miR‐515‐5p controls cancer cell migration through MARK 4 regulation

Article10 February 2016Open Access Transparent process miR-515-5p controls cancer cell migration through MARK4 regulation Olivier E Pardo Corresponding Author Olivier E Pardo Department of Surgery & Cancer, Division of Cancer, Imperial Centre for Translational and Experimental Medicine (ICTEM), Imperial College, Hammersmith Hospital Campus, London, UK Search for more papers by this author Leandro Castellano Leandro Castellano Department of Surgery & Cancer, Division of Cancer, Imperial Centre for Translational and Experimental Medicine (ICTEM), Imperial College, Hammersmith Hospital Campus, London, UK Search for more papers by this author Catriona E Munro Catriona E Munro Department of Surgery & Cancer, Division of Cancer, Imperial Centre for Translational and Experimental Medicine (ICTEM), Imperial College, Hammersmith Hospital Campus, London, UK Search for more papers by this author Yili Hu Yili Hu Department of Oncology, Imperial College Healthcare NHS Trust, Charing Cross Hospital, London, UK Search for more papers by this author Francesco Mauri Francesco Mauri Department of Surgery & Cancer, Division of Cancer, Imperial Centre for Translational and Experimental Medicine (ICTEM), Imperial College, Hammersmith Hospital Campus, London, UK Search for more papers by this author Jonathan Krell Jonathan Krell Department of Surgery & Cancer, Division of Cancer, Imperial Centre for Translational and Experimental Medicine (ICTEM), Imperial College, Hammersmith Hospital Campus, London, UK Search for more papers by this author Romain Lara Romain Lara Department of Surgery & Cancer, Division of Cancer, Imperial Centre for Translational and Experimental Medicine (ICTEM), Imperial College, Hammersmith Hospital Campus, London, UK Search for more papers by this author Filipa G Pinho Filipa G Pinho Department of Surgery & Cancer, Division of Cancer, Imperial Centre for Translational and Experimental Medicine (ICTEM), Imperial College, Hammersmith Hospital Campus, London, UK Search for more papers by this author Thameenah Choudhury Thameenah Choudhury Department of Surgery & Cancer, Division of Cancer, Imperial Centre for Translational and Experimental Medicine (ICTEM), Imperial College, Hammersmith Hospital Campus, London, UK Search for more papers by this author Adam E Frampton Adam E Frampton Department of Surgery & Cancer, Division of Cancer, Imperial Centre for Translational and Experimental Medicine (ICTEM), Imperial College, Hammersmith Hospital Campus, London, UK Search for more papers by this author Loredana Pellegrino Loredana Pellegrino Department of Surgery & Cancer, Division of Cancer, Imperial Centre for Translational and Experimental Medicine (ICTEM), Imperial College, Hammersmith Hospital Campus, London, UK Search for more papers by this author Dmitry Pshezhetskiy Dmitry Pshezhetskiy Department of Surgery & Cancer, Division of Cancer, Imperial Centre for Translational and Experimental Medicine (ICTEM), Imperial College, Hammersmith Hospital Campus, London, UK Search for more papers by this author Yulan Wang Yulan Wang Department of Oncology, Imperial College Healthcare NHS Trust, Charing Cross Hospital, London, UK Search for more papers by this author Jonathan Waxman Jonathan Waxman Department of Surgery & Cancer, Division of Cancer, Imperial Centre for Translational and Experimental Medicine (ICTEM), Imperial College, Hammersmith Hospital Campus, London, UK Search for more papers by this author Michael J Seckl Corresponding Author Michael J Seckl Department of Surgery & Cancer, Division of Cancer, Imperial Centre for Translational and Experimental Medicine (ICTEM), Imperial College, Hammersmith Hospital Campus, London, UK Key Laboratory of Magnetic Resonance in Biological Systems, Wuhan Center for Magnetic Resonance, State Key Laboratory of Magnetic Resonance and Atomic and Molecular Physics, Wuhan Institute of Physics and Mathematics, Chinese Academy of Sciences, Wuhan, China Search for more papers by this author Justin Stebbing Corresponding Author Justin Stebbing Department of Surgery & Cancer, Division of Cancer, Imperial Centre for Translational and Experimental Medicine (ICTEM), Imperial College, Hammersmith Hospital Campus, London, UK Key Laboratory of Magnetic Resonance in Biological Systems, Wuhan Center for Magnetic Resonance, State Key Laboratory of Magnetic Resonance and Atomic and Molecular Physics, Wuhan Institute of Physics and Mathematics, Chinese Academy of Sciences, Wuhan, China Search for more papers by this author Olivier E Pardo Corresponding Author Olivier E Pardo Department of Surgery & Cancer, Division of Cancer, Imperial Centre for Translational and Experimental Medicine (ICTEM), Imperial College, Hammersmith Hospital Campus, London, UK Search for more papers by this author Leandro Castellano Leandro Castellano Department of Surgery & Cancer, Division of Cancer, Imperial Centre for Translational and Experimental Medicine (ICTEM), Imperial College, Hammersmith Hospital Campus, London, UK Search for more papers by this author Catriona E Munro Catriona E Munro Department of Surgery & Cancer, Division of Cancer, Imperial Centre for Translational and Experimental Medicine (ICTEM), Imperial College, Hammersmith Hospital Campus, London, UK Search for more papers by this author Yili Hu Yili Hu Department of Oncology, Imperial College Healthcare NHS Trust, Charing Cross Hospital, London, UK Search for more papers by this author Francesco Mauri Francesco Mauri Department of Surgery & Cancer, Division of Cancer, Imperial Centre for Translational and Experimental Medicine (ICTEM), Imperial College, Hammersmith Hospital Campus, London, UK Search for more papers by this author Jonathan Krell Jonathan Krell Department of Surgery & Cancer, Division of Cancer, Imperial Centre for Translational and Experimental Medicine (ICTEM), Imperial College, Hammersmith Hospital Campus, London, UK Search for more papers by this author Romain Lara Romain Lara Department of Surgery & Cancer, Division of Cancer, Imperial Centre for Translational and Experimental Medicine (ICTEM), Imperial College, Hammersmith Hospital Campus, London, UK Search for more papers by this author Filipa G Pinho Filipa G Pinho Department of Surgery & Cancer, Division of Cancer, Imperial Centre for Translational and Experimental Medicine (ICTEM), Imperial College, Hammersmith Hospital Campus, London, UK Search for more papers by this author Thameenah Choudhury Thameenah Choudhury Department of Surgery & Cancer, Division of Cancer, Imperial Centre for Translational and Experimental Medicine (ICTEM), Imperial College, Hammersmith Hospital Campus, London, UK Search for more papers by this author Adam E Frampton Adam E Frampton Department of Surgery & Cancer, Division of Cancer, Imperial Centre for Translational and Experimental Medicine (ICTEM), Imperial College, Hammersmith Hospital Campus, London, UK Search for more papers by this author Loredana Pellegrino Loredana Pellegrino Department of Surgery & Cancer, Division of Cancer, Imperial Centre for Translational and Experimental Medicine (ICTEM), Imperial College, Hammersmith Hospital Campus, London, UK Search for more papers by this author Dmitry Pshezhetskiy Dmitry Pshezhetskiy Department of Surgery & Cancer, Division of Cancer, Imperial Centre for Translational and Experimental Medicine (ICTEM), Imperial College, Hammersmith Hospital Campus, London, UK Search for more papers by this author Yulan Wang Yulan Wang Department of Oncology, Imperial College Healthcare NHS Trust, Charing Cross Hospital, London, UK Search for more papers by this author Jonathan Waxman Jonathan Waxman Department of Surgery & Cancer, Division of Cancer, Imperial Centre for Translational and Experimental Medicine (ICTEM), Imperial College, Hammersmith Hospital Campus, London, UK Search for more papers by this author Michael J Seckl Corresponding Author Michael J Seckl Department of Surgery & Cancer, Division of Cancer, Imperial Centre for Translational and Experimental Medicine (ICTEM), Imperial College, Hammersmith Hospital Campus, London, UK Key Laboratory of Magnetic Resonance in Biological Systems, Wuhan Center for Magnetic Resonance, State Key Laboratory of Magnetic Resonance and Atomic and Molecular Physics, Wuhan Institute of Physics and Mathematics, Chinese Academy of Sciences, Wuhan, China Search for more papers by this author Justin Stebbing Corresponding Author Justin Stebbing Department of Surgery & Cancer, Division of Cancer, Imperial Centre for Translational and Experimental Medicine (ICTEM), Imperial College, Hammersmith Hospital Campus, London, UK Key Laboratory of Magnetic Resonance in Biological Systems, Wuhan Center for Magnetic Resonance, State Key Laboratory of Magnetic Resonance and Atomic and Molecular Physics, Wuhan Institute of Physics and Mathematics, Chinese Academy of Sciences, Wuhan, China Search for more papers by this author Author Information Olivier E Pardo 1,‡, Leandro Castellano1,‡, Catriona E Munro1,‡, Yili Hu2, Francesco Mauri1, Jonathan Krell1, Romain Lara1, Filipa G Pinho1, Thameenah Choudhury1, Adam E Frampton1, Loredana Pellegrino1, Dmitry Pshezhetskiy1, Yulan Wang2, Jonathan Waxman1, Michael J Seckl 1,3 and Justin Stebbing 1,3 1Department of Surgery & Cancer, Division of Cancer, Imperial Centre for Translational and Experimental Medicine (ICTEM), Imperial College, Hammersmith Hospital Campus, London, UK 2Department of Oncology, Imperial College Healthcare NHS Trust, Charing Cross Hospital, London, UK 3Key Laboratory of Magnetic Resonance in Biological Systems, Wuhan Center for Magnetic Resonance, State Key Laboratory of Magnetic Resonance and Atomic and Molecular Physics, Wuhan Institute of Physics and Mathematics, Chinese Academy of Sciences, Wuhan, China ‡These authors contributed equally to this work *Corresponding author. Tel: +44 2075942814; E-mail: [email protected] *Corresponding author. Tel: +44 2033111421; E-mail: [email protected] *Corresponding author. Tel: +44 203 3118295; E-mail: [email protected] EMBO Reports (2016)17:570-584https://doi.org/10.15252/embr.201540970 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 Abstract Here, we show that miR-515-5p inhibits cancer cell migration and metastasis. RNA-seq analyses of both oestrogen receptor receptor-positive and receptor-negative breast cancer cells overexpressing miR-515-5p reveal down-regulation of NRAS, FZD4, CDC42BPA, PIK3C2B and MARK4 mRNAs. We demonstrate that miR-515-5p inhibits MARK4 directly 3′ UTR interaction and that MARK4 knock-down mimics the effect of miR-515-5p on breast and lung cancer cell migration. MARK4 overexpression rescues the inhibitory effects of miR-515-5p, suggesting miR-515-5p mediates this process through MARK4 down-regulation. Furthermore, miR-515-5p expression is reduced in metastases compared to primary tumours derived from both in vivo xenografts and samples from patients with breast cancer. Conversely, miR-515-5p overexpression prevents tumour cell dissemination in a mouse metastatic model. Moreover, high miR-515-5p and low MARK4 expression correlate with increased breast and lung cancer patients' survival, respectively. Taken together, these data demonstrate the importance of miR-515-5p/MARK4 regulation in cell migration and metastasis across two common cancers. Synopsis miR-515-5p inhibits cancer progression, cell migration and metastasis through its direct target MARK4, a regulator of the cytoskeleton and cell motility. Moreover, reduced miR-515-5p and increased MARK4 levels in metastatic lung and breast cancer correlate with poor patient prognosis. MARK4 down-regulation promotes microtubule polymerisation. Increased cell spreading downstream of miR-515-5p overexpression or MARK4 silencing hinders cell motility and invasiveness. miR-515-5p overexpression or MARK4 silencing prevent organ colonisation by circulating tumour cells. MARK4 inhibitors may represent novel therapeutic agents to control cancer dissemination. Introduction Initially termed MARKL1 (microtubule affinity-regulating kinase-like 1), MARK4 was identified by a cDNA microarray approach to be down-regulated following a decrease in TCF/LEF1 (transcription complex, T-cell factor/lymphoid enhancer-binding factor) activity in hepatocellular carcinoma cell lines. To date, MARK4 expression has been found to be increased in hepatocellular carcinomas and gliomas, suggesting a role for MARK4 in cancer development 12. Using a tandem affinity purification approach, MARK4 was found to interact with a number of proteins linked to the regulation of cell motility, namely 14-3-3 proteins, ARHGEF2 (GEF-H1)—a microtubule-associated exchange factor for Rho GTPases and phosphatase 2A (PP2A), and was previously shown to dephosphorylate TAU and other MAPs controlling their microtubule-binding affinity 34. The identification of these binding partners and MARK4's interaction with three forms of tubulin (α-, β-, γ-tubulin), myosin and actin suggest a clear role for MARK4 in control of the cytoskeleton 56. However, nothing is known about the control of MARK4 expression by, for example, microRNAs. MicroRNAs (miRNAs) are small non-coding RNAs that post-transcriptionally regulate gene expression through binding to the 3′ UTR of their mRNA targets. In 2009, it was shown that more than 50% of known miRNA target genes are located in cancer-associated genomic regions (CAGRs), regions in the genome that are frequently altered in various cancers 7. Therefore, unsurprisingly, the expression of miRNAs is often altered among different cancer sub-types and assessment of the expression of certain miRNAs has allowed for the accurate differentiation between benign and malignant tissues 8. More recently, systemic injection of synthetic tumour suppressor miRNAs has been demonstrated to successfully prevent the growth of metastases in animal models with cancer metastasis 9. This evidence suggests that miRNAs-based therapies could be used to treat metastatic disease or to prevent the formation of metastasis in early-stage disease. miR-515-5p was initially described to be a placenta-specific miRNA involved in foetal growth 10. However, recently, we identified its role as a tumour suppressor in breast cancer 11. We demonstrated that miR-515-5p transcription was directly down-regulated by the oestrogen receptor and that miR-515-5p inhibited breast cell proliferation by inducing apoptosis 11. During this work, a change in MCF-7 and MDA-MB-231 cell morphology was also observed, a finding that we investigate further here. Indeed, we show that miR-515-5p overexpression increases microtubule area not only in breast cancer but also in non-small cell lung cancer (NSCLC) cells and that this correlates with decreased cell migration. We performed an RNA-seq following the overexpression of miR-515-5p in MCF7 and MDA-MB-231 cells, which revealed the down-regulation of five transcripts linked to cell migration: NRAS, FZD4, CDC42BPA, PIK3C2B and MARK4 612131415. These four transcripts were also down-regulated in A549 and H1299 NSCLC cells in response to miR-515-5p overexpression and luciferase reporter constructs, indicating that miR-515-5p directly regulated NRAS, PIK3C2B and MARK4 expression in multiple cancer cells. As the strongest down-regulation of expression by miR-515-5p was seen with MARK4 in both MCF7 and MDA-MB-231 and that this gene had previously been reported to modulate NSCLC cell motility 16, we decided to focus our study on investigating the direct regulation of MARK4 expression by miR-515-5p. We showed that MARK4 silencing reproduces the morphological and cell migration changes observed following miR-515-5p overexpression in both breast and NSCLC cells, demonstrating this to be a consistent phenomenon across two separate tumour types. Conversely, transfection of exogenous MARK4 into miR-515-5p overexpressing cells rescued the observed decrease in cell migration, suggesting that MARK4 down-regulation is a crucial mechanism through which miR-515-5p reduces cell motility. By analysing miR-515-5p expression in tumour samples derived from breast cancer patients, we observed its expression to be inversely correlated with metastasis, while expression of miR-515-5p mimics in A549 NSCLC cells prevented in vivo tumour cell dissemination in a metastatic mouse model. In agreement with metastasis being a poorer prognosis factor for cancer patients, decreased miR-515-5p or increased MARK4 expression was indicative of poorer survival in both metastatic breast and lung cancer. In short, our data demonstrate that miR-515-5p dramatically inhibits cell migration by directly down-regulating MARK4 expression in two different cancer types and suggests a role for miR-515-5p and MARK4 as potential biomarkers in metastatic disease and as possible therapeutic targets. Results miR-515-5p changes cell morphology and inhibits cell migration in breast cancer We initially overexpressed miR-515-5p in breast (MCF7 and MDA-MD-231) and lung (A549 and H1299) cell lines (Appendix Fig S1A) and stained for tubulin to assess cell morphology. We observed an increase in cell area and loss of cell polarity (Fig 1A). The loss of cell polarity was confirmed quantitatively by comparing the cell circularity factor of individual cells in 10 fields of view between control and miR-515-5p overexpressing conditions for the four cell lines (Appendix Fig S2). Overexpression of miR-515-5p significantly increased the circularity factor towards a value of 1, indicating loss of cell elongation. Figure 1. miR-515-5p expression changes the tubulin cytoskeleton and inhibits migration of breast and lung cancer cells A. miR-515-5p expression increases the area of the tubulin cytoskeleton. Tubulin (green) and cell nucleus (blue). Objective x20. Scale bar: 200 μm. B, C. miR-515-5p inhibits random (B) and directed (C) cell migration. The indicated cell lines were transfected with miR-515-5p for 48 h before time-lapse imaging was performed for 18 h (A), or transwell migration assays were performed for 9 h (C). (B, left panel) Plots show overlays of representative trajectories travelled. (B, right panel) The distance of migration was quantified and represented as the mean ± SEM of values normalised to the respective control condition. n ≥ 30 cells tracked per condition. (C, left panel) Representative field of view from the bottom of the transwell chambers. Scale bar is 20 μm. (C, right panel) Data represent percentage of the average number migratory cells per field (n = 5 fields per condition). Data are mean of three experiments ± SEM. P-values were calculated by t-test between miRNA (515-5p) values and their respective non-targeting control miR (NC) values (***P < 0.001). All data shown are representative of experiments performed at least in triplicate. Download figure Download PowerPoint As cell cytoskeleton dynamics and cell polarity are crucial for cell motility, we wished to investigate whether this change in morphology affected cancer cell migration. We analysed the effect of miR-515-5p overexpression in MDA-MB-231, A549 and H1299 on random and directional migration by conducting random and Boyden chamber directional cell migration assays. We observed a sharp decrease in random and directional cell migration in miR-515-5p-transfected cells as compared to their miRVANA non-targeting controls (NC) (Fig 1B and C). This however was not the result of overexpression of this miRNA inducing apoptotic cell death as shown in Appendix Fig S1B for A549 and H2199 cells and previously published by us for MDA-MB-231 11. Hence, miR-515-5p overexpression is able to directly suppress cell migration in these cell lines possibly as a consequence of the observed changes to the cell cytoskeleton. miR-515-5p directly regulates NRAS, MARK4 and PIK3C2B expression To identify which miR-515-5p targets were responsible for its effect on cancer cell migration, we performed an RNA-seq analysis of MCF7 and MDA-MB-231 cells overexpressing miR-515-5p. Interestingly, we found 5 down-regulated transcripts which were predicted to interact with miR-515-5p and have also been implicated in cell migration: NRAS, FZD4, CDC42BPA, PIK3C2B and MARK4 (Fig 2A, Dataset EV1) 612131415. To validate the RNA-seq results, we performed validatory qPCRs for the levels of these five transcripts in MCF7 and MDA-MB-231 cells upon the overexpression of miR-515-5p (Fig 2B and C) and miR-515-5p sponge vectors (Appendix Fig S3A and B), which reduce the levels of miR-515-5p by directly interacting with its mature form. miR-515-5p sponge vectors induced an increase in the levels of the 5 transcripts in MCF7 but not within MDA-MB-231 cells (Appendix Fig S3A and B). This is likely to be because miR-515-5p expression is much lower in MDA-MB-231 than in MCF7 (Appendix Fig S4). Conversely, we observed a significant down-regulation of the expression of all five transcripts in the miR-515-5p-transfected MCF7 cells but only a decrease in N-RAS, PI3KC2B and MARK4 mRNA levels in miR-515-5p-transfected MDA-MB-231 (Fig 2B and C). Interestingly, miR-515-5p dramatically decreased MARK4 mRNA expression (> 95%) in both MCF7 and MDA-MB-231 cells (P < 0.001; Fig 2B and C). Levels of these five transcripts were also decreased in NSCLC cells (A549 and H1299) upon overexpression of miR-515-5p (although not significantly for CDC42BPA in A549 cells), underlying a common molecular function for this miRNA across tumour types (Fig 2D). Figure 2. miR-515-5p regulates mRNAs involved in cell migration A. RNA-seq of MCF7 and MDA-MB-231 transfected with miR-515-5p revealed the down-regulation of five transcripts, NRAS, FZD4, CDC42BPA, PIK3C2B and MARK4. B–D. The effect of miR-515-5p overexpression on NRAS, FZD4, CDC42BPA, PIK3C2B and MARK4 mRNA levels in MCF7 (B), MDA-MB-231(C), A549 and H1299 (D) was determined by qPCR 48 h following transfection of the indicated cell line with non-targeting control miR (NC) or miR-515-5p. Data are displayed as the normalised mean ± SEM of n = 4. P-values were calculated by t-test between miRNA conditions and their respective NC conditions (*P < 0.05; **P < 0.01; ***P < 0.001). E, F. miR-515-5p directly interacts with NRAS, MARK4 and PI3KC2B's 3′ UTR. Relative luciferase activity levels were measured 24 h after co-transfection of MCF-7 (E) and MDA-MB-231 (F) with 3′ UTR-luciferase reporter constructs and either with miR-515-5p or miR-NC. Data shown are normalised mean of three independent experiments ± SEM. Data information: (B–F) P-values were calculated by t-test between miRNA conditions and their respective NC conditions (*P < 0.05; **P < 0.01; ***P < 0.001). Download figure Download PowerPoint We then wished to assess whether the transcripts for N-RAS, MARK4 and PI3KC2B could be directly regulated by the binding of miR-515-5p to their 3′ UTRs. We used a reporter system where luciferase expression was under the control of the 3′ UTRs of our proposed targets. We showed that miR-515-5p negatively regulated the levels of luciferase reporter expression in both MCF7 and MDA231 cells by directly interacting with the 3′ UTR regions of N-RAS, MARK4 and PI3KC2B (P < 0.001; Fig 2D and E). This demonstrated that the mRNAs for these three proteins were true direct targets of miR-515-5p. The specificity of the binding of miR-515-5p to the 3′ UTR regions of MARK4 was demonstrated through mutating both the sites predicted by TargetScan in the seed sequence. Indeed, mutation of both sites in the 3′ UTR prevented miR-515-5p from inhibiting luciferase expression from our reporter vector (Appendix Fig S5). Silencing of MARK4 mimics the effect of miR-515-5p on both random and directional migration As MARK4 was so predominantly down-regulated by miR-515-5p in breast cancer cells (Fig 2B and C), we questioned whether silencing MARK4 would mimic the effect of miR-515-5p on cell morphology and in cell migration assays. As MCF7 cells are known to display poor motility, we did not analyse the effect of either miR-515-5p or MARK4 silencing on MCF7 cell migration 17. Hence, we limited our primary analysis to MDA-MB-231, A549 and H1299 cell lines. Transfection of these cell lines with MARK4-targeting siRNAs led to an increase in the cell area and clustering as can be observed following tubulin staining (Fig 3A). The change in cell area (quantified in Fig 3A, right panel) was accompanied by a loss of cell polarity reminiscent of that observed for miR-515-5p transfection (Fig 1A). We therefore proceeded to study possible associated changes in cell motility. Our previously published cell migration screen had revealed that silencing of MARK4 reduced migration in A549 cells 16, so we used this cell line as an internal control in these assays. Random migration (RM) and Boyden chamber directional migration (DM) assays showed that MARK4 knock-down inhibited cell migration by 48–61%, 55–58% and 54–56% (RM-DM) in MDA-MB-231, A549 and H1299 cell migration, respectively (Fig 3B and C). To further validate the role of MARK4 in the migration of breast cancer cells, we performed random migration experiments in migratory MDA-157 breast cancer cells and found that MARK4 silencing significantly reduced their motility (Appendix Fig S6B). A similar effect was obtained in these cells upon overexpression of miR-515-5p (Appendix Fig S6B) and was accompanied by changes in cell morphology comparable to that observed in MDA-MB-231, A549 and H1299 cells (Appendix Fig S6C). Taken together, these data suggest that MARK4 down-regulation might be the major cause for the decrease in cell migration observed upon miR-515-5p overexpression. Figure 3. MARK4 knock-down mimics the effect of miR-515-5p overexpression A–C. MARK4 siRNA-mediated silencing increases the tubulin cytoskeletal area (A) and reduces random (B) and directed (C) migration in MDA-MB-231 (MDA), A549 and H1299 cells. (A) Data shown are representative of experiments performed in triplicate. Bar graph represents the average ± SEM of n ≥ 50 per technical replicate with three technical replicates per conditions. Cells were transfected either with siRNA targeting MARK4 (M4) or a non-targeting control (NC) for 48 h before time-lapse imaging was performed for 18 h (B) or transwell assay for 9 h (C). (B, top panel) Plots show overlays of representative trajectories described. (B, bottom panel) The migration distance was quantified and represented as the normalised mean ± SEM. (C, top panel) Representative field of view from the bottom side of the transwell membrane. (C, bottom panel) Graphs indicate cell migration expressed as a percentage of the average of migratory cells per field (n = 5 fields per condition). (B–C) Data are mean of three experiments ± SEM. (A–C) P-values were calculated by t-test between the siRNA-transfected and the respective control condition (***P < 0.001). Scale bar: 200 μm. Download figure Download PowerPoint miR-515-5p inhibits cell migration through MARK4 down-regulation To clarify the importance of MARK4 in the cell migration inhibition by miR-515-5p, we transfected MDA-MB-231, A549 and H1299 cells with miR-515-5p, and after 24 h, we overexpressed MARK4 in the transfected cell lines (Appendix Fig S7A). We then analysed whether MARK4 overexpression rescued the effect of miR-515-5p on the random and directional migration of these cell lines (Fig 4A and B). There was no significant difference between the distance travelled by MDA-MB-231, A549 and H1299 cells co-overexpressing miR-515-5p and MARK4 and that of cell lines co-transfected with miR-NC and empty vector control plasmid (EV) in the random migration assays (Fig 4A and Appendix Fig S7B). Similarly, in the directional assay, the expression of MARK4 rescued the decrease in MDA-MB-231, A549 and H1299 cell migration, although this was more prominent for the lung cancer cell lines (Fig 4B and Appendix Fig S7C). Figure 4. MARK4 overexpression rescues the inhibitory effects of miR-515-5p on cell migration A, B. The indicated cell lines were transfected with either miR515-5p or a non-targeting miR (miR-NC) together with a MARK4 expressing or an empty vector (EV) plasmid DNA. MARK4 overexpression rescues the inhibition by miR-515-5p of random (A) and directed (B) migration of the indicated cell lines. (A, left panel) Plots show overlays of representative trajectories described for MDA-MB-231 cells (n = 30). (A, right panel) The migration distance was quantified and represented as the normalised mean ± SEM. (B, left panel) Representative field of view from the bottom side of the transwell membrane for MDA-MB-231. (B, right panel) Graphs indicate cell migration expressed as a percentage of the average of migratory cells per field (n = 5 fields per condition). Data are mean of three experiments ± SEM. P-values were calculated by t-test between the transfected and the respective control condition (EV+miR-NC) (*P < 0.05; **P < 0.01; ***P < 0.001). Scale bar: 200 μm. Download figure Download PowerPoint MARK4 silencing triggers cell cycle arrest In addition to the changes in cell motility, regulation of microtubule dynamics would also be expected to have significant effects on cell division. We therefore compared the cell cycle distribution between control-transfected A549 cells and cells silenced for MARK4. This analysis revealed that MARK4-down-regulated cells accumulated in the G1 phase of the cell cycle while showing a decrease in DNA synthesis (Appendix Fig S8A). This was accompanied by an increase in the levels of p53 as well as its transcriptional target, p21,