miR-125b induces cellular senescence in malignant melanoma
© Nyholm et al.; licensee BioMed Central Ltd. 2014
Received: 1 August 2013
Accepted: 16 April 2014
Published: 24 April 2014
Micro RNAs (miRs) have emerged as key regulators during oncogenesis. They have been found to regulate cell proliferation, differentiation, and apoptosis. Mir-125b has been identified as an oncomir in various forms of tumours, but we have previously proposed that miR-125b is a suppressor of lymph node metastasis in cutaneous malignant melanoma. Our goal was therefore to further examine this theory.
We used in-situ-hybridization to visualise miR-125b expression in primary tumours and in lymph node metastasis. Then using a miRVector plasmid containing a miR-125b-1 insert we transfected melanoma cell line Mel-Juso and then investigated the effect of the presence of a stable overexpression of miR-125b on growth by western blotting, flow cytometry and β-galactosidase staining. The tumourogenicity of the transfected cells was tested using a murine model and the tumours were further examined with in-situ-hybridization.
In primary human tumours and in lymph node metastases increased expression of miR-125b was found in single, large tumour cells with abundant cytoplasm. A stable overexpression of miR-125b in human melanoma cell line Mel-Juso resulted in a G0/G1 cell cycle block and emergence of large cells expressing senescence markers: senescence-associated beta-galactosidase, p21, p27 and p53. Mel-Juso cells overexpressing miR-125b were tumourigenic in mice, but the tumours exhibited higher level of cell senescence and decreased expression of proliferation markers, cyclin D1 and Ki67 than the control tumours.
Our results confirm the theory that miR-125b functions as a tumour supressor in cutaneous malignant melanoma by regulating cellular senescence, which is one of the central mechanisms protecting against the development and progression of malignant melanoma.
Keywordshsa-miR-125b Melanoma Senescence In-situ-hybridization Mel-Juso
Replicative cellular senescence is the principal mechanism limiting proliferation of normal human cells [1, 2]. After approximately 40–60 cell divisions (the Hayflick limit) the cells enter a senescence-associated, irreversible mitotic arrest and exhibit characteristic features comprising flattened morphology with abundant cytoplasm and biochemical markers such as senescence-associated beta-galactosidase (SA-beta-gal), p53 and cell cycle Inhibitors (p16, p21, p27) . Telomere shortening is the primary mechanism of replicative senescence in normal diploid cells .
It has recently been discovered that various forms of DNA and cellular damage may cause premature cellular senescence before achieving the Hayflick limit. Interestingly, activation of oncogenes provides a potent senescence signal (oncogene-induced-senescence, OIS) which is considered to be an early protective mechanism against development of cancer [3, 5–7]. Increased SA-beta-gal staining is seen in a variety of pre-malignant conditions, such as lung adenomas, congenital naevi, benign prostatic hyperplasia and premalignant prostatic intraepithelial neoplasia supporting the role of OIS in early control of malignancy [3, 5, 8, 9]. It has also been observed that progression to the invasive tumour stage is associated with a suppression of OIS [5, 6, 9–11]. Cellular senescence may also be a mechanism inhibiting cancer progression, since senescence-like state can be induced in established tumours and cancer cell lines by a variety of mechanisms including chemotherapeutic agents or radiation (therapy-induced senescence, TIS) [11, 12].
Cutaneous malignant melanoma (MM) is a common, highly aggressive cancer derived from the melanocytes in the skin. MM is an area of high medical need since the metastasis occurs early despite a very low primary tumour mass and metastatic disease is highly resistant to chemotherapy and radiotherapy. MM cells are resistant to apoptosis and the induction of cellular senescence could be explored as a novel approach to therapy .
MicroRNAs (miRNA) are 21–25 nt non-coding RNA molecules, which bind to the 3′UTR of mRNAs conferring translational inhibition or degradation . MiRNAs have been found to regulate cell proliferation, differentiation, and apoptosis [15–17] and they have also been implicated in the regulation of senescence (miR-29, miR-30, miR-34a, miR-34b, miR-34c, miR122 miR-203, miR-205 and miR-217) [18–23] mostly by interfering with either the p53 pathway or the retinoblastoma RB1/E2F function. By an extensive analysis and comparison of miRNA expression levels Kozubek et al. showed that it was possible to distinguish melanoma speciments from benign neavi based on the miRNA signature . This supports the theory that miRNA level may influence the development MM.
By comparing miR expression profiles in metastasizing and non-metastasizing MM we proposed that miR-125b is implicated in the progression of human MM . MiR-125b expression is decreased in the primary cutaneous MM producing sentinel node metastasis comparing to the T-stage-matched non-metastasizing tumours . This has later been confirmed by other groups [27, 28]. Mir-125b is a known oncomir and has been implicated in the pathogenesis of leukemias and B-cell lymphomas, breast cancer, squamous cell carcinoma, urothelial carcinoma, prostate carcinoma and colon cancer [29–35]. In MM it has been shown to be related to the pigmentation level . Kappelmann et al. showed that treatment of MM cells with pre-mir-125b resulted in a strong suppression of cellular proliferation .
We have gathered preliminary evidence that miR-125b may be involved in the regulation of senescence in MM . In this paper we show that upregulation of miR-125b induces senescence and might constitute one of the possible mechanisms of the suppressive effect of miR-125b in MM.
Human MM cell line Mel-Juso (DSMZ, Braunschweig, Germany) was grown in DMEM Glutamax (Invitrogen, Carlsbad, CA) with 10% FBS (Life Technologies, LT) in 37°C and 5% CO2. Transfected Mel-Juso cells were cultured in selection medium, which was the same medium supplemented with 10-μg/mL blasticidin (Invitrogen).
Mel-Juso cells were transfected with a miRVector plasmid containing a miR-125b-1 [UCSC Genome Bioinformatics: uc010rzr.1]  insert and a blasticidin resistance gene (miRVec-125b)  or the control miRNA Vector plasmid with a blasticidin resistance gene but without any insert (miRVec-control) (Source BioScience, LifeSciences, Nottingham, UK) (map and sequence of miR-125b, see Additional file 1: Figure S1). 300.000 cells were seeded out 24 h before transfection to reach a confluency of 70–90%. 1 μg of the plasmid was mixed with 400 μl OPTI-MEM (Invitrogen) and 5 μl Lipofectamine RNAiMax (Invitrogen). The mixture was incubated at room temperature (RT) for 20 min and added to freshly washed Mel-Juso cells in 1600 μl culture medium for 24 h followed by a 14-day-culture in the selection medium in 37°C/5% CO2. miR-125b expression was confirmed using PCR in each clone. Optimisation in the beginning of the study showed that the transfection efficacy in Mel-Juso cells was higher with Lipofectamine RNAiMax than any other transfection reagents. Transfection procedure was done in four replicates for each plasmid and each evaluated using PCR.
For the measurement of BrdU incorporation, Mel-Juso cells transfected with miRVec-125b or the miRVec-control were pulsed for 20 min with 10 μM BrdU, fixed in 70% ethanol for at least 2 h, washed with phosphate-buffered saline (PBS), incubated in 2 N HCl for 30 min, washed in neutralizing buffer (0.2 M Na2B4O7, pH 8.5) and resuspended in dilution buffer (0.5% Tween 20 and 0.5% bovine serum albumin in PBS) prior to the addition of anti-BrdU mouse antibodies (Becton Dickinson, Franklin Lakes, NJ). Goat anti-mouse antibodies labeled with Alexa-Fluor 488 (1:1000; Invitrogen, Carlsbad, CA) were applied as secondary antibodies. DNA was stained with 0.0003% (30–40 μl per samlpe) 7-amino-actinomycin D solution (7AAD; Beckman Coulter, Brea, CA). Samples were analyzed by means of flow cytometry (Beckman-Coulter, Fullerton, CA). BrdU-positive events (FL1) were plotted versus cellular DNA content (FL3).
Tumour formation in mice
Mice experiments were performed in accordance with the national law for the experiments on animals of 14 Jun 2011 (full text available on https://www.retsinformation.dk/Forms/r0710.aspx?id=145380) and supervised by an authorised researcher (CML). All mice work is approved by Dyreforsoegstilsynet (generel permit for our laboratory work in mice, permit number 2012-15-2934-00419). All surgery was performed under sodium pentobarbital anesthesia, and every effort was made to minimize suffering.
Six Male BALB/C nude mice (Taconic, Rye, Denmark), 7 weeks of age, were housed in separate boxes with free access to water and standard laboratory food, in an animal facility at a 12 h light/dark cycle and the temperature 23°- 24°C. Mice were acclimatized for one week before the experiments. Subsequently they were anesthetized with 0.05 ml HypDorm given subcutaneously and 5 x 106 Mel-Juso cells transfected with miRVec-125b vector or the control vector in 0.2 ml PBS were injected subcutaneously into the left or right flank, respectively. Tumours with a diameter of at least 1 mm were mapped separately for each animal and followed until these reached a diameter of 12 mm or for a maximum time of 4 weeks. Animals were killed and tumours were weighed and cut into three parts, which were fixed in RNA Later (for RNA isolation), 4% formalin (for standard histology, immunohistochemistry and in situ hybridization) or were frozen at −80°C (for SA-beta-gal staining). Formalin fixed samples were embedded in paraffin and serial sections were stained with hematoxylin and eosin, Ki-67 and cyclin D1, according to standard histopathology protocols. The quantification of Ki-67 and cyclin D1 expression was done semiquantitatively in each tumour using the following ordinal scale: grade 1 (0%-25% positive tumour cells), grade 2 (26%-50% positive cells), grade 3 (51%-75% positive cells), grade 4 (76%-100% positive cells). Lungs and liver were taken out and cut in to three parts each, which were treated as the tumour samples and checked for distance metastases.
In situ hybridization (ISH) for miR-125b
Formalin-fixed and paraffin-embedded human tissue samples from the archives of the Department of Pathology (Rigshospitalet, Copenhagen, Denmark) were stage T1-T4 cutaneous MM and matched lymph node metastases (14 males and 3 females; mean age 52 years, range 24 to 83 years); mean Breslow depth 1.53 mm (range 0.37 to 4.00 mm); 15 superficial spreading MM and 2 nodular MM. Mouse tumour samples were prepared as described above. Paraffin sections (6 μm) were mounted on SuperFrost®Plus slides (Dako, Glostrup, Denmark) and air-dried for 1–2 h at RT. Sections were deparaffinized in xylene and hydrated through decreasing ethanol concentrations into PBS. Proteinase-K treatment 15 μg/ml in PK-buffer (5 mM Tris–HCl, pH 7.4, 1 mM EDTA, 1 mM NaCl) was performed at 37°C for 8 min in a volume of 300 μL in a Dako hybridizer (Dako). Sections were washed twice in sterile PBS and immediately dehydrated through an increasing gradient of ethanol solutions. Aliquots of 3 different Mercury LNA miRNA Detection Probes; hsa-miR-125b, scrambled probe, and U6 snRNA (Exiqon, Vedbaek, Denmark) were then denatured by heating to 90°C for 4 min and diluted to 40 nM, 40 nM, and 1 nM, respectively, in a formamide-free ISH buffer (Exiqon). 50 μL probe mixture was hybridized with the tissue sections in the hybridizer at 55°C for 60 min. The slides were placed at RT in 5× saline-sodium citrate (SSC) (Invitrogen) and stringency washes were performed for 5 min each at 55°C in 5× SSC (one wash), 1× SSC (two washes) and 0.2× SSC (two washes). The sections were then washed in PBS and blocked with DIG Wash and Block Buffer Set (Roche, Mannheim, Germany). Alkaline phosphatase (AP)-conjugated anti-DIG (Roche) was diluted 1:800 in the blocking solution and incubated for 60 min at RT. Slides were washed twice with PBS containing 0.1% Tween-20. Ready to use tablets (Roche) of 4-nitro-blue tetrazolium chloride (NBT) and 5-brom-4-chloro-3′-indolyl-phosphate (BCIP) substrate were dissolved in aqueous 0.2 mM levamisole. Slides were incubated for 120 min at 30°C to develop the dark-blue NBT-formazan precipitate. Sections were washed twice for 5 min in KTBT buffer (50 mM Tris–HCl, 150 mM NaCl, 10 mM KCl), and then twice in water, dehydrated in the ethanol gradient and mounted.
Monolayers of Mel-Juso cells transfected with miRVec-125b or the miRVec-control after a 24-h culture in 4-chamber glass (Nunc, Rochester, NY) or cryostat tissue sections from mice mounted on SuperFrost®Plus slides were washed twice in PBS and fixed in 0.5% glutaraldehyde (pH = 7.0) for 15 min at RT. The samples were washed twice with 20 ml 1 mM MgCl2in PBS (pH = 6.0) and stained with the SA-beta-gal staining solution containing 1 mg/ml of 5-bromo-4-chloro-3-indolyl beta-galactopyranocid, 4% dimethylformamide, 0.0012 mM potassium ferrocyanide and 1 mM MgCl2 in PBS (pH = 6.0) for 4 h (Mel-Juso) or overnight (melanocytes) at 37°C [3, 5]. Percentage of positive cells was evaluated blindely by one of the investigators (RG).
Cells were washed in PBS and lysed in the sample buffer 0.5 M Tris–HCl pH 6.8; 5% glycerol; 10% SDS; DTT 0.2 M) supplemented with protease inhibitor cocktail (Roche). Equal amounts of protein were separated by a 4-8% and 12% Bis-Tris gel electrophoresis at 200 V followed by electrophoretic transfer to a nitrocellulose membrane (Bio-Rad Laboratories, Hercules, CA). Membranes were blocked for 1 h at 4°C with Li-Cor blocking agent (Li-Cor, Lincoln, NE) before incubation with the primary antibodies against β-actin (mouse) (Sigma Aldrich, St. Louis, MO), p16 (mouse) (BD Pharmingen, San Diego, CA), p21 (mouse) (Dako), p27 (mouse) (Santa Cruz Biotechnology, Santa Cruz, CA) or p53 (rabbit) (Dako) overnight at 4°C. Subsequently, they were incubated for 1 h with the appropriate secondary antibodies labeled with 800IR dye (anti-rabbit) (Li-Cor), Alexa Fluor 680 (anti-mouse or anti-rat) both from Molecular Probes (Invitrogen). Protein bands were detected and quantified with the infrared Odyssey imaging System (Li-Cor). Quantified intensities were adjusted to the relevant actin intensity and control and sample was then compaired.
10.000 Mel-Juso cells transfected with miRVec-125b or the miRVec-control were seeded in 10 cm petri dishes containing 10 ml selection medium and cultured for 10–14 days until the appearance of macroscopically visible colonies. The plates were washed, fixed in paraformaldehyde for 24 h and stained with crystal violet 0.05% (Sigma Aldrich) for 20 min before washing in water. Plates were photographed (microscope Olympus IX 70 and camera Nikon D60) and colonies were counted manually.
Cell viability and apoptosis assessment
1.500.000 Mel-Juso cells transfected with miRVec-125b or the miRVec-control were seeded in 10 cm Petri dishes containing 10 ml selection medium, the medium changed after 24 h and all (floating and adherent) cells collected after another 24 h. Unfixed cells were stained simultaneously with FITC-Annexin-V and PI, according to the manufacturer’s protocol (Beckman-Coulter, Fullerton, CA) and analyzed by means of flow cytometry (Beckman-Coulter, Fullerton, CA) as described previously .
RNA isolation and real-time q-PCR (RT-qPCR)
Mel-Juso cells transfected with miRVec-125b or the miRVec-control were collected and washed twice in PBS. Murine tumour tissue samples were homogenized using a TissueLyzer II (Qiagen, Valencia, CA, USA) and processed as previously described . Small RNAs were purified using the mirVana Isolation Kit (Ambion, Foster City, CA) and the PureLink RNA Micro to Midi Kit (Invitrogen) according to the manufacturer’s instructions. The concentration of RNA was measured spectrophotometrically using NanoDrop ND-1000 (Thermo Scientific, Wilmington, DE), and RNA integrity was confirmed with Agilent 2100 Bioanalyzer using Agilent Nano RNA kit (Agilent Technologies, Santa Clara, CA). miR-125b expression was measured in the samples containing the same amount of RNA with quantitative qRT-PCR assay (TaqMan TM microRNA Reverse Transcription kit-4366596, and TaqMan Universal PCR Master mix-4324018, Applied Biosystems, Foster City, CA) and validated primer sets (Applied Biosystems) according to the manufacturer’s instructions. MiR-191 was used as a reference for the normalization of qRT-PCR-data. The q-PCR was performed in triplicates using a 7900HT Fast Real-Time PCR System (Applied Biosystems).
DNA was extracted from transfected Mel-Juso cell lines containing either miRVec-125b or miRVec-control using QIAamp DNA mini-Kit (Qiagen) in accordance with the manufacturer’s protocol. PCR was made with reagents from Life Technologies as follows: Master mix (10% Gene amp PCR buffer II, 2.5 mM MgCl2, 400 μM dNTP, 0.2 μM of each primer and 1U Ampli Taq gold) was prepared and 7.2 μL was mixed with 16.8 μL of ddH2O and 1 μL of 5 ng/μL DNA (template). PCR was setup on an Eppendorf MasterCycler gradient cycler (Hamburg, Germany) with the following program: 95°C 10 min, then 35 cycles of: 95°C 30 s, 62°C 30 s and 72°C 1.5 min; ended with 72°C 10 min and 4°C hold. The PCR product was visually inspected on a 2.2% agarose gel on the Flashgel system (Lonza, Basel, Switzerland). The remaining PCR product was purified on Qiaquick columns (Qiagen) according to manufacturer’s specifications and eluted in 50 μL buffer. Four μL of purified PCR product were used as template in the sequencing reaction.
Sequencing was performed in duplicates with the BigDye V.1.1 cycling sequencing kit (Life Technologies) Samples consisted of 4 μL of Ready reaction mix, 2 μL of BigDye buffer, 3.2 pmol of primer (forward-GCGTTTAAACTTAAGCTTGGTACCGAGC, reverse-CATTCCCCCCTTTTTCTGGAGAC), 4 μL of template and 20 μL of ddH2O. Reactions were performed with forward and reverse primers in duplicate with the following program: 96°C 1 min, then 25 cycles of: 96°C 10s, 50°C 5 s and 60°C 4 min, ending with 4°C hold. PCR products were purified with Centrisep columns (Princeton Separations, Adelphia, NJ) according to the manufacturer’s recommendations. Two μL of purified sequencing product were mixed with 8 μL of HiDi formamide and run on an ABI3130xl DNA Sequencer (Life Technologies) with the following specifications: Array 36 cm, polymer POP4, injection voltage 3 kV, injection time 7 s, run voltage 15 kV and run time 1700 sec. One of the reverse duplicates was removed due to low quality. Results were analyzed and assembled with Sequencher v.5 (Gene Codes Corporation, Ann Arbor, MI, USA). Differences in the chromatograms were visually inspected. Low quality regions resulting in differences were removed. Sequence ends had to be cropped due to low reaction quality.
All numeric results are given as mean ± SD unless stated otherwise. t-test was used for intergroup comparison. P < 0.05 was considered significant.
miR-125b is expressed in human primary cutaneous MM and lymph node metastases
miR-125b inhibits proliferation and induces senescence in human melanoma line Mel-Juso
Mel-Juso cell line is established from the primary tumour of a 58-year-old woman with MM in 1977. This line has wildtype BRAF, is poorly differentiated  and exhibits intermediate invasiveness . Transfection with miRVec-125b resulted in an 8.0 ± 1.13-fold upregulation of miR-125b by RT-qPCR compared to the miRVec-control transfected cells. After the experiments were completed, the miR-125b insert was sequenced to confirm that no mutation had occurred in the miR-125b sequence of the insert throughout the experimental period. For full insert consensus sequence, see Additional file 1: Figure S2.
To evaluate whether apoptosis was responsible for the apparent lesser growth observed in the colony forming assay we compared the proportions of annexin-positive cells in the miRVec-125b transfected cells with the controls. We found no evidence of apoptosis in either group (95.5% ±0.7% annexin-negative, viable cells in the miRVec-125b transfected cell line versus 94.7% ±1.2% in the control; p = 0.46).
miR-125b expression in Mel-Juso cells induces senescence and reduces proliferation in a murine tumour model
This study documented that stable, ectopic expression of miR-125b induced cellular senescence in a MM cell line Mel-Juso, both in vitro and in a xenotransplantation model in mice in vivo. The results were surprising, since miR-125b is generally considered to be an oncogene and in a transgenic mouse model miR-125b overexpression causes myeloid leukemia and B-cell malignancies [46, 47]. However, our and other’s preliminary evidence suggested that miR-125b has an anticancer effect in MM [26, 28, 36, 41] and its decreased expression correlated with the metastatic potential of primary cutaneous MM. In the present study we substantiated this hypothesis using a model of stably transfected Mel-Juso cell line with the miRVec-125b vector previously developed by Voorhoeve et al. . We were able to demonstrate an increase in senescence markers (SA-beta-gal, p27, p21, p53) and decreased proliferation with a G0/G1 arrest in miRVec-125b-transfected cells as compared with the control. miRVec-125b -transfected cells were tumourigenic in vivo, but the tumours showed an increased rate of senescence and decreased amount of proliferating cells, as measured by Ki67 and cyclin D1 staining. Together with the results of ISH showing that miR-125b is expressed in primary human MM and lymph node metastases, the data indicate that miR-125b is implicated in the regulation of senescence in human MM.
The mechanism by which miR-125b overexpression promotes cellular senescence is unknown. MiR-125b may negatively regulate the p53 tumour suppressor gene and Bak1  but such a downregulation has not been seen in Mel-Juso cells in our experiments (data not shown). Other known targets are BMF and Lin28a  but they are unlikely to be involved in cellular senescence in MM. In general, the functional role of miR-125b seems to be cell specific, since in some types of cancer (e.g. urothelial carcinoma) this miR seems to acts as a tumour suppressor .
MiR-dependent induction of cellular senescence in MM has recently and independently been demonstrated by other researchers for miR-203 and miR-205 in cutaneous MM and for miR-34a in uveal MM [23, 25, 50]. A common biochemical pathway in these cases seems to be mediated by E2F and downregulation of Akt. This is an unlikely mechanism in the case of miR-125b since the screening for the effect of miR-125b overexpression on Akt, Stat, mTOR did not show any changes in the expression level in MM cell lines and E2F2 and E2F3 proteins are not expressed in Mel-Juso cells (data not shown).
In this study we used ISH to evaluate the miR-125b expression locally in the tissue. We observed that the ISH on human tissue samples showed a strong nucleic concentration of miR-125b. This has been surprising since the conventional theory requires the cytoplasmic presence of miRNA for its proper function [51–53]. However, the studies on the compartmentalization of miR-125b have been done with cell lines and not tissue samples, like in our study. On the other hand, our ISH staining on the murine tumours showed a more pronounced cytoplasmic staining. One explanation of this could be that the miR-125b distribution in the cell is different between cells from a cultivated cell line and cells from tissue samples. Kobuzek et al.  showed that the miR-125b signal in MM is different in tissue and cell lines. Another explanation could be that the specific distribution varies from tissue to tissue. This is supported by the fact that ISH staining for miR-125b on other tissues have showed that in some tissues there is a clear staining of the nuclei [54–56] while in others the staining is mainly cytoplasmatic [57, 58]. This will have to be validated in further studies. It is finally possible, that nuclear miR-125b have a distinct biological role, as previously suggested for other miRNA species.
Another limitation of this study is that the functional role of miR-125b has only been investigated in Mel-Juso cells and it remains to be seen whether the same is valid for other MM cell lines and primary melanocytes. These experiments were attempted but failed due to massive apoptotic response caused by miRVec-125b plasmid. It is known that Mel-Juso cells are very resistant to apoptosis, which enabled us to achieve a stable miR-125b overexpression. Second, we focused primarily on the effect of the sustained overexpression of miR-125b in MM cells. The use of inducible miR-125b vectors will be helpful to elucidate the effect of acute changes in miR-125b levels.
Our results confirm the theory that miR-125b function as a tumour supressor in cutaneous malignant melanoma by regulating cellular senescence, which is one of the central mechanisms protecting against the development and progression of malignant melanoma.
In view of the recent developments in the use of miRNA mimics and inhibitors for therapy, it is conceivable that miR-125b would be utilized for the treatment of MM by inducing senescence of cancer cells. However, as exemplified by our murine MM model, miR-125b induces senescence focally in the tumours and the effect on tumour mass is negligible. It is known that miRNAs act primarily as switches and amplifiers of the cellular signaling pathways and their physiological effect is rarely very strong . It is therefore our goal for future research to identify which signaling pathways of therapeutic relevance are targeted by miR-125b and devise strategies by which mir-125b overexpression may amplify the effect of anticancer drug.
In situ hybridization
We thank Eva Hoffman, Omid Niazi and Pia Eriksen for their technical help with the experiments. miRVectors were kindly donated by prof. Anders Lund, Biotech Research and Innovation Centre, University of Copenhagen, Denmark. The study was supported by research grants from the following foundations: Aage Bang Foundation, Birgit and Svend Igor Pock-Steens Foundation and The Danish Cancer Society.
- Hayflick L, MOORHEAD PS: The serial cultivation of human diploid cell strains. Exp Cell Res 1961, 25: 585–621. 10.1016/0014-4827(61)90192-6View ArticlePubMedGoogle Scholar
- Hayflick L: The cell biology of aging. J Invest Dermatol 1979, 73: 8–14. 10.1111/1523-1747.ep12532752View ArticlePubMedGoogle Scholar
- Michaloglou C, Vredeveld LC, Soengas MS, Denoyelle C, Kuilman T, van der Horst CM, Majoor DM, Shay JW, Mooi WJ, Peeper DS: BRAFE600-associated senescence-like cell cycle arrest of human naevi. Nature 2005, 436: 720–724. 10.1038/nature03890View ArticlePubMedGoogle Scholar
- Harley CB, Futcher AB, Greider CW: Telomeres shorten during ageing of human fibroblasts. Nature 1990, 345: 458–460. 10.1038/345458a0View ArticlePubMedGoogle Scholar
- Collado M, Gil J, Efeyan A, Guerra C, Schuhmacher AJ, Barradas M, Benguria A, Zaballos A, Flores JM, Barbacid M, Beach D, Serrano M: Tumour biology: senescence in premalignant tumours. Nature 2005, 436: 642. 10.1038/436642aView ArticlePubMedGoogle Scholar
- Collado M, Serrano M: The power and the promise of oncogene-induced senescence markers. Nat Rev Cancer 2006, 6: 472–476. 10.1038/nrc1884View ArticlePubMedGoogle Scholar
- Collado M, Serrano M: Senescence in tumours: evidence from mice and humans. Nat Rev Cancer 2010, 10: 51–57. 10.1038/nrc2772View ArticlePubMedPubMed CentralGoogle Scholar
- Choi J, Shendrik I, Peacocke M, Peehl D, Buttyan R, Ikeguchi EF, Katz AE, Benson MC: Expression of senescence-associated beta-galactosidase in enlarged prostates from men with benign prostatic hyperplasia. Urology 2000, 56: 160–166. 10.1016/S0090-4295(00)00538-0View ArticlePubMedGoogle Scholar
- Majumder PK, Grisanzio C, O’Connell F, Barry M, Brito JM, Xu Q, Guney I, Berger R, Herman P, Bikoff R, Fedele G, Baek WK, Wang S, Ellwood-Yen K, Wu H, Sawyers CL, Signoretti S, Hahn WC, Loda M, Sellers WR: A prostatic intraepithelial neoplasia-dependent p27 Kip1 checkpoint induces senescence and inhibits cell proliferation and cancer progression. Cancer Cell 2008, 14: 146–155. 10.1016/j.ccr.2008.06.002View ArticlePubMedPubMed CentralGoogle Scholar
- Braig M, Lee S, Loddenkemper C, Rudolph C, Peters AH, Schlegelberger B, Stein H, Dorken B, Jenuwein T, Schmitt CA: Oncogene-induced senescence as an initial barrier in lymphoma development. Nature 2005, 436: 660–665. 10.1038/nature03841View ArticlePubMedGoogle Scholar
- Serrano M: Shifting senescence into quiescence by turning up p53. Cell Cycle 2010, 9: 4256–4257. 10.4161/cc.9.21.13785View ArticlePubMedPubMed CentralGoogle Scholar
- Gewirtz DA, Holt SE, Elmore LW: Accelerated senescence: an emerging role in tumor cell response to chemotherapy and radiation. Biochem Pharmacol 2008, 76: 947–957. 10.1016/j.bcp.2008.06.024View ArticlePubMedGoogle Scholar
- Giuliano S, Ohanna M, Ballotti R, Bertolotto C: Advances in melanoma senescence and potential clinical application. Pigment Cell Melanoma Res 2010, 24: 295–308.View ArticleGoogle Scholar
- Pasquinelli AE: MicroRNAs and their targets: recognition, regulation and an emerging reciprocal relationship. Nat Rev Genet 2012, 13: 271–282.PubMedGoogle Scholar
- Bartel DP: MicroRNAs: genomics, biogenesis, mechanism, and function. Cell 2004, 116: 281–297. 10.1016/S0092-8674(04)00045-5View ArticlePubMedGoogle Scholar
- Farh KK, Grimson A, Jan C, Lewis BP, Johnston WK, Lim LP, Burge CB, Bartel DP: The widespread impact of mammalian MicroRNAs on mRNA repression and evolution. Science 2005, 310: 1817–1821. 10.1126/science.1121158View ArticlePubMedGoogle Scholar
- Kato M, Slack FJ: microRNAs: small molecules with big roles - C. elegans to human cancer. Biol Cell 2008, 100: 71–81. 10.1042/BC20070078View ArticlePubMedGoogle Scholar
- He X, He L, Hannon GJ: The guardian’s little helper: microRNAs in the p53 tumor suppressor network. Cancer Res 2007, 67: 11099–11101. 10.1158/0008-5472.CAN-07-2672View ArticlePubMedGoogle Scholar
- Martinez I, Cazalla D, Almstead LL, Steitz JA, DiMaio D: miR-29 and miR-30 regulate B-Myb expression during cellular senescence. Proc Natl Acad Sci U S A 2011, 108: 522–527. 10.1073/pnas.1017346108View ArticlePubMedGoogle Scholar
- Menghini R, Casagrande V, Cardellini M, Martelli E, Terrinoni A, Amati F, Vasa-Nicotera M, Ippoliti A, Novelli G, Melino G, Lauro R, Federici M: MicroRNA 217 modulates endothelial cell senescence via silent information regulator 1. Circulation 2009, 120: 1524–1532. 10.1161/CIRCULATIONAHA.109.864629View ArticlePubMedGoogle Scholar
- Inui M, Martello G, Piccolo S: MicroRNA control of signal transduction. Nat Rev Mol Cell Biol 2010, 11: 252–263.View ArticlePubMedGoogle Scholar
- Benhamed M, Herbig U, Ye T, Dejean A, Bischof O: Senescence is an endogenous trigger for microRNA-directed transcriptional gene silencing in human cells. Nat Cell Biol 2012, 14: 266–275. 10.1038/ncb2443View ArticlePubMedGoogle Scholar
- Noguchi S, Mori T, Otsuka Y, Yamada N, Yasui Y, Iwasaki J, Kumazaki M, Maruo K, Akao Y: Anti-oncogenic microRNA-203 induces senescence by targeting E2F3 in human melanoma cells. J Biol Chem 2012, 287: 11769–11777. 10.1074/jbc.M111.325027View ArticlePubMedPubMed CentralGoogle Scholar
- Kozubek J, Ma Z, Fleming E, Duggan T, Wu R, Shin DG, Dadras SS: In-depth characterization of microRNA transcriptome in melanoma. PLoS One 2013, 8: e72699. 10.1371/journal.pone.0072699View ArticlePubMedPubMed CentralGoogle Scholar
- Dar AA, Majid S, De SD, Nosrati M, Bezrookove V, Kashani-Sabet M: miRNA-205 suppresses melanoma cell proliferation and induces senescence via regulation of E2F1 protein. J Biol Chem 2011, 286: 16606–16614. 10.1074/jbc.M111.227611View ArticlePubMedPubMed CentralGoogle Scholar
- Glud M, Rossing M, Hother C, Holst L, Hastrup N, Nielsen FC, Gniadecki R, Drzewiecki KT: Downregulation of miR-125b in metastatic cutaneous malignant melanoma. Melanoma Res 2010, 20: 479–484. 10.1097/CMR.0b013e32833e32a1View ArticlePubMedGoogle Scholar
- Kim KH, Bin BH, Kim J, Son ED, Park PJ, Choi H, Kim BJ, Yu SJ, Kang H, Kang HH, Cho EG, Lee TR: Novel inhibitory function of miR-125b in Melanogenesis. Pigment Cell Melanoma Res 2013, 27: 140–144.View ArticlePubMedGoogle Scholar
- Kappelmann M, Kuphal S, Meister G, Vardimon L, Bosserhoff AK: MicroRNA miR-125b controls melanoma progression by direct regulation of c-Jun protein expression. Oncogene 2013, 32: 2984–2991. 10.1038/onc.2012.307View ArticlePubMedGoogle Scholar
- Guan Y, Yao H, Zheng Z, Qiu G, Sun K: MiR-125b targets BCL3 and suppresses ovarian cancer proliferation. Int J Cancer 2010, 128: 2274–2283.View ArticleGoogle Scholar
- Huang L, Luo J, Cai Q, Pan Q, Zeng H, Guo Z, Dong W, Huang J, Lin T: MicroRNA-125b suppresses the development of bladder cancer by targeting E2F3. Int J Cancer 2010, 128: 1758–1769.View ArticleGoogle Scholar
- Hui AB, Lenarduzzi M, Krushel T, Waldron L, Pintilie M, Shi W, Perez-Ordonez B, Jurisica I, O’Sullivan B, Waldron J, Gullane P, Cummings B, Liu FF: Comprehensive MicroRNA profiling for head and neck squamous cell carcinomas. Clin Cancer Res 2010, 16: 1129–1139. 10.1158/1078-0432.CCR-09-2166View ArticlePubMedGoogle Scholar
- Baffa R, Fassan M, Volinia S, O’Hara B, Liu CG, Palazzo JP, Gardiman M, Rugge M, Gomella LG, Croce CM, Rosenberg A: MicroRNA expression profiling of human metastatic cancers identifies cancer gene targets. J Pathol 2009, 219: 214–221. 10.1002/path.2586View ArticlePubMedGoogle Scholar
- Schaefer A, Jung M, Mollenkopf HJ, Wagner I, Stephan C, Jentzmik F, Miller K, Lein M, Kristiansen G, Jung K: Diagnostic and prognostic implications of microRNA profiling in prostate carcinoma. Int J Cancer 2010, 126: 1166–1176.PubMedGoogle Scholar
- Veerla S, Lindgren D, Kvist A, Frigyesi A, Staaf J, Persson H, Liedberg F, Chebil G, Gudjonsson S, Borg A, Mansson W, Rovira C, Hoglund M: MiRNA expression in urothelial carcinomas: important roles of miR-10a, miR-222, miR-125b, miR-7 and miR-452 for tumor stage and metastasis, and frequent homozygous losses of miR-31. Int J Cancer 2009, 124: 2236–2242. 10.1002/ijc.24183View ArticlePubMedGoogle Scholar
- Calin GA, Sevignani C, Dumitru CD, Hyslop T, Noch E, Yendamuri S, Shimizu M, Rattan S, Bullrich F, Negrini M, Croce CM: Human microRNA genes are frequently located at fragile sites and genomic regions involved in cancers. Proc Natl Acad Sci U S A 2004, 101: 2999–3004. 10.1073/pnas.0307323101View ArticlePubMedPubMed CentralGoogle Scholar
- Glud M, Manfe V, Biskup E, Holst L, Dirksen AM, Hastrup N, Nielsen FC, Drzewiecki KT, Gniadecki R: MicroRNA miR-125b induces senescence in human melanoma cells. Melanoma Res 2011, 21: 253–256. 10.1097/CMR.0b013e328345333bView ArticlePubMedGoogle Scholar
- UCSC genoma bioinfromatics. 2013. http://genome-euro.ucsc.edu/
- Voorhoeve PM, Ie SC, Schrier M, Gillis AJ, Stoop H, Nagel R, Liu YP, van DJ, Drost J, Griekspoor A, Zlotorynski E, Yabuta N, De VG, Nojima H, Looijenga LH, Agami R: A genetic screen implicates miRNA-372 and miRNA-373 as oncogenes in testicular germ cell tumors. Cell 2006, 124: 1169–1181. 10.1016/j.cell.2006.02.037View ArticlePubMedGoogle Scholar
- Kamstrup MR, Gjerdrum LM, Biskup E, Lauenborg BT, Ralfkiaer E, Woetmann A, Odum N, Gniadecki R: Notch1 as a potential therapeutic target in cutaneous T-cell lymphoma. Blood 2010, 116: 2504–2512. 10.1182/blood-2009-12-260216View ArticlePubMedGoogle Scholar
- Holst LM, Kaczkowski B, Gniadecki R: Reproducible pattern of microRNA in normal human skin. Exp Dermatol 2010, 19: e201-e205. 10.1111/j.1600-0625.2009.01049.xView ArticlePubMedGoogle Scholar
- Holst LM, Kaczkowski B, Glud M, Futoma-Kazmierczak E, Hansen LF, Gniadecki R: The microRNA molecular signature of atypic and common acquired melanocytic nevi: differential expression of miR-125b and let-7c. Exp Dermatol 2011, 20: 278–280. 10.1111/j.1600-0625.2010.01163.xView ArticlePubMedGoogle Scholar
- Ziegler-Heitbrock HW, Munker R, Johnson J, Petersmann I, Schmoeckel C, Riethmuller G: In vitro differentiation of human melanoma cells analyzed with monoclonal antibodies. Cancer Res 1985, 45: 1344–1350.PubMedGoogle Scholar
- Wach F, Eyrich AM, Wustrow T, Krieg T, Hein R: Comparison of migration and invasiveness of epithelial tumor and melanoma cells in vitro. J Dermatol Sci 1996, 12: 118–126. 10.1016/0923-1811(95)00470-XView ArticlePubMedGoogle Scholar
- Fidler IJ: Tumor heterogeneity and the biology of cancer invasion and metastasis. Cancer Res 1978, 38: 2651–2660.PubMedGoogle Scholar
- Hoek KS, Schlegel NC, Brafford P, Sucker A, Ugurel S, Kumar R, Weber BL, Nathanson KL, Phillips DJ, Herlyn M, Schadendorf D, Dummer R: Metastatic potential of melanomas defined by specific gene expression profiles with no BRAF signature. Pigment Cell Res 2006, 19: 290–302. 10.1111/j.1600-0749.2006.00322.xView ArticlePubMedGoogle Scholar
- Enomoto Y, Kitaura J, Hatakeyama K, Watanuki J, Akasaka T, Kato N, Shimanuki M, Nishimura K, Takahashi M, Taniwaki M, Haferlach C, Siebert R, Dyer MJ, Asou N, Aburatani H, Nakakuma H, Kitamura T, Sonoki T: Emu/miR-125b transgenic mice develop lethal B-cell malignancies. Leukemia 2011, 25: 1849–1856. 10.1038/leu.2011.166View ArticlePubMedGoogle Scholar
- Bousquet M, Harris MH, Zhou B, Lodish HF: MicroRNA miR-125b causes leukemia. Proc Natl Acad Sci U S A 2010, 107: 21558–21563. 10.1073/pnas.1016611107View ArticlePubMedPubMed CentralGoogle Scholar
- Le MT, Teh C, Shyh-Chang N, Xie H, Zhou B, Korzh V, Lodish HF, Lim B: MicroRNA-125b is a novel negative regulator of p53. Genes Dev 2009, 23: 862–876. 10.1101/gad.1767609View ArticlePubMedPubMed CentralGoogle Scholar
- Chaudhuri AA, So AY, Mehta A, Minisandram A, Sinha N, Jonsson VD, Rao DS, O’Connell RM, Baltimore D: Oncomir miR-125b regulates hematopoiesis by targeting the gene Lin28A. Proc Natl Acad Sci U S A 2012, 109: 4233–4238. 10.1073/pnas.1200677109View ArticlePubMedPubMed CentralGoogle Scholar
- Yan D, Zhou X, Chen X, Hu DN, Dong XD, Wang J, Lu F, Tu L, Qu J: MicroRNA-34a inhibits uveal melanoma cell proliferation and migration through downregulation of c-Met. Invest Ophthalmol Vis Sci 2009, 50: 1559–1565.View ArticlePubMedGoogle Scholar
- Jeffries CD, Fried HM, Perkins DO: Nuclear and cytoplasmic localization of neural stem cell microRNAs. RNA 2011, 17: 675–686. 10.1261/rna.2006511View ArticlePubMedPubMed CentralGoogle Scholar
- Ohrt T, Muetze J, Svoboda P, Schwille P: Intracellular localization and routing of miRNA and RNAi pathway components. Curr Top Med Chem 2012, 12: 79–88. 10.2174/156802612798919132View ArticlePubMedGoogle Scholar
- Liao JY, Ma LM, Guo YH, Zhang YC, Zhou H, Shao P, Chen YQ, Qu LH: Deep sequencing of human nuclear and cytoplasmic small RNAs reveals an unexpectedly complex subcellular distribution of miRNAs and tRNA 3′ trailers. PLoS One 2010, 5: e10563. 10.1371/journal.pone.0010563View ArticlePubMedPubMed CentralGoogle Scholar
- Gu Y, Sun J, Groome LJ, Wang Y: Differential miRNA expression profiles between the first and third trimester human placentas. Am J Physiol Endocrinol Metab 2013, 304: E836-E843. 10.1152/ajpendo.00660.2012View ArticlePubMedPubMed CentralGoogle Scholar
- Vacchi-Suzzi C, Hahne F, Scheubel P, Marcellin M, Dubost V, Westphal M, Boeglen C, Buchmann-Moller S, Cheung MS, Cordier A, De BC, Deurinck M, Frei M, Moulin P, Oakeley E, Grenet O, Grevot A, Stull R, Theil D, Moggs JG, Marrer E, Couttet P: Heart structure-specific transcriptomic atlas reveals conserved microRNA-mRNA interactions. PLoS One 2013, 8: e52442. 10.1371/journal.pone.0052442View ArticlePubMedPubMed CentralGoogle Scholar
- Manfe V, Biskup E, Willumsgaard A, Skov AG, Palmieri D, Gasparini P, Lagana A, Woetmann A, Odum N, Croce CM, Gniadecki R: cMyc/miR-125b-5p signalling determines sensitivity to bortezomib in preclinical model of cutaneous T-cell lymphomas. PLoS One 2013, 8: e59390. 10.1371/journal.pone.0059390View ArticlePubMedPubMed CentralGoogle Scholar
- Xu N, Zhang L, Meisgen F, Harada M, Heilborn J, Homey B, Grander D, Stahle M, Sonkoly E, Pivarcsi A: MicroRNA-125b down-regulates matrix metallopeptidase 13 and inhibits cutaneous squamous cell carcinoma cell proliferation, migration, and invasion. J Biol Chem 2012, 287: 29899–29908. 10.1074/jbc.M112.391243View ArticlePubMedPubMed CentralGoogle Scholar
- Arora A, Guduric-Fuchs J, Harwood L, Dellett M, Cogliati T, Simpson DA: Prediction of microRNAs affecting mRNA expression during retinal development. BMC Dev Biol 2010, 10: 1. 10.1186/1471-213X-10-1View ArticlePubMedPubMed CentralGoogle Scholar
- The pre-publication history for this paper can be accessed here:http://www.biomedcentral.com/1471-5945/14/8/prepub
This article is published under license to BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly credited.