Exosomal miR-9 inhibits angiogenesis by targeting MDK and regulating PDK/AKT pathway in nasopharyngeal carcinoma
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Exosomes are small vesicles containing a wide range of functional proteins, mRNA and miRNA. Exosomal miRNAs from cancer cells play crucial roles in mediating cell-cell communication and tumor-microenvironment cross talk, specifically in enabling metastasis and promoting angiogenesis. We focused on miR-9 that was identified as a tumor suppressor previously in nasopharyngeal carcinoma (NPC) tumorigenesis.
Differential centrifugation, transmission electron microscopy and nanoparticle tracking analysis were used to isolate and identify exosomes. Quantitative PCR and western blotting analysis were used to detect miR-9, pri-miR-9, CD63, TSG101, MDK, P70S6K P-Ser424 and PDK1 P-Ser241 expression. Laser confocal microscopy was used to trace exosomal miR-9 secreted by NPC cells into HUVECs. The effect of exosomal miR-9 on cell migration and tube formation of HUVECs in vivo and vitro was assessed by using migration assay, tube formation assay and matrigel plug assay, respectively. Bioinformatics analysis and luciferase reporter assay were utilized to confirm the binding of exosomal miR-9 to the 3′untranslated region (3′-UTR) of MDK, while Phosphorylation Array was performed to identify AKT Pathway in HUVECs treated with exosomal miR-9. Furthermore, Immunohistochemistry (IHC) and in situ hybridization (ISH) was used to detected miR-9, CD31 and MDK expression in human NPC tumor samples.
NPC cells transfected with miR-9-overexpressing lentivirus, released miR-9 in exosomes. Exosomal miR-9 directly suppressed its target gene - MDK in endothelial cells. Mechanistic analyses revealed that exosomal miR-9 from NPC cells inhibited endothelial tube formation and migration by targeting MDK and regulating PDK/AKT signaling pathway. Additionally, the level of MDK was upregulated in NPC tumor samples and was positively correlated with microvessel density. Notably, the level of exosomal miR-9 was positively correlated with overall survival, and MDK overexpression was positively associated with poor prognosis in NPC patients, suggesting the clinical relevance and prognostic value of exosomal miR-9 and MDK.
Taken together, our data identify an extracellular anti-angiogenic role for tumor-derived, exosome-associated miR-9 in NPC tumorigenesis and prompt further investigation into exosome-based therapies for cancer treatment.
KeywordsExosome miR-9 Angiogenesis MDK Nasopharyngeal carcinoma
- 3′-UTR: 3′
Human umbilical vein endothelial cell
Nasopharyngeal carcinoma (NPC) is one of common malignant tumors in Southeast Asia, especially in Southern China, with a high rate of local invasion and locoregional lymphatic metastasis . Cervical nodal metastasis is a frequent clinical feature in NPC, occurring in roughly three-quarters of all patients . Because of the improvement of radiotherapy and chemotherapy, excellent local control has generally been achieved, but distant failure is a key challenge, and the treatment of metastatic NPC remains disappointing. Therefore, better understanding of the pathogenesis and metastatic mechanisms is essential for the development of novel therapeutics for NPC.
Exosomes are small vesicles ranging in size between 30 and 150 nm, which have recently been recognized as critical players of intercellular communication . Recent studies have revealed that exosomes play crucial roles in mediating tumor-microenvironment cross talk through the horizontal transfer of proteins, mRNAs and microRNAs (miRNAs) . Tumor-derived exosomes contribute to the development of immunosuppression and promote tumor growth and metastasis. By stimulating neoangiogenesis, activating mesenchymal stroma cells, and remodeling extracellular matrix, they contribute to the formation of a pre-metastatic niche and establish suitable microenvironments in distant metastatic sites [5, 6]. Therefore, these vesicles represent a rich source of novel biomarkers for cancer diagnosis and prognosis.
Pathological angiogenesis is a hallmark of cancer. In addition to the role of exosomes in promoting tumor metastasis, new evidence supports the idea that exosomal miRNAs stimulate angiogenesis by facilitating neoplasia-to-endothelial cell communication. It has been shown that miRNA-enclosed exosomes derived from cancer cells could interact with endothelial cells and thereby stimulate endothelial cell proliferation, migration and tube formation . Umezu et al. found that the exosome, derived from K562 cells with enforced miR-92a expression, did not affect the growth of human umbilical vein endothelial cell line (HUVECs) but did enhance endothelial cell migration and tube formation . Zhang et al. also showed that THP-1 cells selectively packaged miR-150 into microvesicles and actively secreted them into the extracellular environment, and extracellular miR-150 could then enter endothelial cells and enhance cell migration . However, no functional evidence of exosomal miRNAs has been documented in NPC angiogenesis.
Our previous studies found that ectopic expression of miR-9 dramatically inhibited tumor growth and metastasis in NPC , and plasma miR-9 might serve as a useful biomarker to predict NPC metastasis and to monitor tumor dynamics . However, another study found that tumor-secreted miR-9 promoted endothelial cell migration and angiogenesis by activating the JAK-STAT pathway in colorectal cancer and melanoma . These findings suggested that the contribution of miR-9 to angiogenesis seemed controversial. Here, we investigated the role of exosomal miR-9 in NPC angiogenesis.
Clinical samples and cell culture
The NPC biopsy specimens and serum samples (n = 110) were collected from Nanfang Hospital between 2007 and 2015. All samples were pathologically reassessed. The clinical characteristics of the study participants were presented in Additional file 1: Table S1. None of the patients had received radiotherapy or chemotherapy before biopsy and blood sampling. The clinical classification for NPC was based on the seventh edition UICC/AJCC staging system. All patients were treated with a uniform protocol of image-guided intensity modulated radiotherapy or/and cisplatin-based concurrent chemotherapy following induction chemotherapy according to National Comprehensive Cancer Network (NCCN) Guidelines. Healthy donors were recruited, and normal biopsies of the nasopharynx were obtained from every donor and used as healthy control. Informed consent was obtained from all individuals, and the research protocols were approved by the Ethics Committee of Nanfang Hospital. The approval number is NCT01171235. The median follow-up was 60 months. The 5-year overall survival rate was 71.8%.
Human NPC cell lines (5-8F and CNE1), HUVECs and human embryonic kidney 293 T cells (HEK293T) were purchased from the Cell Bank of the Chinese Academy of Sciences (Shanghai, China). An immortalized nasopharyngeal epithelial cell NP69 was kindly provided by cancer center of Sun Yat-sen University. NPC cell lines were cultured in RPMI1640 supplemented with 10% heat-inactivated exosome depleted fetal bovine serum (Gibco) at 37 °C in a humidified atmosphere containing 5% CO2. HUVEC cells were cultured in endothelial basal medium (EBM-2) supplemented with 10% fetal bovine serum. NP69 was cultured in Keratinocyte-SFM (Invitrogen) supplemented with bovine pituitary extract (BD Biosciences). HEK293T cells were cultured in Dulbecco’s modified Eagle’s medium (DMEM; Invitrogen). The PDK1 inhibitor OSU-03012 was purchased from Selleckchem (Houston, TX).
Exosomes isolation, transmission electron microscopy and nanoparticle tracking analysis
Exosomes were isolated from the plasma of healthy controls or NPC patients and from cell culture medium by differential centrifugation as previously described . Briefly, collected culture supernatants or plasma samples were differentially centrifuged at 300×g, 1200×g, and 10,000×g for 3 h, and the supernatant was filtered and ultracentrifuged at 110,000×g for 3 h (all steps were performed at 4 °C). Exosomes were collected from the pellet and resuspended in phosphate-buffered saline (PBS).
Exosomes were prepared and fixed as described previously . The samples were observed with a transmission electron microscope (H-7650, HITACHI, Japan) at an acceleration voltage of 80 kV.
Measurements for nanoparticle tracking analysis (NTA) were performed using the Nanosight NS3000 system (Nanosight, Amesbury, United Kingdom) equipped with a blue laser (405 nm). Exosomes were illuminated by the laser, and their movement under Brownian motion was recorded in 9 s sample videos which were analyzed with NTA analytical software (version 2.3, Nanosight). The capture settings and analysis settings were done manually according to the manufacturer’s instructions.
Cy3-labeled pre-miR miRNA trasnfection, exosomes labeling and confocal microscopy
Pre-miR miRNA precursor (hsa-miR-9, Ambion, Austin, TX) was labeled with Label IT siRNA Tracker Cy3 Kit, according to the manufacturer’s instructions (Mirus, Madison, WI, USA). 5-8F cells were transfected with 100 nM of Cy3-labeled pre-miR miRNA precursor using HiPerFect (Qiagen, Dusseldorf, Germany). After incubation for a day, the culture medium was collected and used for exosome preparation. The exosomes including Cy3-miR-9 were labeled using the green lipophilic fluorescent dye PKH67 (Sigma-Aldrich, St. Louis, MO, USA). The PKH67-labeled exosomes including Cy3-miR-9 were incubated with cultured HUVEC cells. After incubation, HUVEC cells were washed and visualized under confocal microscopy (FV10i, Olympus, Japan). 4′6-diamidino-phenylidole (DAPI, Abbott, USA) was used for nuclear staining.
Migration assay and tube formation assay
The migration ability of HUVEC cells was tested with BD Transwell invasion assay inserts, according to the manufacturer’s protocol. HUVEC cells were placed in the upper chamber of a Transwell coated with Matrigel. The chamber was placed in a 24-well culture dish. After incubation, the cells that did not invade through the pores were removed by a cotton swab. Cells on the lower surface of the membrane were fixed with 4% paraformaldehyde, stained with 0.5% crystal violet and counted.
The formation of capillary-like structures was analyzed as described previously . HUVEC cells (2 × 104 cells/well) were plated on top of Matrigel (300 μL/well) and treated with exosomes (600 μL of exosome fraction/well) derived from 5-8F/con or 5-8F/miR-9 cells. The total tube area was quantified as mean relative tube length obtained from image analysis of five random microscopic fields using Image J software (http://rsb.info.nih.gov/nih-image).
Matrigel plug assay
Matrigel plug assays were performed as described elsewhere . Briefly, 4 × 106 HUVEC cells were incubated with exosomes derived from 5-8F/miR-9 cells cells or 5-8F/con cells. The cell suspensions were mixed with 500 μl of Matrigel-reduced growth factor (BD Matrigel 356,230) at a ratio of 1:1. Then the cell suspensions were injected subcutaneously in the dorsal region of nude mice (female, 7–8 weeks old, BALB/c). Plugs were recovered 2 weeks later and then embedded in OCT, cryostat sectioned, and stained by hematoxylin and eosin. The vessel area was quantified as mean relative tube length obtained from image analysis of six random microscopic fields using Image J software. For all the experiments, animal handling and experimental procedures were approved by the Animal Experimental Ethics Committee of Southern Medical University.
Dual luciferase reporter assay
A 300-bp fragment of MDK 3′ untranslated regions (UTR) including wild type (wt) or mutant (mt) miR-9 binding sites were cloned downstream of the firefly luciferase gene in pGL3 vector (Promega). For reporter assays, HUVEC cells were firstly co-transfected with wt or mt MDK 3′ UTR vector and the control vector pRL-CMV [(cytomegalovirus) coding for Renilla luciferase, Promega) using lipofectamine 2000 reagent (Invitrogen). Then the cells were incubated with exosomes extracted from miR-9-overexpressing NPC cells or control cells. Luciferase activity was measured using the Dual-Luciferase Reporter Assay System (Promega, Madison, WI, USA).
The cells were lysed in lysis buffer (Roche, Penzberg, Germany), and equal amounts of protein were separated by SDS-PAGE. The exosome pellets isolated from the same amount of culture medium (10 mL) were lysed in 200 μL of lysis buffer (Roche), and the same amounts of lysate were loaded in each lane of the gels. Immunoblots were probed with antibodies directed against MDK (rabbit monoclonal anti-Midkine, Abcam, Cambridge, MA, USA), P70S6K P-Ser424 (rabbit polyclonal anti-S6 K1, Abcam), or PDK1 P-Ser241 (rabbit polyclonal anti-PDK1, Abcam). CD63 (rabbit monoclonal anti-CD63, Abcam) and TSG101 (rabbit monoclonal anti-TSG101, Abcam) were used as exosomal markers.
The human and mouse AKT Pathway Phosphorylation Array and MAPK Pathway Phosphorylation Array were performed according to the manufacture’s instructions (RayBiotech, Norcross, GA, USA).
Immunohistochemistry (IHC) and in situ hybridization (ISH)
For quantification of microvessel density (MVD) in NPC tumor samples, the number of blood vessels staining positive for CD31 (1:100 dilution, Abcam, Cambridge, MA, USA) was recorded in ten random fields at 200 magnification. The expression of MDK in NPC tumor samples was examined with IHC as previously described . The antibody was purchased from Abcam (rabbit monoclonal anti-Midkine, Abcam). In situ detection of miR-9 on formalin-fixed paraffin-embedded samples was essentially performed with a miRCURY LNA™ miR-9 detection probe, using an ISH optimization kit (Exiqon, Vedbaek, Denmark) . A scramble-miR probe (Exiqon) was performed as negative control.
Plasmids, transfection and lentivirus transduction
The lentiviral vector for delivery of pre-miR-9 was described previously . The vector for cDNA delivery of MDK was purchased from GeneCopoeia. The production, purification, and titration of lentivirus were performed as described by Tiscornia et al. . Transient transfection of miR-9 mimic or inhibitor (Ambion, Thermo Fisher Scientific) was performed using Lipofectamine 2000 reagent (Invitrogen), and transient transfection of miR-9 mimic or control into exosomes was performed using Exo-fect exosome transfection kit (System Biosciences). To inhibit MDK expression, siRNA against MDK (Invitrogen) was used. Transient transfection of siMDK was performed using Lipofectamine 2000.
RNA isolation, reverse transcription and quantitative real-time PCR
Isolation of cellular and exosomal miRNAs was performed using TRIzol reagent (Invitrogen) and Total Exosome RNA and Protein Isolation Kit (Invitrogen) respectively as described previously . MiR-9 expression in both cells and exosomes was performed using a TaqMan microRNA reverse transcription kit and TaqMan microRNA assay kit (Applied Biosystems, Foster City, CA) according to the manufacturer’s instructions. To measure the mRNA levels of MDK and pri-miR-9, total RNA was reversely transcribed using ImProm-II Reverse Transcription System (Promega). Quantitative real-time PCR (qPCR) was performed using SYBR Green PCR master mix (Applied Biosystems). All samples were normalized to internal controls and fold changes were calculated through relative quantification .
All statistical analyses were conducted using SPSS 19.0 software (SPSS, Chicago, IL, USA). Data were presented as Mean ± SD of at least three independent experiments. Two-tailed Student’s t test was used for comparisons of two independent groups. Comparisons of multiple independent groups were analyzed using One-way ANOVA followed by a Student-Newman-Keuls test. Survival curves were plotted by the Kaplan-Meier method and compared by the log-rank test. The relationships between CD31 expression and miR-9 or MDK expression were explored by Spearman’s correlation. A two-sided P < 0.05 was considered statistically significant.
miR-9 is reduced in exosomes derived from cultured NPC cells and plasma samples
Exosomal miR-9 is delivered from NPC cells into endothelial cells
Exogenous miR-9 inhibits recipient HUVEC cell migration and tube formation
To further confirm the anti-angiogenic role of exosomal miR-9, we performed an in vivo matrigel plug assay to detect the newly-formed blood vessels in the transplanted gel plugs in nude mice (Fig. 3c). The relative tube length of the neovessels line in Matrigel plugs was quantified (Fig. 3d). The data revealed that the plugs containing the exosomes derived from 5-8F/miR-9 significantly reduced the relative tube length compared with control (Fig. 3e). These additional experiments provided evidence of important roles for exosomal miR-9 derived from NPC cells on angiogenesis in vivo.
Exosomal miR-9 derived from NPC cells targets MDK and regulates PDK/AKT signaling pathway
To examine MDK expression in NPC cells, we transfected miR-9 mimic or negative control into 5-8F and CNE1 cells. The results showed that transfection of miR-9 mimic could reduce the expression of MDK in NPC cells (Fig. 4g). In contrast, MDK protein was not detected in exosomes derived from 5-8F and CNE1 cells (Additional file 4: Figure S3). The reduction of MDK expression in NPC cells might be induced by endogenous miR-9, and MDK protein of NPC cells did not transfer to the other cells via exosomes. These data indicated that exosomal (exogenous) miR-9 interacted with the MDK 3′UTR to exert translational repression in HUVEC cells. Moreover, direct knockdown of MDK in HUVEC by siRNA (efficient interference of MDK expression was shown in Additional file 5: Figure S4) significantly inhibited cell migration (Fig. 4h) and tube formation (Fig. 4i). To elucidate whether the anti-angiogenic effect of exosomal miR-9 was mediated by repression of MDK in HUVEC cells, we performed a MDK rescue assay. HUVEC cells were infected with LV-MDK for 72 h and followed by treatement with miR-9-ovexpressing exosome. We showed that ectopic expression of miR-9 significantly reversed MDK-induced promotion of cell migration and tube formation (Additional file 6: Figure S5).
Expression of miR-9 is negatively correlated with microvessel density in NPC
MDK is overexpressed and positively correlated with microvessel density in NPC
This is the first study to elucidate NPC cells to endothelial cells communication via an exosomal miRNA, which might be associated with the angiogenic activity of endothelial cells. Firstly, we visualized the transfer of NPC cell-derived miR-9 to HUVECs. Secondly, we found that exogenous miR-9 via exosomal transport could function as an endogenous miRNA in endothelial cells. Finally, we proved that cell-to-cell communication via exosomal miR-9 affected endothelial migration and tube formation through targeting MDK and regulating PDK/AKT signaling pathway. These data indicate that miRNAs originating from cancer cells can regulate the angiogenic activity of endothelial cells.
Mounting evidence suggests the key role of tumor-derived exosomes in modulating angiogenesis in many cancers, such as melanoma [19, 20], leukemia , breast cancer [22, 23]. Tumor-derived exosomes release a specific content of miRNAs, mRNAs, and proteins in endothelial cells, therefore modifying angiogenesis. Several reports find that miR-9 functions as a regulator of angiogenesis, and this miRNA has a dual function of pro- or anti-angiogenetic activity by the cooperation of individual target genes in endothelial cells. One study showed that microvesicles-derived miR-9 effectively reduced SOCS5 expression and activated JAK-STAT pathway, which promoted endothelial cell migration and tumour angiogenesis. This pro-angiogenetic role of miR-9 was proved in several cancers, including lung cancer, melanoma, pancreatic cancer, glioblastoma and colorectal cancer . Qu et al. indicated that miR-9 overexpression promoted osteoblast differentiation and angiogenesis via the AMPK signaling pathway . Another research found that miR-9 coupled neurogenesis and brain angiogenesis through the inhibition of Tlx and Onecut transcription factors regulating neuronal VEGF-A expression . Although regulation of proliferation, epithelial-mesenchymal transition, invasion, and tumor angiogenesis attributed to miR-9 is studied in a variety of cancer types , the effect of miR-9 on angiogenesis in NPC have never been elucidated. Our data showed that exosomal miR-9 enhanced recipient HUVEC cell migration and tube formation. Additionally, we found that the plasma exosomal miR-9 level was positively correlated with overall survival in NPC patients, suggesting the clinical relevance and prognostic value of tumor-derived exosomes.
Midkine (MDK) is a heparin-binding growth factor prominently expressed in embryonic tissues but down-regulated to negligible levels in healthy tissues of adults, which is considered to be a “pan-cancer” biomarker [27, 28, 29]. Overexpression of MDK were observed in many cancers [29, 30, 31], including lung cancer, ovarian carcinoma and thyroid carcinoma. However, the status and function of MDK have never been documented in NPC tumorigenesis. In this study, we found that MDK was a new target gene of miR-9 in endothelial cells. Previous studies showed that MDK activated both the Akt and ERK1/2 pathways, upregulated the expression of several cell-cycle-related proteins and promoted gastric cancer cell survival and growth . Another study found that MDK activated AKT and ERK pathways and induced the Adriamycin resistance in gastric cancer cell . Our results indicated that the inhibitory effect of exosomal miR-9 on endothelial cell was mediated by targeting MDK and regulating PDK1/AKT pathway. For the first time, we illustrated the link between MDK and PDK1/AKT pathway in tumor angiogenesis. More importantly, we found that MDK was overexpressed and positively correlated with microvessel density in NPC, and high MDK expression was positively associated with poor prognosis in NPC, suggesting the valuable potential of MDK as a prognostic biomarker.
This study has several limitations. Tumor angiogenesis is known to be modulated by multiple kinds of factors, including cytokines, chemokines, and growth factors derived from tumor microenvironment . Exosomes carry various molecules, including proteins, mRNAs and miRNA. Thus, further studies are needed to understand how soluble factors and/or exosomal contents affect miR-9 in the tumor microenvironment in NPC. There is another issue that needs to be addressed. The role of MDK in NPC tumorigenesis is required to be further elucidated in future. Most importantly, the clinical relevance of exosomal miR-9 and MDK expression in NPC patients awaited further validation on larger sizes of samples.
Taken together, this study demonstrated that exosomal miR-9 inhibited angiogenesis by targeting MDK and through PDK/AKT pathway in NPC. Given the molecular and biological complexity of angiogenesis, a better understanding of how cancer-derived exosomes participate in this process represents an important challenge, which can open new paths for the development of novel and effective anti-angiogenic drugs.
Taken together, our data identify exosomal miR-9 derived from NPC cells inhibits angiogenesis by targeting MDK and regulating PDK/AKT pathway in nasopharyngeal carcinoma. The findings of this study suggest that miR-9 and MDK may be valuable as novel targets for the treatment of human nasopharyngeal carcinoma.
This work was funded by National Natural Science Foundation of China (81301849 to J.L. and 81472535 to X.P.L.) and Natural Science Foundation of Guangdong Province, China (2014A030310032 to X.X.).
Availability of data and materials
The datasets used and analyzed during the current study are available from the corresponding authors on reasonable request.
JL, QHL and XPL conceived and designed the research. JL, QHL, XL, XX, YQD and BZ performed the experiments. JL, QHL, JJT, FW and XHP analyzed and interpreted the data. JL and QHL drafted and wrote the paper. JL and QHL was responsible for the main work of the study and contributed equally to this work. All authors read and approved the final manuscript.
Ethics approval and consent to participate
The study was conducted in accordance with the Declaration of Helsinki principles. Informed consent was obtained from all individuals, and the research protocols were approved by the Ethics Committee of Nanfang Hospital Affiliated to Southern Medical University and the Animal Experimental Ethics Committee of Southern Medical University.
Consent for publication
The authors declare that they have no competing interests.
Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.
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