microRNA-214 contributes to melanoma tumour progression through suppression of TFAP2C
2011; Springer Nature; Volume: 30; Issue: 10 Linguagem: Inglês
10.1038/emboj.2011.102
ISSN1460-2075
AutoresElisa Penna, Francesca Orso, Daniela Cimino, Tenaglia Enrico, Antonio Lembo, Elena Quaglino, Laura Poliseno, Adele Haimovic, Simona Osella‐Abate, Cristiano De Pittà, Eva Pinatel, Michael Stadler, Paolo Provero, Maria Grazia Bernengo, Iman Osman, Daniela Taverna,
Tópico(s)RNA Research and Splicing
ResumoArticle5 April 2011free access microRNA-214 contributes to melanoma tumour progression through suppression of TFAP2C Elisa Penna Elisa Penna Molecular Biotechnology Center (MBC), University of Torino, Torino, Italy Department of Oncological Sciences, University of Torino, Torino, Italy Search for more papers by this author Francesca Orso Francesca Orso Molecular Biotechnology Center (MBC), University of Torino, Torino, Italy Department of Oncological Sciences, University of Torino, Torino, Italy Center for Complex Systems in Molecular Biology and Medicine, University of Torino, Torino, Italy Search for more papers by this author Daniela Cimino Daniela Cimino Molecular Biotechnology Center (MBC), University of Torino, Torino, Italy Department of Oncological Sciences, University of Torino, Torino, Italy Center for Complex Systems in Molecular Biology and Medicine, University of Torino, Torino, Italy Search for more papers by this author Enrico Tenaglia Enrico Tenaglia Molecular Biotechnology Center (MBC), University of Torino, Torino, Italy Department of Oncological Sciences, University of Torino, Torino, Italy Search for more papers by this author Antonio Lembo Antonio Lembo Molecular Biotechnology Center (MBC), University of Torino, Torino, Italy Department of Genetics, Biology and Biochemistry, University of Torino, Torino, Italy Search for more papers by this author Elena Quaglino Elena Quaglino Molecular Biotechnology Center (MBC), University of Torino, Torino, Italy Department of Clinical and Biological Sciences, University of Torino, Torino, Italy Search for more papers by this author Laura Poliseno Laura Poliseno Department of Dermatology, New York University Medical Center, New York, NY, USA Search for more papers by this author Adele Haimovic Adele Haimovic Department of Dermatology, New York University Medical Center, New York, NY, USA Search for more papers by this author Simona Osella-Abate Simona Osella-Abate Department of Biomedical Science and Human Oncology, Ist. Dermatologic Clinic, University of Torino, Torino, Italy Search for more papers by this author Cristiano De Pittà Cristiano De Pittà Department of Biology and C.R.I.B.I.-Biotechnology Center, University of Padova, Padova, Italy Search for more papers by this author Eva Pinatel Eva Pinatel Molecular Biotechnology Center (MBC), University of Torino, Torino, Italy Department of Genetics, Biology and Biochemistry, University of Torino, Torino, Italy Search for more papers by this author Michael B Stadler Michael B Stadler Friederich Miescher Institute, Basel, Switzerland Search for more papers by this author Paolo Provero Paolo Provero Molecular Biotechnology Center (MBC), University of Torino, Torino, Italy Department of Genetics, Biology and Biochemistry, University of Torino, Torino, Italy Search for more papers by this author Maria Grazia Bernengo Maria Grazia Bernengo Department of Biomedical Science and Human Oncology, Ist. Dermatologic Clinic, University of Torino, Torino, Italy Search for more papers by this author Iman Osman Iman Osman Department of Dermatology, New York University Medical Center, New York, NY, USA Search for more papers by this author Daniela Taverna Corresponding Author Daniela Taverna Molecular Biotechnology Center (MBC), University of Torino, Torino, Italy Department of Oncological Sciences, University of Torino, Torino, Italy Center for Complex Systems in Molecular Biology and Medicine, University of Torino, Torino, Italy Search for more papers by this author Elisa Penna Elisa Penna Molecular Biotechnology Center (MBC), University of Torino, Torino, Italy Department of Oncological Sciences, University of Torino, Torino, Italy Search for more papers by this author Francesca Orso Francesca Orso Molecular Biotechnology Center (MBC), University of Torino, Torino, Italy Department of Oncological Sciences, University of Torino, Torino, Italy Center for Complex Systems in Molecular Biology and Medicine, University of Torino, Torino, Italy Search for more papers by this author Daniela Cimino Daniela Cimino Molecular Biotechnology Center (MBC), University of Torino, Torino, Italy Department of Oncological Sciences, University of Torino, Torino, Italy Center for Complex Systems in Molecular Biology and Medicine, University of Torino, Torino, Italy Search for more papers by this author Enrico Tenaglia Enrico Tenaglia Molecular Biotechnology Center (MBC), University of Torino, Torino, Italy Department of Oncological Sciences, University of Torino, Torino, Italy Search for more papers by this author Antonio Lembo Antonio Lembo Molecular Biotechnology Center (MBC), University of Torino, Torino, Italy Department of Genetics, Biology and Biochemistry, University of Torino, Torino, Italy Search for more papers by this author Elena Quaglino Elena Quaglino Molecular Biotechnology Center (MBC), University of Torino, Torino, Italy Department of Clinical and Biological Sciences, University of Torino, Torino, Italy Search for more papers by this author Laura Poliseno Laura Poliseno Department of Dermatology, New York University Medical Center, New York, NY, USA Search for more papers by this author Adele Haimovic Adele Haimovic Department of Dermatology, New York University Medical Center, New York, NY, USA Search for more papers by this author Simona Osella-Abate Simona Osella-Abate Department of Biomedical Science and Human Oncology, Ist. Dermatologic Clinic, University of Torino, Torino, Italy Search for more papers by this author Cristiano De Pittà Cristiano De Pittà Department of Biology and C.R.I.B.I.-Biotechnology Center, University of Padova, Padova, Italy Search for more papers by this author Eva Pinatel Eva Pinatel Molecular Biotechnology Center (MBC), University of Torino, Torino, Italy Department of Genetics, Biology and Biochemistry, University of Torino, Torino, Italy Search for more papers by this author Michael B Stadler Michael B Stadler Friederich Miescher Institute, Basel, Switzerland Search for more papers by this author Paolo Provero Paolo Provero Molecular Biotechnology Center (MBC), University of Torino, Torino, Italy Department of Genetics, Biology and Biochemistry, University of Torino, Torino, Italy Search for more papers by this author Maria Grazia Bernengo Maria Grazia Bernengo Department of Biomedical Science and Human Oncology, Ist. Dermatologic Clinic, University of Torino, Torino, Italy Search for more papers by this author Iman Osman Iman Osman Department of Dermatology, New York University Medical Center, New York, NY, USA Search for more papers by this author Daniela Taverna Corresponding Author Daniela Taverna Molecular Biotechnology Center (MBC), University of Torino, Torino, Italy Department of Oncological Sciences, University of Torino, Torino, Italy Center for Complex Systems in Molecular Biology and Medicine, University of Torino, Torino, Italy Search for more papers by this author Author Information Elisa Penna1,2,‡, Francesca Orso1,2,3,‡, Daniela Cimino1,2,3, Enrico Tenaglia1,2, Antonio Lembo1,4, Elena Quaglino1,5, Laura Poliseno6, Adele Haimovic6, Simona Osella-Abate7, Cristiano De Pittà8, Eva Pinatel1,4, Michael B Stadler9, Paolo Provero1,4, Maria Grazia Bernengo7, Iman Osman6 and Daniela Taverna 1,2,3 1Molecular Biotechnology Center (MBC), University of Torino, Torino, Italy 2Department of Oncological Sciences, University of Torino, Torino, Italy 3Center for Complex Systems in Molecular Biology and Medicine, University of Torino, Torino, Italy 4Department of Genetics, Biology and Biochemistry, University of Torino, Torino, Italy 5Department of Clinical and Biological Sciences, University of Torino, Torino, Italy 6Department of Dermatology, New York University Medical Center, New York, NY, USA 7Department of Biomedical Science and Human Oncology, Ist. Dermatologic Clinic, University of Torino, Torino, Italy 8Department of Biology and C.R.I.B.I.-Biotechnology Center, University of Padova, Padova, Italy 9Friederich Miescher Institute, Basel, Switzerland ‡These authors contributed equally to this work *Corresponding author. Department of Oncological Sciences, Molecular Biotechnology Center (MBC), University of Torino, Via Nizza, 52, 10126 Torino, Italy. Tel.: +39 011 670 6497; Fax: +39 011 670 6432; E-mail: [email protected] The EMBO Journal (2011)30:1990-2007https://doi.org/10.1038/emboj.2011.102 There is a Have you seen? (May 2011) associated with this Article. PDFDownload PDF of article text and main figures. Peer ReviewDownload a summary of the editorial decision process including editorial decision letters, reviewer comments and author responses to feedback. ToolsAdd to favoritesDownload CitationsTrack CitationsPermissions ShareFacebookTwitterLinked InMendeleyWechatReddit Figures & Info Malignant melanoma is fatal in its metastatic stage. It is therefore essential to unravel the molecular mechanisms that govern disease progression to metastasis. MicroRNAs (miRs) are endogenous non-coding RNAs involved in tumourigenesis. Using a melanoma progression model, we identified a novel pathway controlled by miR-214 that coordinates metastatic capability. Pathway components include TFAP2C, homologue of a well-established melanoma tumour suppressor, the adhesion receptor ITGA3 and multiple surface molecules. Modulation of miR-214 influences in vitro tumour cell movement and survival to anoikis as well as extravasation from blood vessels and lung metastasis formation in vivo. Considering that miR-214 is known to be highly expressed in human melanomas, our data suggest a critical role for this miRNA in disease progression and the establishment of distant metastases. Introduction The ability of tumours to acquire malignancy and spread in their host organism is one of the main issues in cancer treatment, as metastasis formation accounts for >90% of human cancer deaths. Nevertheless, the understanding of the molecular mechanisms that regulate metastatic dissemination remains fragmentary. The cascade of events that lead to metastasis is a complex multi-step process by which primary tumour cells acquire the ability to detach and invade adjacent tissues, intravasate, survive in the systemic circulation and translocate through the vasculature, adhere to the walls of capillaries of distant organs, extravasate in the parenchyma and finally proliferate in secondary tumours (Gupta and Massague, 2006). It is urgent to identify and characterize the genetic and epigenetic changes occurring during tumour progression. Several protein-coding genes involved in malignancy have been identified and characterized (Steeg, 2006; Nguyen et al, 2009). More recently, abnormalities in non-coding genes, such as microRNAs (miRs), have also been found to contribute to tumourigenesis (Croce, 2009; Valastyan and Weinberg, 2009). miRs are small endogenous non-coding RNAs able to post-transcriptionally downregulate expression of specific target genes by binding to the 3′UTRs of their mRNAs causing destabilization, degradation or translation inhibition (Filipowicz et al, 2008; Bartel, 2009). The ability of miRs to achieve simultaneous fine tuning of numerous different target genes makes them fundamental regulators of cellular signalling and implicates them in tumour progression (Inui et al, 2010). Several miRs, such as miR-21, miR-10b, miR-373 and 520c, miR-126, miR-335, miR-31, miR-200, miR-151 and miR-9, have already been reported to regulate tumour progression and metastasis (Valastyan and Weinberg, 2009; Ma et al, 2010). Malignant melanoma is one of the most aggressive human cancers (Parkin et al, 2005) which progresses very rapidly via specific steps characterized by defined molecular alterations. Melanomas arise when the melanocytes of the epidermis become transformed and start to proliferate abnormally, leading to radial and vertical growth phases and subsequent spreading all over the body (Melnikova and Bar-Eli, 2008). The transition from the non-invasive to the invasive and metastatic stage is accompanied by gain of function of a number of transcription factors such as CREB/ATF-1, ATF-2, NFκB, SNAIL and STATs, while the loss of the AP-2 transcription factors (TFAP2) positively correlates with malignancy. At the same time, alterations in the repertoire of adhesion molecules, including MCAM-MUC18, E-cadherin, N-cadherin and several integrins, as well as changes in genes involved in angiogenesis, invasion and survival, such as VEGF, bFGF, IL-8, c-KIT, EGFR, MMP2 and PAR-1, are linked to the acquirement of higher metastatic potential (Melnikova and Bar-Eli, 2008). Several miRs, including miR-137, miR-221/222, miR-182 and miR-34a, have already been found to be involved in melanoma progression by regulating key genes such as c-KIT, MITF, FOXO3, ITGB3, CCND1 and p27Kip1 (Mueller and Bosserhoff, 2009). It now becomes fundamental to unravel how miRs control melanoma aggressiveness. We identified a new pathway, coordinated by miR-214 and including TFAP2C, ITGA3 as well as multiple surface molecules, which controls melanoma metastasis dissemination by increasing migration, invasion, extravasation and survival of melanoma cells. Results miR-214 is upregulated in a metastatic melanoma model To assess a potential correlation between deregulation of miRs and melanoma malignancy, a miR profiling, which will be presented elsewhere (Cimino et al, unpublished), was performed in a melanoma progression model (Xu et al, 2008). The model consisted of a poorly metastatic A375 parental cell line (A375P) and its four highly metastatic variants, MA-1, MA-2, MC-1 and MC-2 derived by repeated passages in mice. Among the modulated miRs, miR-214 was found to be differentially expressed comparing metastatic (high) versus parental (low) cells in culture, as shown by qRT–PCR (Figure 1A and B). miR-214 showed a very strong enhancement of expression in samples derived from in vivo lung metastases following tail vein injections of MA-2 cells in immunodeficient mice (Figure 1B), suggesting an influence of the microenvironment for high expression. Induction of miR-214 expression in vivo was also observed in subcutaneous tumours derived from different melanoma cell lines expressing low miR-214 in culture (WK-Mel, GR4-Mel, 1300-Mel, Dett-Mel, SK-Mel-173, SK-Mel-197) (Supplementary Figure S1A). Other miRs, previously found to be involved in melanoma, such as miR-34a, miR-221, miR-222 and miR-137, also showed some differential expression in this system, however, not as pronounced as miR-214 changes (Figure 1A). When we extended expression analysis for miR-137 to other melanoma malignant cell lines it resulted to be overexpressed in some of them, such as WK-Mel, GR4-Mel, SK-Mel-173 and SK-Mel-197 compared with A375P. Instead no expression was detected in 1300-Mel, Dett-Mel and SK-Mel-187 cells (Supplementary Figure S1B). Some miRs were poorly expressed or did not show differential expression in our A375P isogenic model, including miR-210, which we used as a control (Figure 1C). miR-210 was expressed to some extent in most of the melanoma cells analysed although often at a low level (Supplementary Figure S1C). Importantly, miR-214 copy number gain was found in the genome of A375P, its MA-2 and MC-1 variants and in other melanoma cells, such as GR4-Mel, Dett-Mel, SK-Mel-103 and SK-Mel-187, as measured by genomic qRT–PCR (Supplementary Figure S1D) and SNP (not shown) analyses. Figure 1.miR-214 modulates cell migration and invasion. (A–C) Expression levels of the indicated miRs were evaluated in A375P cells or in its metastatic variants MA-1, MA-2, MC-1, MC-2 or in a pool of MA-2-derived lung metastases (MA-2 mets) by qRT–PCR. Results are shown as fold changes (mean±s.e.m.) relative to A375P cells, normalized on U44 RNA level. (D–M) Wound healing motility (D, E) or transwell migration or matrigel invasion (F–K) or adhesion on fibronectin, laminin or collagen (L, M) assays for cells either transfected with the indicated miR precursors or inhibitors or their negative controls (pre- and anti-miR or control) or stably transduced with pWPT-empty or miR-214 overexpression vectors. Results are shown as mean±s.e.m. of the reciprocal of the wound size (motility assay) or of the area covered by migrated or adherent cells (migration, invasion and adhesion assays). Two or three independent experiments were performed in triplicate and results were either shown as representative ones (A–C) or pooled together (D–M). *P<0.05; **P<0.01; ***P<0.001. Download figure Download PowerPoint miR-214 expression enhances cell movement The more pronounced expression of miR-214 in metastatic cells prompted us to investigate the potential pro-metastatic role of miR-214 by analysing cell movement following miR-214 expression modulations. We stably or transiently overexpressed miR-214 in the miR-214-empty, poorly motile A375P cells and in the MA-2 metastatic variant, expressing an intermediate endogenous level of miR-214 (see Figure 1B), as well as in other melanoma cells, such as 1300-Mel, SK-Mel-187, WK-Mel and GR4-Mel, expressing low to undetectable miR-214 in culture (see Supplementary Figure S1A), by lentiviral infections with miR-214 expression or empty vectors (pWPT-miR-214 or pLemiR-214 or empty) or by transfections with miR-214 precursors or negative controls (pre-miR-214 or control). Conversely, we silenced miR-214 in the highly motile and invasive MC-1 and MC-2 variants following transfection with specific miR-214 antisense inhibitors or negative controls (anti-miR-214 or control). The efficacy of miR-214 modulations was tested by qRT–PCRs; miR-214 expression was increased up to 200 000- or 500-fold, respectively, in pre-miR-214 transiently transfected cells and stable infected cells (Supplementary Figure S2A–C) and almost completely silenced following miR-214 inhibition (Supplementary Figure S2D). Significantly, miR-214 overexpression by pre-miR-214 transfection in MA-2 cells enhanced cell motility, migration and invasion as evaluated by wound healing assays (Figure 1D) or transwell assays in presence or absence of matrigel (Figure 1F and G) compared with negative controls. Migration and invasion were also increased 2–3-fold in stable miR-214-overexpressing MA-2 cells compared with cells containing the empty vector (Figure 1H and I). Moreover, miR-214 overexpression was sufficient to promote a significant increase in migration and/or invasion in poorly motile A375P cells (pWPT-miR-214, Supplementary Figure S3A) and in other unrelated melanoma cell lines, such as 1300-Mel, SK-Mel-187, WK-Mel and GR4-Mel (pre-miR-214, Supplementary Figure S3E–J). The effects observed on cell movement in the A375P isogenic model were specific for miR-214, since no significant variation in migration or invasion was observed following transient miR-210 overexpression (Figure 1F and G). In line with these results, transient miR-214 downregulation in MC-1 or MC-2 cells, following transfection with anti-miR-214, led to a 50% reduction in motility in a wound healing assay (Figure 1E, MC-1) and a 40–80% decrease in migration and matrigel invasion in transwell assays (Figures 1J and K, MC-1; Supplementary Figure S3B and C, MC-2). miR-214 functions were also evaluated by stable silencing in MC-1 cells using miR-214-specific sponges cloned in the 3′UTR of the green fluorescent protein (GFP) gene (see Materials and methods). MC-1 cells were transduced with pLenti-CMV-GFP-Puro-sponge1 (pLenti-sponge1) or pLenti-CMV-GFP-Puro-sponge3 (pLenti-sponge3) or pLenti-CMV-GFP-Puro (pLenti-empty) vectors and the efficacy of each sponge was evaluated by measuring the expression of the GFP in a western blot (WB) analysis (Supplementary Figure S2E). In presence of pLenti-sponge1 or pLenti-sponge3 GFP expression was highly decreased, indicating the efficient binding of miR-214 on the complementary sequences in the 3′UTR of the GFP. As shown in Supplementary Figure S3D, stable miR-214 silencing by sponge1 or sponge3 resulted in impairment of MC-1 cell migration in vitro (transwell assays), compared with control cells. Interestingly, miR-214 overexpression (pre-miR-214) was also able to significantly increase cell migration and matrigel invasion in other tumour cells, including human MDA-MB-231 or murine 4T1 mammary epithelial cancer cell lines (Supplementary Figures S2A and S3K–N). When we looked for the involvement of miR-214 in cell adhesion we observed significant adhesion alterations on different extracellular matrices (ECMs). In particular, transient miR-214 overexpression in A375P cells improved adhesion on fibronectin, laminin and collagen (1.5–3-fold increase), while miR-214 silencing in MC-1 cells consistently resulted in comparable adhesion defects on these matrices (Figure 1L and M). Taken together, these results show that miR-214 significantly enhances in vitro cell movement and modulates adhesion, suggesting that it might facilitate metastasis formation by favouring tumour cell invasion and adhesion to the surrounding ECM. miR-214 expression enhances metastasis formation in vivo Because of its ability to induce motility and invasion in vitro, we asked whether miR-214 could influence cell movement and metastasis formation in vivo. Thus, miR-214-overexpressing MA-2 cells or miR-214-silenced MC-1 cells were injected into the tail vein of immunodeficient SCID mice and the number of lung metastases was evaluated 7 weeks later. A significantly higher number of macroscopic lung metastases could be observed for miR-214-overexpressing (pWPT-miR-214) MA-2 cells when compared with control (pWPT-empty) cells (Figure 2A). To investigate whether miR-214 was able to promote or regulate metastasis formation in the poorly metastatic parental cells, miR-214-overexpressing (pWPT-miR-214) or control (pWPT-empty) A375P cells were injected into SCID mice. Metastasis formation was analysed 9 weeks later and no macroscopic metastases were found on the lung surface for the two groups of mice. However, histological analyses revealed that 3 out of 10 mice injected with miR-214-overexpressing cells contained small metastatic formations in their lungs, while no micrometastases were found in control mice (Supplementary Figure S4). In contrast, miR-214-silenced MC-1 cells (by anti-miR-214 transfection) were significantly impaired in their ability to seed lung metastases and formed fewer lesions than the control (anti-control) cells (Figure 2B). High miR-214 expression levels were also able to enhance lung metastasis formation from a primary tumour. In fact, when the 4T1 mammary epithelial cancer cells, transduced with a lentiviral vector encoding for the turbo red-fluorescent protein (tRFP) alone (pLemiR-empty) or for miR-214 plus tRFP (pLemiR-214), were injected in mammary fat pad of female BALB/c mice, a significantly higher distribution of red-fluorescent micrometastasis in the lungs was observed for miR-214-overexpressing cells compared with controls, 20 days after injection (Supplementary Figure S2C; Figure 2C). However, miR-214-overexpressing cells gave rise to similar sized primary tumours as control cells (not shown). miR-214 enhancement effects on in vitro cell movement and in vivo metastasis formation are directly ascribed to the metastatic ability of the cells and not to proliferation effects. In fact, miR-214 overexpression (pre-miR-214) in A375P and MA-2 cells or miR-214 silencing (anti-miR-214) in MC-1 cells did not influence in vitro proliferation compared with controls (Figure 3A and B). Moreover, anchorage-independent growth in soft agar was not affected, since the number and the size of colonies for stable miR-214-overexpressing (pWPT-miR-214) A375P and MA-2 cells were comparable with controls (pWPT-empty) (Figure 3C and D). Furthermore, miR-214 did not influence primary tumour growth. In fact, when miR-214-overexpressing (pWPT-miR-214) or control (pWPT-empty) A375P or MA-2 cells were subcutaneously injected in the flanks of nude mice, the final tumour weight was similar for the different samples (Figure 3E and F). Figure 2.miR-214 enhances metastasis formation in vivo. (A, B) Metastasis formation in the lungs of SCID mice, 7 weeks after tail vein injection of MA-2 or MC-1 cells, stably transduced with pWPT-empty or miR-214 overexpression vectors (A) or transfected with miR-214 inhibitors or negative controls (anti-miR-214 or control) (B). Total number of macroscopic metastases per lung is shown as box and whiskers plots with median and minimum/maximum (n=5 mice per group). (C) Lung metastasis formation 3 weeks after injection of 4T1 cells stably transduced with pLemiR-tRFP (pLemiR-empty) or pLemiR-miR-214 overexpression (pLemiR-214) vectors in the mammary fat pad of BALB/c mice. Number of red-fluorescent micrometastasis per lung is shown as mean±s.e.m. for n=10 mice per group. Representative whole lung (a, b, e, f, i and j) and H&E staining (c, d, g, h, k and l) pictures are shown; bar=1 mm and 100 μm, respectively. Two independent experiments were performed and representative ones are shown (A, B) or results were pooled together (C). *P<0.05; **P<0.01. Download figure Download PowerPoint Figure 3.miR-214 does not affect cell and tumour growth. Proliferation (A, B) or anchorage-independent growth (C, D) or primary tumour growth 4 weeks after subcutaneous injection in the two flanks of nude mice (E, F) of cells transfected with miR-214 precursors or inhibitors or their negative controls (pre- and anti-miR-214 or control) or stably transduced with pWPT-empty or miR-214 overexpression vectors. Results are shown as mean±s.e.m. of the optical density (OD) (A, B) or of the area covered by colonies (C, D) or as tumour weight (dots or squares) and mean for n=15 (E) or 17 (F) mice per group. Two to four independent experiments were performed (in triplicate for A–D) and results were either shown as representative ones (A–D) or pooled together (E, F). Download figure Download PowerPoint miR-214 modulates extravasation and survival To assess the involvement of miR-214 in extravasation and survival, we combined in vitro and in vivo experiments. We first simulated transendothelial migration in vitro by seeding CMRA-labelled (red) miR-214-overexpressing (pre-miR-214) or control (pre-control) A375P or MA-2 cells in the upper chambers of fibronectin-coated transwells, covered by a confluent GFP-transduced human umbilical vein endothelial cell (HUVEC) monolayer. The remodelling of the endothelium and the consequent migration of melanoma cells in the lower chamber of the transwell were then evaluated (Figure 4A). The interaction of melanoma and endothelial cells induced the formation of fenestrations in the HUVEC monolayer. In the absence of melanoma cells (panel a) or in the presence of melanoma cell-conditioned medium (not shown), the HUVEC monolayer remains intact, suggesting the requirement of a direct cell–cell contact. Interestingly, the spaces formed in the endothelium were larger in the presence of miR-214-overexpressing A375P or MA-2 cells, compared with those formed with control cells (panels b, c, f and g) and, consequently, an increased number of cells migrated through the endothelial cells (panels d, e, h and i). In conclusion, miR-214 overexpression in A375P and MA-2 cells resulted in a 2–3-fold more efficient transendothelial migration. To evaluate the ability of miR-214 to influence extravasation in vivo, we first demonstrated that cell extravasation in the mouse lungs occurs within 48 h following injection of any metastatic variant in the blood circulation. For this, CMRA-labelled (red) negative control-transfected MC-1 (Figure 4B) or MA-2 (not shown) cells were injected in the tail vein of nude mice and their localization in the lungs was evaluated at different times. Two hours after injection, a high percentage of cells was located inside or associated with the lung blood vessels (Figure 4B, panel a), although some cells were already visible in the parenchyma (not shown). Forty-eight hours after injection, most of the cells present in the lungs appeared to be dispersed in the parenchyma (Figure 4B, panel b). To evaluate the role of miR-214 in the regulation of extravasation, CMRA-labelled miR-214-overexpressing A375P or MA-2 cells (pLemiR-214 or pre-miR-214, respectively) or silenced (anti-miR-214) MC-1 cells were injected via tail vein in nude mice and their ability to persist in the lungs was quantitated 48 h later. Two- or four-fold increased extravasation was observed, respectively, for miR-214-overexpressing A375P and MA-2 cells (Figure 4C, panels c, d, g and h), while about a 50% reduction in extravasation ability was observed for miR-214-silenced MC-1 cells (Figure 4C, panels k and l), compared with controls. This was not the consequence of a different lodging in the lung microvasculature, evaluated 2 h post-injection, since no difference was observed between miR-214-modulated cells and negative controls (Figure 4C, panels a, b, e, f, i and j). In vivo extravasation experiments were also performed with CMRA-labelled MC-1 cells stably transduced with specific miR-214 sponge (pLenti-sponge3) or empty (pLenti-empty) vectors as in Supplementary Figure S2E. In line with transient anti-miR-214 experiments, MC-1 cells expressing pLenti-sponge3 showed a highly reduced (80–90%) ability to extravasate compared with control cells, 48 h post-injection, as measured in the red (CMRA) or green (GFP) channel (Supplementary Figure S5A and B). No difference in lodging was observed at 2 h. In addition to transendothelial migration and extravasation ability, resistance to apoptosis was evaluated following miR-214 expression modulation in cells kept in the absence of adhesion (anoikis) and serum for 72 h, by AnnexinV–FITC and TMRM staining and cytofluorimetric analyse
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