewer metabolically active cells than cells transfected with the negative control. Data at each time point are expressed as the mean ± SEM of the results obtained from triplicates in one experiment. Results represent those obtained in three experiments.

Figure 3.

 

Transfection of miR-34a reduced the migration of uveal melanoma cells. Transwell migration assay of uveal melanoma cell lines was performed. Uveal melanoma cells M23 and SP6.5 were transfected with miR-34a or a negative control (NC) for 24 hours and plated on culture inserts in DMEM containing 20 ng/mL HGF to assess the number of migrating cells. The number of cells that migrated through the pores was quantified by counting 10 independent visual fields with the use of a 20× microscope objective. Results are expressed as the mean ± SEM for the data obtained from three independent assays. *Differences in cell migration between miR-34a and NC-transfected cells were significant (P < 0.01).

Figure 4.

 

Predicted miR-34a binding sites in c-Met 3′ UTR. (A) Specific locations of the binding sites (red) and c-Met (MET) 3′ UTR (blue). Alignment between the predicted miR-34a target sites and miR-34a. The conserved 7-bp “seed” sequence for miR-34a:mRNA pairing is indicated. (B) Diagram depicting the pMIR luciferase reporter constructs, containing a CMV promoter, which was used to verify the putative miR-34a binding sites. (C) HEK293 cells were cotransfected with miR-34a, pLuc-MET 3′UTR, along with a pRL-SV40 reporter plasmid. After 24 hours, luciferase activity was measured. Values are presented as relative luciferase activity after normalization to Renilla luciferase activity. Data are expressed as mean ± SEM of the results obtained from three independent experiments. Luc, luciferase. *Differences in luciferase activity between miR-34a and negative control–transfected cells were significant (P < 0.01).

Figure 5.

 

Introduction of miR-34a downregulated the expression of c-Met in uveal melanoma cells. (A) Northern blot analysis of miR-34a expression in uveal melanoma cells M23 and SP6.5 after transfection with miR-34a or a negative control. (B) c-Met expression levels in M23 and SP6.5 cells after transfection with miR-34a were determined by Western blot analysis. Compared with the negative control miRNA, miR-34a expression dramatically reduced the levels of c-Met in both cell lines. GAPDH was used as an internal control. (C) Immunofluorescence staining of c-Met in M23 and SP6.5 uveal melanoma cells. M23 and SP6.5 cells plated on glass coverslips were transfected with either miR-34a or a negative control (NC). Cells were fixed with paraformaldehyde and stained for either c-Met (red) or with propidium iodide (DAPI) as a nuclear counterstain (blue).

Figure 6.

 

Introduction of miR-34a downregulated phosphorylated Akt and cell cycle–related proteins. M23 and SP6.5 cells were transfected with miR-34a or a negative control. Cell lysates were prepared and used for Western blot analysis with phosphorylated-Akt (p-Akt), total Akt, phosphorylated-ERK1/2 (p-ERK1/2), total ERK1/2, phosphorylated-Rb (p-Rb), phosphorylated-cdc2 (p-cdc2), E2F1, and E2F3 antibodies. GAPDH was used as a loading control. (A) miR-34a downregulated the expression of p-Akt but not of total Akt or Erk1/2 and its phosphorylated counterpart. (B) With regard to cell cycle-related proteins, p-Rb, p-cdc2 and E2F3 were downregulated by miR-34a.