This Article Abstract Full Text (PDF) Alert me when this article is cited Alert me if a correction is posted Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Bemis, L. T. Articles by Robinson, W. A. Search for Related Content PubMed PubMed Citation Articles by Bemis, L. T. Articles by Robinson, W. A.

MicroRNA-137 Targets Microphthalmia-Associated Transcription Factor in Melanoma Cell Lines

¹ Division of Medical Oncology and ² Department of Dermatology, University of Colorado Denver School of Medicine, Aurora, Colorado

Requests for reprints: William A. Robinson, Division of Medical Oncology, University of Colorado Denver School of Medicine, Campus Box 8117, P.O. Box 6511, Aurora, CO 80045. E-mail: William.Robinson{at}uchsc.edu.

	Abstract

Top Abstract Introduction Materials and Methods Results Discussion References

 
Micropthalmia-associated transcription factor (MITF) is the master regulator of melanocyte development, survival, and function. Frequent alteration in the expression of MITF is detected in melanoma, but the mechanism(s) underlying the alteration in expression have not been completely determined. In these studies, we have identified microRNA-137 (miR-137) as a regulator of MITF expression. The genomic locus of miR-137 at chromosome 1p22 places it in a region of the human genome previously determined to harbor an allele for melanoma susceptibility. Here, we show that expression of mature miR-137 in melanoma cell lines down-regulates MITF expression. Further, we have identified a 15-bp variable nucleotide tandem repeat located just 5' to the pre-miR-137 sequence, which alters the processing and function of miR-137 in melanoma cell lines. [Cancer Res 2008;68(5):1362–8]

	Introduction

Top Abstract Introduction Materials and Methods Results Discussion References

 
Malignant melanoma represents a significant public health burden and is increasing in incidence at a greater rate than any other cancer type (1). The molecular mechanisms involved in the development of melanoma are complex and not entirely clear. In an attempt to discern regions of the genome that might harbor genes contributing to the development of melanoma, several studies have characterized regions of the genome that are lost or amplified in melanoma cell lines and in families with a high risk for melanoma (2). One such region located on chromosome 1p22 harbors a gene or genes that are responsible for increasing melanoma risk but are not yet identified (3). With the identification of microRNAs (miRNA) as important regulators of gene expression, we hypothesized that a miRNA at 1p22 might be regulating target genes associated with a risk of developing melanoma.

MiRNAs are located throughout the genome and may be transcribed as noncoding primary miRNA (pri-miRNA) transcripts. The pri-miRNAs are processed into precursor miRNA (pre-miRNA) and finally into the mature and functional miRNA of 21 to 25 nucleotides (4). The mature miRNAs function by binding to the 3' untranslated region (3'UTR) of multiple target mRNAs. The binding is usually imperfect and results in an inhibition of translation of the target protein or degradation of the target mRNA (4–6). Numerous studies characterizing the expression profiles of miRNAs in cancer have shown the down-regulation of miRNA expression in cancer cell lines as compared with normal tissues (7–9). Furthermore, the recent identification of altered expression of miRNAs in a variety of cancer sites has confirmed that this is yet another class of molecules capable of functioning as tumor suppressors or oncogenes (10).

The first confirmation of specific mutations identified in miRNAs in cancer cells but not in normal cells came from the work by Calin and Croce (11) on B-cell chronic lymphocytic leukemia (CLL). A variety of alterations were identified in two miRNAs in B cells from CLL patients, and these mutations were not found in B cells from healthy participants. The mutations altered either the pri-miRNA or the pre-miRNA. Characterization of such mutations suggests that these miRNAs function as tumor suppressors in CLL (11) because the mutations resulted in lower expression of the specific miRNAs thereby allowing the increased expression of an oncogene, Bcl-2.

The alterations in miRNAs that are reported for CLL (11) led us to question if a miRNA or multiple miRNAs might contain alterations in melanoma. MiRNA expression profiles have been delineated in a large variety of cancer sites (7–9); however, these studies included few melanoma samples. Thus, we initially conducted a survey of miRNAs and their chromosomal location to determine if any were located in regions of the human genome suspected to harbor genes contributing to melanoma but as yet unidentified. As noted above, one commonly reported region of the human genome thought to harbor a gene contributing to melanoma susceptibility is a region of chromosome 1 (12). This chromosomal region contained one miRNA according to the miRNA database at Sanger Center³ (13, 14). Interestingly, this miRNA, microRNA 137 (miR-137), was also suspected to bind to the 3'UTR of the key regulator of melanogenesis, micropthalmia-associated transcription factor (MITF), as indicated in the miRGen database⁴ (15). MITF is the "master regulator" of melanocyte cell growth, maturation, apoptosis, and pigmentation (16). We undertook studies to confirm the predicted role of miR-137 in the regulation of MITF. Additionally, we identified a variable nucleotide tandem repeat (VNTR) in the pri-miRNA-137 that alters the function of miR-137 in melanoma cells.

	Materials and Methods

Top Abstract Introduction Materials and Methods Results Discussion References

 
miRNA computational predictions. The publicly accessible database miRBase³ was consulted to identify miRNA located on chromosome 1 in the region predicted to harbor a gene involved in melanoma. The data bases TargetScan 3.1⁵ (17, 18) and miRgen⁴ (15) were used to identify potential targets for miR-137 regulation in melanoma. MiRNA binding predictions were confirmed by manually analyzing the MITF 3'UTR for binding sites of miR-137. The publicly available MFold software was used to predict the secondary structure of miR-137 (19).

Cell lines and growth conditions. The human melanoma cell lines WM1617, WM852, WM793, and A375 were chosen based on previous studies of the expression of MITF. We had previously determined that WM852, WM1617, and WM793 express MITF (data not previously reported), and others had shown the lack of expression of MITF in A375 (20). All melanoma cell lines were cultured in RPMI medium supplemented with 10% fetal bovine serum, 1% penicillin/streptomycin, and 50 mmol/L L-glutamine at 37°C and 5% CO₂. In addition, the cell line WM852 was treated with 100 ng/mL -melanocyte stimulating hormone (-MSH; Sigma-Aldrich) for 8 h. Cells were transfected with eGFP-C1, GFP-MITF-3'UTR, GFP-MITF-3'UTR-7mer, GFP-MITF-3'UTR-8mer, and GFP-MITF-3'UTR-double, with or without 5 nmol/L miRIDIAN miR-137 mimic (Dharmacon), using Qiagen Effectene Transfection Reagent. Quantitative reverse transcription-PCR (RT-PCR) of green fluorescent protein (GFP) was used to verify transfection.

Western blot analysis. Cells were harvested by rinsing with PBS and lysed with lysis buffer (1% SDS, 1 mmol/L EDTA, 10 mmol/L Tris-HCl, pH 7.4), which was heated to 90°C. The samples were briefly sonicated and centrifuged at 14,000 rpm for 15 min at 4°C. The antibodies used in this study included MITF (clone D5), GFP (clone GFP01; Lab Vision), and pan-actin (4968; Cell Signaling). Protein samples were resolved on a 10% SDS polyacrylamide gel, transferred onto a nitrocellulose membrane, and blocked in 5% dried milk in PBS/0.0025% Tween 20. Membranes were incubated in the appropriate primary antibody overnight, followed by incubation with a secondary antibody (horseradish peroxidase–conjugated antimouse). Proteins were detected with enhanced luminol/peroxide buffer SuperSignal West Femto (Pierce) on a Chemidoc (Bio-Rad) imaging system and quantitated with the Quantity One software (4.5.0 basic, Bio-Rad).

PCR amplification of genomic DNA. Genomic DNA was prepared from cell lines with the Qiagen DNeasy Tissue kit (Qiagen) and 50 to 100 ng were amplified using GoGreen Taq as directed (Promega). The primers for miR-137 were miR-137 forward, 5'-GCAGCAAGAGTTCTGGTGGC, and miR-137 reverse, 5'-TGGAACCAGTGCGAAAACAC. Following PCR amplification, the products were electrophoresed on a 3% agarose gel, and then bands were excised and extracted with the Qiagen Gel extraction kit (Qiagen). The samples were directly sequenced on an AB3700 automated sequencer in the Core Laboratory of the University of Colorado Cancer Center using the same primers as used for PCR. All sequencing reactions were done in both the forward and reverse directions. Alignments and mutation analysis were conducted using BLAST (National Center for Biotechnology Information) software.

RNA isolation, cDNA synthesis, and quantitative RT-PCR. Total RNA was isolated using the miRNeasy Mini Kit (Qiagen). For miRNA message analysis, the cDNA was synthesized using the NCode miRNA First-Strand cDNA Synthesis Kit (Invitrogen). Briefly, this step adds a polyadenylate tail to the miRNA in the total RNA samples. For all other mRNA analyses, cDNA was synthesized using the SuperScript II Reverse Transcriptase Kit (Invitrogen). The amount of miRNA was monitored with Platinum SYBR Green qPCR SuperMix-UDG reagent (Invitrogen). Quantitative PCR was done under the following thermocycler conditions: 50°C for 2 min, 95°C for 10 min, and 40 cycles of 95°C for 15 s and 57°C for 60 s. Quantitative RT-PCR was used to determine the expression levels of the target genes MITF, GFP, and β-actin as internal control (21). Expression of the mature miR-137 and U6 as internal control was conducted with the NCode kit (Invitrogen). Results are expressed as relative quantitation as previously described (22). Primers were, for primary miR-137, 5'-CAAGGCTTGTTAACACTGTAAC (forward) and 5'-TCTGTCAATGTCTGAATAAATG (reverse); MITF exon 9–10, 5'-CAGGAACTTGAAATGCAGGCTCGA (forward); MITF exon 10, 5'-GATCAGTGACACCGACGGGAGAA (reverse); β-actin, 5'-ATCCACGAAACTACCTTCAACTC (forward) and 5'-GAGGAGCAATGATCTTGATCTTC (reverse); miR-137 mature, 5'-TATTGCTTAAGAATACGCGTAG (forward); U6, 5'-CGCAAGGATGACACGCAAATTCGT (forward); and GFP, 5'-CGACAAGCAGAAGAACGGCATCAA (forward) and 5'-AACTCCAGCAGGACCATGTGAT (reverse).

GFP-MITF-3'UTR reporter constructs. The 3'UTR of MITF is encoded in exon 10. A cDNA encoding the most 1,143 bp of the 3'UTR was purchased from American Type Culture Collection (522701) and a fragment from the vector EcoRI to NotI encompassing the insert was ligated into pCR2.1-TOPO (Invitrogen) for convenient restriction digestion. The insert was moved to pEGFP-C1 (Clontech Laboratories) at the EcoRI and ApaI sites. Mutation of the miR-137 binding site in the 3'UTR of MITF was created using the QuickChange Stratagene method. Primers for quick-change mutations were MITF 7-mer, 5'-CCTGCTGTTGGATGCAGAACTAATTTCTGTATGGTCCATA (forward) and 5'-TATGGACCATACAGAAATTAGTTCTGCATCCAACAGCAGG (reverse); MITF 8-mer, 5'-GTTTTTAAACAATAAAGAACTAAGAACAAATACAA (forward) and 5'-TTGTATTTGTTCTTAGTTCTTTATTGTTTAAAAAC (reverse). Melanoma cells were then transfected using the lipophilic reagent Effectene (Qiagen) with no vector, the GFP vector, or the GFP-MITF-3'UTR constructs. GFP expression was monitored by Western blot analysis and quantitative RT-PCR.

Pri-miR-137 vector construction. The pre-miR-137 sequence is located within the non–protein-coding RNA gene BRAMY2014205 and we believe this gene to be the pri-miR-137 sequence. The BRAMY2014205 clone was purchased from the Department of Biotechnology of National Institute of Technology and Evaluation, Japan (AK094607/FLJ37288/BRAMY2014205). The cDNA was prepared at the Institute of Medical Science of the University of Tokyo (IMS-UT) and Helix Research Institute, Inc., by the "oligo-capping" method and cloned into the pME18SFL3 vector. We reconstructed the VNTR region by a complex cloning strategy to prevent changes in base pairs that might be necessary for the function of the primary miR-137. First, a restriction fragment from the original cDNA clone (BRAMY2014205) starting with EcoRI and going to the internal NotI was gel purified and ligated into pT7T3-PAC vector at EcoRI and NotI. Then this vector was restricted at the internal BamHI and NotI and the 12 repeats from a PCR product derived from WM852 genomic DNA were ligated into this plasmid to create the 12-VNTR region. A fragment from this repaired vector was then ligated into the original vector at EcoRI and NotI, creating the plasmid with 12 VNTRs. The plasmid with 3 VNTRs is considered wild-type whereas the 12-VNTR plasmid has 12 repeats obtained from the WM852 cell line.

Northern blot analysis. RNA samples from cell lines were analyzed by Northern blot. Briefly, 2.0 µg of total RNA were mixed with an equal volume of formamide and heated to 65°C for 10 min. The samples were electrophoresed in a 15% urea-Tris-borate EDTA gel (Bio-Rad) and transferred onto GeneScreen Plus membrane (Perkin-Elmer). The membrane was UV cross-linked with 120-mJ energy and baked at 80°C for 1 h. The miR-137 probe (5'-CTACGCGTATTCTTAAGCAATA-3') and U6 snRNA loading control probe (5'-TGTGCTGCCGAAGCGAGCAC-3') were ³²P labeled at their 5' end using T4 polynucleotide kinase (New England Biolabs). The probes were hybridized to the membrane and exposed on X-ray film at –80°C.

	Results

Top Abstract Introduction Materials and Methods Results Discussion References

 
In initial studies, we used computational methods to identify miRNAs located in the 1p22 chromosomal region that might regulate genes important in melanocyte development and function (13, 14). One miRNA was detected in this location, miR-137, located between 92 and 100 Mb on chromosome 1 (Fig. 1A ). We then queried if genes known to be important in melanocyte regulation and the development of melanoma were potential targets of miR-137. TargetScan (17, 18) ranked MITF as a likely target for regulation by miR-137 with two binding sites in the 3'UTR (Fig. 1B). As noted, MITF is considered the master regulator of melanogenesis and is known to be misregulated in melanoma (16).

View larger version (34K): [in this window] [in a new window] [Download PPT slide]   Figure 1. Identification of putative miRNA regulators in melanoma. A, miR-137 locus showing the primary, precursor, and mature miR-137 at chromosome 1p22, a region of common loss of heterozygosity in melanoma (chromosome 1: 98284214–98284315 [–]). The primary transcript for miR-137 is capped and polyadenylated (2,381 bp), and the precursor miR-137 is encoded in exon 3 (102 bp). The mature miR-137 (22 bp) is processed from the pre-miR-137. The seed binding region is italicized. B, MITF is a target of miR-137 as predicted by TargetScan. Shown are the seed binding regions located in the most 3' region of the UTR. The mutated seed bases, designated by letter a, were mutated to C, and C was mutated to A to confirm binding of miR-137 to these regions. C, relative comparison of MITF and miR-137 expression in four melanoma cell lines by protein expression and quantitative RT-PCR. Western blot analysis of MITF protein expression equalized to β-actin is shown based on densitometry of MITF and β-actin expressed as the ratio of MITF to β-actin. mRNA expression of MITF by quantitative RT-PCR is expressed as relative quantitation equalized to β-actin. miR-137 expression was quantified by the NCode method (Invitrogen) equalized to U6 RNA and expressed as relative quantitation (described in Materials and Methods).

Despite the highly variable protein expression of MITF in melanoma cell lines, the mRNA expression of MITF was similar for both WM852 and A375. This finding suggested the presence of altered regulation of MITF possibly by a miRNA. However, when we queried if this could be due to increased miR-137 expression in A375 cells as compared with WM852 (Fig. 1C), we found just the opposite. The NCode method for quantitative RT-PCR was applied to quantify expression of the mature miR-137 in melanoma cell lines. In contrast to our hypothesis, WM852 had increased miR-137 as compared with A375 (Fig. 1C, NCode).

Mutations in miRNAs identified from CLL patient samples alter the ability of the miRNA to down-regulate target genes. Thus, we queried if mutations in either a binding site for miR-137 or in the miR-137 itself could have occurred in the WM852 cell line. To assess whether mutations exist in miR-137 and/or its target gene MITF in WM852, we examined the molecular integrity of the binding sites for miR-137 in the MITF-3'UTR and the pre-miR-137 in 12 melanoma cell lines. Genomic DNA from 12 melanoma cell lines was prepared and amplified by PCR. No mutations were found in the putative miR-137 binding sites of the MITF 3'UTR in any of the cell lines examined. We next examined the integrity of miR-137 itself. Primers designed for amplification of miR-137 included the entire pre-miRNA (pre-miR-137) and at least 20 bp on either side. When the PCR products were separated on an agarose gel, there was a dramatic size difference in two of the cell lines, WM1617 and WM852 (Fig. 2A ). A VNTR was identified 6 bp 5' to the pre-miR-137. The reference DNA sequence confirmed our finding with a reported three repeats (VNTR) of 15 bases each at this location (GenBank no. AK094607). In Fig. 2C, the VNTR copy number is included for both alleles when heterozygous alleles were detected. There were no mutations in the 22-bp sequence of the mature miR-137 in these cell lines. There were, however, differences in the number of VNTRs found in melanoma cell lines with the two highest repeats being in cell lines that express MITF (Figs. 1C and 2C).

View larger version (33K): [in this window] [in a new window] [Download PPT slide]   Figure 2. VNTRs of 15 bp were identified 5' to the pre-miR-137. A, PCR products from amplification of the pre-miR-137 region from melanoma cell lines showing different numbers of VNTRs [WM1617 (5, 10), WM852 (12), WM793 (3), A375 (3)] when electrophoresed on a 3% agarose gel. B, sequence of the VNTR repeat region from cell lines A375 and WM852. The VNTR is TAGCAGCGGCAGCGG with the repeat regions underlined. Three repeats are found in A375 and 12 repeats in WM852. The existing restriction sites used to construct the miRNA expression plasmid containing 12 VNTRs are in italics. This sequence has been submitted to GenBank with accession no. 920034. C, VNTR 5' to the pre-miR-137 in melanoma cell lines. The alleles are expressed as the number of VNTRs. When the allele is heterozygous for VNTR number, both VNTR numbers are included.

In these studies, the GFP protein expression was dramatically reduced in the A375 cell line that received GFP-MITF-3'UTR as compared with GFP empty vector, whereas the mRNA expression for GFP remained comparable or higher (Fig. 3A ). In contrast, transfection of the GFP constructs into the WM852 cell line suppressed the GFP protein expression through the MITF-3'UTR to a lesser extent (Fig. 3B). To further evaluate the regulation of MITF through its 3'UTR, we cotransfected these same cells with a miR-137 mimic, the GFP control vector, and the GFP-MITF-UTR and examined GFP expression. Addition of the miR-137 mimic to A375 and WM852 cells had an added effect on reducing GFP expression through the GFP-MITF-3'UTR in A375 and was able to dramatically suppress GFP-MITF-3'UTR expression in WM852 (Fig. 3). These studies gave strong evidence that miR-137 regulates MITF expression through its 3'UTR and suggested that the WM852 cell line with 12 VNTRs in the pri-miR-137 could not as efficiently shut down GFP protein expression through the MITF-3'UTR. However, with addition of a functional miR-137 mimic, expression was dramatically reduced in the GFP-MITF-3'UTR–transfected WM852 cell line (Fig. 3B). Further confirmation that miR-137 regulates MITF by binding to the 3'UTR was accomplished by mutating the binding sites in the 3'UTR. When the binding site that contains the 8 bp of homology in the seed region (8-mer) was mutated, it no longer showed reduced expression by mimics to miR-137. In contrast, when the binding site that contains the 7 bp of homology in the seed region (7-mer) was mutated, there was no observable difference between the wild-type UTR. The double mutant in which both binding sites were mutant was similar in expression to the 8-mer mutant alone (Fig. 3C).

View larger version (30K): [in this window] [in a new window] [Download PPT slide]   Figure 3. MITF is regulated through its 3'UTR. A, A375 cells were transfected with GFP or GFP-MITF-3'UTR plasmids with or without a mimic of miR-137. Shown is GFP protein expression measured by Western blot analysis and compared with a loading control of β-actin. GFP protein expression was down-regulated in cells containing the MITF-3'UTR and further down-regulated in cells with the MITF-3'UTR and the miR-137 mimic. Quantitative RT-PCR (qRT-PCR) of GFP compared with β-actin confirmed transfection. B, WM852 cells, which express high levels of MITF, were transfected as described above and examined for GFP protein expression by Western blot analysis and compared with a loading control of β-actin. The expression of the GFP-MITF-3'UTR decreased dramatically with the addition of mimic to miR-137. GFP proteins generated from the GFP-MITF-3'UTR plasmid are slightly smaller than the empty vector control because of an altered stop codon. C, A375 cells were transfected with GFP, GFP-MITF-3'UTR, GFP-MITF-7mer, GFP-MITF-8mer, GFP-MITF-double mutant plasmids, and miR-137 mimic. GFP protein expression was down-regulated in those cells with a functional MITF-3'UTR. Mutation of the 8-mer binding site in the GFP-MITF-3'UTR (GFP-MITF-8mer or GFP-MITF-double) blocked the suppression of GFP-MITF-3'UTR construct. Quantitative RT-PCR of GFP compared with β-actin confirmed transfection; however, as previously shown, GFP-MITF-UTR message is also reduced, suggesting that the mimic causes degradation of the message as well as the protein.

View larger version (32K): [in this window] [in a new window] [Download PPT slide]   Figure 4. Twelve VNTRs in pri-miR-137 alter processing as compared with 3 VNTRs. A, VNTRs in the primary miRNA transcript of miR-137 alter the secondary structure of the pri-miR-137. MFold was used to predict the secondary structure of miR-137 with either the 12 VNTRs as identified in the cell line WM852 or with 3 VNTRs as observed in the cell line A375. Arrow, the region containing the VNTRs. B, transfection of the full-length miR-137 followed by Northern blot analysis of miR-137 shows a reduced ability to process miR-137 when it contains 12 VNTRs as compared with 3 VNTRs. A375 cells with three repeats of the VNTR in miR-137 were transfected with control vector, 3-repeat VNTR, or 12-repeat VNTR and probed for miR-137 expression. The 12-repeat shows a reduced processing of miR-137 to the mature size. Northern blot analysis was not sensitive enough to detect the native mature miR-137; however, quantitative RT-PCR showed that miR-137 mature sequence is present in A375. C, the full-length cDNA of miR-137 with 12 VNTRs is less capable of down-regulating MITF. Shown is MITF protein expression after transfection of WM852 cells with control vector, 3-VNTR-miR-137, and 12-VNTR-miR-137. The 3 VNTR–containing cDNA of miR-137 is able to block MITF protein expression.

To confirm that the VNTR 5' to the pre-miR-137 alters its ability to down-regulate MITF expression, we transfected the melanoma cell line WM852 (12 VNTRs) with a vector expressing the full-length cDNA of miR-137 (3 VNTRs) and examined protein expression of MITF by Western blot analysis. WM852 was chosen because of its high expression of MITF and because it has no naturally occurring 3-VNTR repeat region (Fig. 2C). As shown in Fig. 4, the transient transfection with a plasmid encoding miR-137 with 3 VNTRs resulted in suppression of MITF protein expression compared with the plasmid that contained the 12 VNTRs in the primary miR-137 (Fig. 4C). These data indicate that the processing and function of miR-137 are altered by VNTR number and subsequently effect MITF expression.

Although fair skin is a well-known risk factor for skin cancer, the increased risk cannot be simply explained by pigmentary phenotype (26). UV light stimulates sun tanning by inducing the expression of -MSH by the keratinocytes, which then stimulates the melanocytes to express MITF (27). Sun tanning is thought to protect the skin from DNA damage largely by activation of MITF (27) and its downstream action of stimulating melanogenesis (27). Based on our finding that miR-137 regulates the expression of MITF in melanoma cells, we examined the possibility that miR-137 might also be regulated by -MSH (Fig. 5 ). The consistent down-regulation of miR-137 was observed in the WM852 cell line, suggesting that miR-137 down-regulation may be an important component of the sun tanning response. This will be the focus of further investigation because of the critical link between sun exposure and melanoma.

View larger version (10K): [in this window] [in a new window] [Download PPT slide]   Figure 5. MiR-137 expression is altered in response to -MSH. Stimulation of WM852 cells with -MSH for 8 h results in the down-regulation of miR-137. miR-137 expression was quantified by the NCode method (Invitrogen) equalized to U6 RNA and expressed as relative quantitation (described in Materials and Methods). Bars, SE; the experiment was repeated in triplicate.

	Discussion

Top Abstract Introduction Materials and Methods Results Discussion References

In the present studies, we have shown, as predicted, that miR-137 regulates MITF protein expression and that an alteration occurs in some melanoma cell lines, preventing proper miR-137 function. We expect that miR-137 is not the only miRNA regulating MITF because other miRNAs are also predicted to regulate this important transcriptional regulator. Bioinformatics calculations indicate that there are at least six other miRNAs that may be important in this regard. How all of these interplay in the regulation of this or any other gene is only now being studied. Clearly, miR-137 is one of the regulators of MITF and is located at a melanoma hotspot in the human genome.

MiRNAs can be embedded in various regions including intronic and exonic sequences. The factors controlling their regulation are beginning to be elucidated but are not yet clear. miR-137 is embedded in a larger expressed but noncoding mRNA, which is thought to be the primary miRNA transcript. This transcript is then folded and cleaved to a pre-miRNA and further processed into the mature 22-bp miR-137. As a part of these studies, we have described a 15-bp VNTR in the primary miRNA of miR-137 and present data that alterations in the number of tandem repeats in this area interfere with mature miR-137 processing and function. Exactly how this might occur is currently under study in our laboratory. In previous studies, other investigators have examined the effect(s) of sequence variations in pre-miRNAs and primary miRNAs (24, 25). Most of these variations found in patient samples were single-base substitutions and led to predicted changes in the folding structure of pre-miRNAs. Despite altered folding predictions, little effect on the processing and expression of the mature miRNAs was found in vivo. Here we describe a much larger change in the pri-miRNA in a melanoma cell line that seems to effect expression and function of the mature form. An alteration of this nature has not been reported with other miRNAs. In preliminary studies, changes in VNTR number have been found not only in melanoma cells but also in other melanocytic disorders. Further understanding of the miR-137 VNTR may have important bearing on our understanding of MITF regulation in melanoma and other human pigmentary disorders and the protective aspects of sun tanning.

	Acknowledgments

 
Grant support: Harry J. Lloyd Charitable Trust award (L.T. Bemis).

The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

	Footnotes

 
³ http://microrna.sanger.ac.uk/

⁴ http://www.diana.pcbi.upenn.edu/miRGen/v3/miRGen.html

⁵ http://www.targetscan.org

Received 7/30/07. Revised 12/ 6/07. Accepted 1/ 3/08.

	References

Top Abstract Introduction Materials and Methods Results Discussion References

 

1. Jemal A, Siegel R, Ward E, Murray T, Xu J, Thun MJ. Cancer statistics, 2007. CA Cancer J Clin 2007;57:43–66.[Abstract/Free Full Text]
2. Goldstein AM, Chan M, Harland M, et al. High-risk melanoma susceptibility genes and pancreatic cancer, neural system tumors, and uveal melanoma across GenoMEL. Cancer Res 2006;66:9818–28.[Abstract/Free Full Text]
3. Walker GJ, Indsto JO, Sood R, et al. Deletion mapping suggests that the 1p22 melanoma susceptibility gene is a tumor suppressor localized to a 9-Mb interval. Genes Chromosomes Cancer 2004;41:56–64.[CrossRef][Medline]
4. Lee Y, Jeon K, Lee JT, Kim S, Kim VN. MicroRNA maturation: stepwise processing and subcellular localization. EMBO J 2002;21:4663–70.[CrossRef][Medline]
5. Chen K, Rajewsky N. The evolution of gene regulation by transcription factors and microRNAs. Nat Rev Genet 2007;8:93–103.[Medline]
6. Pillai RS, Bhattacharyya SN, Filipowicz W. Repression of protein synthesis by miRNAs: how many mechanisms? Trends Cell Biol 2007;17:118–26.[Medline]
7. Calin GA, Croce CM. MicroRNA signatures in human cancers. Nat Rev Cancer 2006;6:857–66.[CrossRef][Medline]
8. Garzon R, Fabbri M, Cimmino A, Calin GA, Croce CM. MicroRNA expression and function in cancer. Trends Mol Med 2006;12:580–7.[CrossRef][Medline]
9. Lu J, Getz G, Miska EA, et al. MicroRNA expression profiles classify human cancers. Nature 2005;435:834–8.[CrossRef][Medline]
10. Zhang B, Pan X, Cobb GP, Anderson TA. microRNAs as oncogenes and tumor suppressors. Dev Biol 2007;302:1–12.[CrossRef][Medline]
11. Calin GA, Croce CM. Genomics of chronic lymphocytic leukemia microRNAs as new players with clinical significance. Semin Oncol 2006;33:167–73.[CrossRef][Medline]
12. Gillanders E, Juo SH, Holland EA, et al. Localization of a novel melanoma susceptibility locus to 1p22. Am J Hum Genet 2003;73:301–13.[CrossRef][Medline]
13. Griffiths-Jones S. The microRNA Registry. Nucleic Acids Res 2004;32:D109–11.[Abstract/Free Full Text]
14. Griffiths-Jones S, Grocock RJ, van Dongen S, Bateman A, Enright AJ. miRBase: microRNA sequences, targets and gene nomenclature. Nucleic Acids Res 2006;34:D140–4.[Abstract/Free Full Text]
15. Megraw M, Sethupathy P, Corda B, Hatzigeorgiou AG. miRGen: a database for the study of animal microRNA genomic organization and function. Nucleic Acids Res 2007;35:D149–55.[Abstract/Free Full Text]
16. Levy C, Khaled M, Fisher DE. MITF: master regulator of melanocyte development and melanoma oncogene. Trends Mol Med 2006;12:406–14.[CrossRef][Medline]
17. Lewis BP, Burge CB, Bartel DP. Conserved seed pairing, often flanked by adenosines, indicates that thousands of human genes are microRNA targets. Cell 2005;120:15–20.[CrossRef][Medline]
18. Lewis BP, Shih IH, Jones-Rhoades MW, Bartel DP, Burge CB. Prediction of mammalian microRNA targets. Cell 2003;115:787–98.[CrossRef][Medline]
19. Zuker M. Mfold web server for nucleic acid folding and hybridization prediction. Nucleic Acids Res 2003;31:3406–15.[Abstract/Free Full Text]
20. Garraway LA, Widlund HR, Rubin MA, et al. Integrative genomic analyses identify MITF as a lineage survival oncogene amplified in malignant melanoma. Nature 2005;436:117–22.[CrossRef][Medline]
21. Merrick DT, Haney J, Petrunich S, et al. Overexpression of vascular endothelial growth factor and its receptors in bronchial dysplasia demonstrated by quantitative RT-PCR analysis. Lung Cancer 2005;48:31–45.[CrossRef][Medline]
22. Livak KJ, Schmittgen TD. Analysis of relative gene expression data using real-time quantitative PCR and the 2(–C(T)) Method. Methods 2001;25:402–8.[CrossRef][Medline]
23. Grimson A, Farh KK, Johnston WK, Garrett-Engele P, Lim LP, Bartel DP. MicroRNA targeting specificity in mammals: determinants beyond seed pairing. Mol Cell 2007;27:91–105.[CrossRef][Medline]
24. Calin GA, Ferracin M, Cimmino A, et al. A MicroRNA signature associated with prognosis and progression in chronic lymphocytic leukemia. N Engl J Med 2005;353:1793–801.[Abstract/Free Full Text]
25. Diederichs S, Haber DA. Sequence variations of microRNAs in human cancer: alterations in predicted secondary structure do not affect processing. Cancer Res 2006;66:6097–104.[Abstract/Free Full Text]
26. April CS, Barsh GS. Distinct pigmentary and melanocortin 1 receptor-dependent components of cutaneous defense against ultraviolet radiation. PLoS Genet 2007;3:e9.[CrossRef][Medline]
27. Cui R, Widlund HR, Feige E, et al. Central role of p53 in the suntan response and pathologic hyperpigmentation. Cell 2007;128:853–64.[CrossRef][Medline]
28. Landgraf P, Rusu M, Sheridan R, et al. A mammalian microRNA expression atlas based on small RNA library sequencing. Cell 2007;129:1401–14.[CrossRef][Medline]
29. Thomson JM, Newman M, Parker JS, Morin-Kensicki EM, Wright T, Hammond SM. Extensive post-transcriptional regulation of microRNAs and its implications for cancer. Genes Dev 2006;20:2202–7.[Abstract/Free Full Text]

This article has been cited by other articles:

				M. Kitago, S. R. Martinez, T. Nakamura, M.-S. Sim, and D. S.B. Hoon Regulation of RUNX3 Tumor Suppressor Gene Expression in Cutaneous Melanoma Clin. Cancer Res., May 1, 2009; 15(9): 2988 - 2994. [Abstract] [Full Text] [PDF]

This Article Abstract Full Text (PDF) Alert me when this article is cited Alert me if a correction is posted Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Bemis, L. T. Articles by Robinson, W. A. Search for Related Content PubMed PubMed Citation Articles by Bemis, L. T. Articles by Robinson, W. A.