e- ISSN 0976 - 3651 Print ISSN 2229 - 7480
International Journal of Biological
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Pharmaceutical Research
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DIFFERENTIAL EXPRESSION PROFILES OF MICRORNAS
INVOLVED IN MALIGNANT MELANOCYTE PIGMENTATION
Petra El Hajj
1, Mohammad Krayem
1, Hussein Fayyad-Kazan
2, Bassam Badran
3, Ghanem
Ghanem
1, Fabrice Journe
11
Laboratory of Oncology and Experimental Surgery, Institut Jules Bordet, Université Libre de Bruxelles, 1 Rue Heger-Bordet, 1000 Brussels, Belgium.
2Laboratory of Experimental Hematology, Institut Jules Bordet, Université Libre de Bruxelles, 1 Rue Heger-Bordet, 1000
Brussels, Belgium.
3
Department of Biochemistry, Lebanese University, Rafic Campus, 1003 Hadath-Beirut, Lebanon.
ABSTRACT
Pigmentation as well as pigmentation-associated genes have been recently correlated with poor clinical outcome of metastatic melanoma patients. MicroRNA regulation of melanogenic genes emerges as an alternative mechanism controlling pigmentation. In our study, we have identified and compared miRNA expression profiles in different melanoma cell lines before and after stimulation of pigmentation. The latter was achieved using different mechanisms: by tyrosine, the melanogenesis main substrate, by vemurafenib, a mutated BRAF inhibitor that moderates excessive degradation of MITF, and by forskolin, a stimulator of cAMP pathway. We could identify a series of highly downregulated miRNAs and another of upregulated ones having potential target involved in pigmentation as revealed by in silico analyses. Our study pointed out miRNAs that target CREB and MITF, the main genes activating pigment formation, and, interestingly, others implicated in melanosome transport, bringing new insights into the understanding of the regulation of pigmentation in melanoma and possibly in melanocytes.
Key Words: MicroRNAs, melanoma cell lines, pigmentation. INTRODUCTION
Proteins associated with pigmentation, melanosomal proteins in particular, have been, and still being used, as melanoma markers and targets for therapy, and amelanotic melanomas are usually considered of poor progonosis. Conversely, promotion of melanogenesis may reflect disease progression. Indeed, melanin and melanin intermediates have been used as markers of melanoma progression, particularly 5-S-cysteinyldopa (Wakamatsu et al., 2002), TYRP1 (Journe et al., 2011; El Hajj et al., 2013), Corresponding Author
Fabrice Journe
Email: [email protected]
TYRP2/DCT (Mandruzzato et al., 2006) and pigmentation presence in sentinel lymph nodes (van Lanschot et al., 2014) were found to correlate with poor clinical outcome. Germline MC1R polymorphisms are related to altered pigment production and increased risk for melanoma (Landi et al., 2006). MSH can be overproduced by malignant melanocytes on one hand and MC1R are overexpressed as compared to the normal melanocyte on the other, so that an auto/paracrine loop may continuously stimulate melanogenesis (Loir et al., 1997) through cAMP pathway activation and MITF synthesis.
There is an overwhelming data on the regulation of pigmentation through the literature (Yamaguchi et al., 2007) that includes many signaling pathways all acting on
MITF that is considered essential to initiate and promote melanogenesis. For example, activating BRAF and NRAS mutations negatively affect pigmentation through a sustained activation of the MAPK pathway leading to MITF excessive degradation by the proteasome (Zhao et al., 2011). This can be amplified by low phosphatase activity of PTEN (e.g. mutation) and promotion of PI3K/AKT signaling that can inhibit the MITF regulator GSK-3β within Wnt/β-catenin pathway (Terragni et al., 2011). Pigmentation score of melanoma tissues taken from patients before and during treatment with BRAF inhibitors increased in half of the cases (Long et al., 2013).
MicroRNA regulation of melanogenic genes has recently been described and emerges as an alternative mechanism controlling pigmentation. MicroRNAs (miRNAs) are small (about 22 nucleotide-long) non-coding endogenous RNAs which function through complementary base pairing with mRNAs to either regulate mRNA degradation or modulate protein translation (Bartel, 2004). Individual miRNAs are able to affect the expression of hundreds of genes. It is predicted that one third of all mRNAs are targeted and regulated by miRNAs. MicroRNAs can be expressed in a tissue-specific and developmental stage tissue-specific manner and play an important role in regulating biological processes such as development, proliferation, differentiation and apoptosis.
Only few studies have focused on the role played by miRNAs in the regulation of pigmentation in melanocytes or melanoma cells. Notably, miR-155 has been identified as a powerful regulator of TYRP1 (Li et al., 2012), while 137 (Dong et al., 2012) and miR-145 (Dynoodt et al., 2013) repress the expression of MITF and its downstream targets TYR, TYRP1 and TYRP2/DCT. In this context, a recent review by Mione et al. summarizes the activities of miRNAs in melanocytes and melanomas (Mione & Bosserhoff, 2015).
In the current study, in order to identify a group of miRNAs acting together to promote pigmentation, we aimed to compare miRNA expression profiles in different melanoma cell lines before and after stimulation of pigmentation through different key mechanisms.
MATERIAL AND METHODS
Effectors
Vemurafenib (PLX4032) and forskolin (both from Selleck Chemicals, Houston, TX, USA) were dissolved in DMSO and stored at -20°C. L-Tyrosine (from Sigma-Aldrich, Saint-Louis, MO, USA) was dissolved in NaOH and stored at -20°C.
Melanoma cell lines and culture conditions
Human melanoma cell lines were from lymph node metastases. HBL line is WTBRAF/WTNRAS while MM050 and MM074 lines are V600EBRAF/WTNRAS. Mutations in BRAF and NRAS were assessed as
previously reported (Herraiz et al., 2012). Cell lines were maintained in HAM-F10 medium (Lonza, Rockland, ME, USA) supplemented with 5% heat-inactivated fetal calf serum, 5% heat-inactivated newborn calf serum and with L-glutamine, penicillin, kanamycin and streptomycin at standard concentrations (Sigma-Aldrich) and a humidified atmosphere of 5% CO2 at 37°C. Importantly, HAM-F10
medium has the lowest tyrosine concentration compared to others usually used to culture malignant melanocytes, namely RPMI and DMEM. This keeps melanogenesis at the lowest level of activation. Lines are regularly checked for mycoplasma contamination using MycoAlert® Mycoplasma Detection Kit (Lonza).
Stimulation of cell pigmentation
HBL, MM050 and MM074 cell lines were plated at 1.5x106 cells in 100 mm Petri dish with HAM-F10 medium. One day after plating, medium was replaced by fresh one containing 250 μM tyrosine alone for HBL line, supplemented with 1 µM vemurafenib for MM074 line, or 1 µM vemurafenib and 10 µM forskolin for MM050 line. Cells were cultured for 48 hours or longer as described in results.
Total RNA extraction
Total RNA, including microRNAs, was extracted by using miRVana miRNA Isolation Kit (Ambion, Austin, TX, USA) following the manufacturer’s protocol with the addition of an acid/phenol/chloroform, the aqueous phase containing the RNA portion was mixed with ethanol and passed through a filter cartridge for 15 sec at 10,000 g, then the filter is washed several times and the RNA is eluted with RNase-free water in a final volume of 50 μl. RNA concentration was evaluated using a NanoDropTM 1000 spectrophotometer (Thermo Scientific, Wilmington, DE, USA). Total RNA integrity control was assessed based on the RNA profile using the Agilent RNA 6000 Nano Kit and the quality control in miRNA analysis was assessed using the Agilent Small RNA Kit both generated by a Bioanalyzer 2100 (Agilent Technologies, Santa Clara, CA, USA). RNA extracts were used for mRNA (RT-qPCR) and miRNA (Taqman Low Density Array) quantification (see below).
mRNA RT-qPCR
MITF (forward: 5’- GGCCTCACCATCA GCAACTC-3’, reverse: 5’- TCTCTTTGGCCAGTGCTCTTG-3’), TYR (forward: 5’-GCAAAGCATAC CATCAGCTCA-3’, reverse: 5’-GCAGTGCATCCA TTGACACAT-3’), TYRP1 (forward: 5’-AGCCCTCAGTATCCCCATGAT-3’, reverse: 5’-CCCGGACAAAGTGGTTCTTTT-3’), TYRP2 (forward: 5’-GAGTACACCCCGACTACCGTGA-3’, reverse: 5’-GGCGTCCTGGTCCTAATAATG-3’), and 18S (forward: 5’-GCGGCGGAAAATAGCCTTTG-3’, reverse: 5’-GATCACACGTTCCACCTCATC-3’) (Life Technologies, Gent, Belgium). The amplification was performed on a LightCycler 480 System (Roche Diagnostics GmbH, Mannheim, Germany) using an initial activation step (95°C for 10 min) followed by 40 cycles of amplification (95°C for 15 sec and 60°C for 60 sec). Relative quantification was calculated by normalizing the Ct (cycle threshold) of the four genes with the Ct of 18S (loading control) using the 2-∆∆Ct calculation method.
Taqman Low Density Array (TLDA) for miRNA profiling and data analysis
A two-step procedure was performed to profile the miRNAs in control and treated melanoma cell lines. First 300 ng of total RNA was subjected to RT (reverse transcription) using a TaqMan® microRNA Reverse Transcription Kit (#4366596; Applied Biosystems, Ghent, Belgium) and Megaplex RT primers (Human Pool A, #4399966; Applied Biosystems, Ghent, Belgium) following the manufacturer’s protocol, allowing simultaneous reverse transcription of 361 mature human miRNAs. RT was performed on a T100 thermal cycler (BioRad) with the following cycling conditions: 40 cycles of 16°C for 2 min, 42°C for 1 min and 50°C for 1 sec, followed by a final step of 80°C for 5 min to inactivate the reverse transcriptase. After the amplification step, the products were diluted with RNase-free water, combined with TaqMan gene expression Master Mix and then loaded into TaqMan Human MicroRNA Array A (#4398965; Applied Biosystems, Gent, Belgium), which is a 384-well formatted plate and real-time PCR-based microfluidic card with embedded TaqMan primers and probes in each well for the 361 different mature human miRNAs.
Quantitative PCR was performed according to the manufacturer’s instructions on an ABI PRISM 7900HT sequence detection system (Applied Biosystems, Gent, Belgium) with the following cycling conditions: 50°C for 2 min, 94.5°C for 10 min followed by 40 cycles of 95°C for 30 sec and 59.7°C for 1 min. The Ct (cycle threshold) was automatically given by SDS 2.3 software (Applied Biosystems, Ghent, Belgium). Using the Data assist v3.01 we obtained the fold changes in miRNAs calculated by the equation 2−ΔΔCt relative to control. The U6 transcript embedded in the TaqMan Human MicroRNA Arrays was used as normalization signal as it sorted as the best endogenous control. MicroRNAs of Ct above 35 were excluded for analysis. MicroRNAs were considered
differentially regulated if at least two-fold up or downregulation was observed.
MicroRNAs target prediction
Computer-based programs were used to predict potential targets sites for the up and downregulated miRs in the 3’UTR of mRNA of selected pigmentation genes. We searched in TargetScan 6.2 (www.targetscan.org), miRanda (www.microrna.org/microrna/home.do) and PicTar web interface to find potential target genes playing roles in pigmentation. Table 1 lists important genes involved in melanogenesis and their key features according to Universal Protein Resource (www.uniprot.org).
RESULTS
Evaluation of cell pigmentation
Visual pigmentation of the melanoma cell pellets exposed to tyrosine, vemurafenib and forskolin was monitored and documented by photos obtained at 48 hours and 14 days (Fig.1). MM074 cells become lightly pigmented after 48 hours and highly black after 14 days of treatment. By contrast, the pigmentation of HBL and MM050 cells was obvious only at 14 days after treatment.
We confirmed these observations by RT-qPCR of mRNA expression levels of four key genes involved in melanogenesis. In all cell lines, MITF, TYR, TYRP1 and TYRP2/DCT mRNA levels were significantly increased after treatments for 48 hours (Fig.2). The effect of the different treatments on levels of gene expression was particularly critical in MM050 cells with TYRP1 increase by 46 fold. Of note, the expression of pigmentation genes have been compared at 24, 48 hours and 7 days after treatment initiation and the maximum expression of all genes was found at 48 hours (data not shown).
Profiling of miRNAs in pigmented cells
Identification of putative miRNA target genes linked to pigmentation
Up and downregulated miRNAs were then interrogated for potential target genes implicated in melanogenesis pathway. Thus, we used two strategies using computer algorithms TargetScan 6.2 (http://www.targetscan.org/), miRanda (micro.org) and PicTar web interface. First, we looked for genome-wide predictions of target sequences of differentially expressed miRNAs and further assessed if these target genes may be involved in melanogenesis. Second, we search for all miRNAs predicted to bind a selected list of genes involved
in melanogenesis (Table 1) and further examined if these miRNAs were among ones differentially expressed in our microarray analysis. Hence, we identified few genes that may be targeted by miRNAs and that are involved in the production of pigments (MITF, TYRP1, CREB1/5, Sox9, calnexin) as well as in the transport of melanosome to keratinocytes (Rab27, kinesin, myosin 5a). Among these genes, MITF and TYRP1, whose mRNAs were upregulated after tyrosine/vemurafenib/forskolin treatment, were the most possible targets of the pigmentation-associated miRNAs identify in our study.
Figure 1. Pellets of melanoma cells treated with tyrosine, vemurafenib and forskolin showing the stimulation of pigmentation after 48 hours (MM074) and 14 days (HBL and MM050).
Figure 2. Normalized gene expression levels (fold changes) of MITF, TYR, TYRP1 and TYRP2 mRNA in HBL, MM074 and MM050 melanoma cell lines exposed or not to tyrosine (Tyr), vemurafenib (vemu) and forskolin (FSK) for 48 hours. The mRNA expression level of each gene was significantly increased after treatment. The data show means ± SEM of 4 replicates of PCR amplification from 2 independent experiments.
Table 1. List of proteins involved in melanogenesis (pigmentation process).
Protein Key features Functions in melanogenesis
MITF Basic helix-loop-helix
leucine-zipper transcription factor Induces the expression of several pigmentation genes
TYR (tyrosinase) Copper monooxygenase type I transmembrane glycoprotein
Catalyzes the first 2 steps in the conversion of tyrosine to melanin
TYRP1 (tyrosinase-related protein 1)
Type I transmembrane
glycoprotein Melanin synthesis, stabilizing of tyrosinase TYRP2 (tyrosinase-related
protein 2) or DCT (DOPA chrome tautomerase)
Type I transmembrane
glycoprotein Eumelanin synthesis MC1R
(melanocortin 1 receptor) G protein-coupled receptor
Initiates the signaling cascade that leads to the production of the eumelanin
POMC (proopiomelanocortin) Polypeptide hormone precursor Cleaved to the two agonists of MC1R (α-MSH and ACTH)
ACTH (adrenocorticotropic
hormone) Polypeptide tropic hormone Agonists of MC1R CREB (cAMP-responsive
element binding protein)
Leucine-zipper transciption
factor Signaling from MC1R to MITF SOX9/SOX10
(sex determining region Y-box9/10)
HMG-box containing
transcription factors Regulate MITF
Pmel17/gp100/silver protein Type I transmembrane glycoprotein
Generates internal matrix fibers that define the transition from Stage I to Stage II melanosome
Fscn1 (fascin homolog 1) Actin-binding proteins Organizing filamentous actin into bundles crucial for dendrite formation
OCA1
(ocular albinism type 1) G-protein-coupled receptor Promotes early stages of melanosome biogenesis MelanA/MART1 (melanoma
antigen recognized by T cells 1) Type III membrane protein Promotes early stages of melanosome biogenesis OCA2 12 membrane spanning
transport protein Regulates the relative acidity of melanosomes Myosin 5a ATP-dependent motor protein Mediates melanosome binding to actin
Rab27A Ras-related GTPase Mediates melanosome binding to actin Mlph (melanophilin) Rab effector protein Mediates melanosome binding to actin
Rab38 Ras-related GTPase Post-Golgi trafficking of melanogenic enzymes Rab32 Ras-related GTPase Post-Golgi trafficking of melanogenic enzymes
Kinesin and dynein ATP-dependent motor proteins Facilitate the movement of melanosomes by promoting their attachment to microtubules
Calnexin Calcium-binding protein Proper folding and assembly of melanogenesis-related proteins in the ER
Table 2. miRNAs identified as down or upregulated in pigmented melanoma cell lines compared to non-pigmented lines by Data Assist analysis, fold-changes and potential target genes using TargetScan and miRBase. Potential target genes of miRNAs playing roles in pigmentation identified using TargetScan 6.2, miRanda and PicTar web interface.
Downregulated miRNAs in pigmented cells
Fold-changes expression
pigmented/non-pigmented ratio Potential target genes
hsa-miR-628-5p 0.02 CREB5
hsa-miR-518f 0.04
hsa-miR-22 0.07 MITF
hsa-miR-194 0.21 MITF
hsa-miR-367 0.35 CREB1, kinesin, myosin 5a
hsa-miR-10b 0.35
Upregulated miRNAs in pigmented cells
Fold-changes expression
pigmented/non-pigmented ratio Potential target genes
hsa-miR-211 2.0 MITF
hsa-miR-186 2.2 MITF, kinesin, calnexin, Rab27,
SOX9, SOX10
hsa-miR-508 2.2 MITF, myosin 5a
hsa-miR-324-3p 2.4
hsa-miR-510 2.4
hsa-miR-192 4.9 CREB5, kinesin
DISCUSSION
In this study, we compared miRNA expression profiles associated to pigmentation, stimulated through activation of three different mechanisms in three selected melanoma cell line models. We then identified differentially expressed miRNAs before and after stimulation and used them for in silico search for associated target genes.
The three cell lines were exposed to higher tyrosine concentration, the substrate of pigmentation key enzyme TYR, as the lines were cultured in Ham-F10 medium which contains low tyrosine. Of note, the latter alone was able to stimulate melanogenesis in the wild type BRAF HBL line, suggesting that these cells have no identified alterations in the pigmentation process. By contrast, in V600EBRAF MM074 line, the mutant BRAF inhibitor vemurafenib was required to lower MAPK activity and, hence, limit the excessive degradation of constitutive phosphorylated MITF, thus leading to pigmentation. Indeed, it was reported that inhibition of mutant BRAF by interfering RNA increased pigmentation in melanoma cells (Rotolo et al., 2005). Moreover, in the
V600EBRAF MM050 line, forskolin was required to
increase MITF levels in addition to vemurafenib. In fact, forskolin activates adenylate cyclase which increases the intracellular levels of cAMP, stimulating CREB and promoting MITF transcription (Herraiz et al., 2011).
Few studies pointed out the role of miRNAs in the regulation of pigmentation, mainly in animal models or human melanocytes. A study by Kennell et al. reported that miR-8 is a positive regulator of pigmentation in Drosophila melanogaster but the mechanism of such regulation is not known yet (Kennell et al., 2012). Dynoodt et al. identified miRNAs interfering with the pigmentation process (Dynoodt et al., 2013). They performed miRNA profiling on mouse melanocytes exposed to UV irradiation and forskolin, sorted 16 miRNAs differentially expressed in treated versus untreated cells, and identified miR-145 as strongly downregulated by treatment. They showed that overexpression or depletion of miR-145 in cells revealed, respectively, decreased or increased expression of many pigmentation-related genes, including Sox9, MITF, TYR, TYRP1, myosin-5a, Rab27a, and Fscn1, suggesting a key role for miR-145 in regulating melanogenesis. In addition, Jian et al. reported that miR-340 plays a key role in
regulating UV-induced dendrite formation and melanosome transport (Jian et al., 2014). In another study, Yan et al. reported a novel regulatory mechanism for skin pigmentation in fish and identified 13 miRNAs differentially expressed between red and white skin (Yan et al., 2013). They found that miR-429 is a potential regulator of melanogenesis as its silencing results in an obvious change in skin melanin content. The post-transcriptional regulation of Foxd3 by miR-429 could affect the expression of MITF and its downstream genes, including TYR, TYRP1, and TYRP2/DCT, which in turn determine skin color in fish. Two additional studies from the same group reported a pivotal role for miR-203 in human melanoma cells through reducing melanosome transport and promoting melanogenesis by targeting kinesin superfamily protein 5b (Noguchi et al., 2014b), and through negative regulating the CREB/MITF/Rab27a pathway and inhibiting cell growth (Noguchi et al., 2014a). Finally, another study reported that the expression of miR-125b in human melanoma cell lines was inversely correlated to pigment levels and that a miR-125b mimic decreased the expression of pigmentation-related genes and melanin content, suggesting that miR-125b could be a novel inhibitory factor of melanogenesis (Kim et al., 2014).
the involvement of miRNAs in melanogenesis, notably by targeting CREB and MITF, and highlight new miRNAs that could play roles in melanosome transport. Functional analyses by transfection strategies using e.g. pre-miR or antago-miR are required to better define the exact role played these miRNAs in the regulation of melanoma pigmentation.
Altogether, these data document the complexity of cell pathways that control melanogenesis and identify novel miRNAs that are potential additional modulators of melanoma pigmentation. The understanding of the regulation of pigmentation in melanoma is indeed of clinical relevance in order to explain why pigment or melanogenic markers are associated with a poor prognosis and to examine if manipulating pigmentation in melanoma
could be used as an additional therapeutic strategy. Indeed, as more than 150 genes have been identified to affect pigmentation, the inhibition of one particular gene does not appear as an effective strategy to impact pigmentation, while the dysregulation of multiple pigmentation-associated genes under the control of one miRNA might offer a more promising and realistic clinical approach.
ACKNOWLEDGEMENTS
This study received financial support from the "MEDIC Foundation", "Les Amis de l'Institut Bordet" and the "Fondation Lambeau-Marteaux". Petra El Hajj is the recipient of a fellowship from the Lebanese National Council for Scientific Research and the Lebanese University.
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