FEBS Letters 589 (2015) 756–765
journal homepage: www.FEBSLetters.org
miR-10a controls glioma migration and invasion through regulating epithelial–mesenchymal transition via EphA8 Yan Yan a,⇑,1, Qin Wang a,1, Xiao-Ling Yan a,1, Yi Zhang b, Wei Li a, Fan Tang a, Xu Li a, Ping Yang a a b
Department of Clinical Laboratory, Tianjin Huan Hu Hospital, Tianjin Key Laboratory of Cerebral Vessels and Neural Degeneration, Tianjin, China Tianjin Life Science Research Center, Department of Microbiology, School of Basic Medical Sciences, Tianjin Medical University, Tianjin, China
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Article history: Received 4 January 2015 Revised 3 February 2015 Accepted 4 February 2015 Available online 12 February 2015 Edited by Tamas Dalmay Keywords: MicroRNAs Glioma Eph tyrosine kinase receptor A8 Migration Invasion Epithelial–mesenchymal transition
a b s t r a c t MicroRNAs (miRNAs) play a critical role in the development of cancers. However, the role of miRNAs in glioma is still poorly understood. In this study, we demonstrate that microRNA-10a (miR-10a) promotes cell migration and invasion by negatively regulating the expression of Eph tyrosine kinase receptor A8 (EphA8). Ectopic expression of EphA8 counteracts the promotion of migration and invasion induced by miR-10a. We further demonstrate that miR-10a and EphA8 regulate epithelial–mesenchymal transition (EMT) to affect cell migration and invasion. Collectively, we unveil a branch of the miR-10a/EphA8 pathway that regulates the progression of glioma. Ó 2015 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved.
1. Introduction Malignant gliomas are the most common primary tumors of the central nervous system. They are characterized by high invasion, migration and proliferation abilities. Grade IV astrocy-tomas are known as glioblastoma multiforme (GBM). Despite aggressive surgery combined with radiation, chemotherapy, and biological therapy, the median survival is only 12–15 months for patients with GBM [1]. Many factors influence the effectiveness of glioma therapies, including rapid and invasive tumor growth. Hence, there is a recognized need for new approaches based on increased understanding of the biological and molecular nature of these tumors. MicroRNAs (miRNAs) are endogenous, small, non-coding RNAs that regulate gene expression by antisense complementarity to specific mRNA. MiRNAs have been demonstrated to play critical roles in the biologic processes such as cell migration, invasion, proliferation, apoptosis, stress resistance, and cell identity [2–5]. Until
Abbreviations: miRNA, microRNA; miR-10a, microRNA-10a; EphA8, Eph tyrosine kinase receptor A8; EGFP, enhanced green fluorescence protein; UTR, untranslated region; qRT-PCR, quantitative real-time polymerase chain reaction; EMT, epithelial–mesenchymal transition ⇑ Corresponding author. at: No. 122 Qi-Xiang-Tai Road, Tianjin 300060, China. E-mail address: [email protected] (Y. Yan). 1 These authors contributed equally to this work.
now, many miRNAs have been reported to regulate glioma development, including miR-335 [6], miR-218 [7], miR-15b, miR-152 [8], miR-92b [9], miR-145 [10], and miR-124 [11], et al. However, the effect of miR-10a on glioma has not been reported. Our previous studies have demonstrated that miR-10a was involved in metastatic process in hepatoma cells [12]. Thus, we investigated whether miR-10a also contributed to the metastatic behavior of glioma cells. Ephrin receptors comprise the largest known family of receptor tyrosine kinases (RTKs), which interact with their ligands, ephrins. Recently, compelling evidence suggested that expression of ephrin ligands and receptors is often reduced in advanced-stage tumors and regulates tumor growth, metastasis and angiogenesis by altering cell proliferation, motility, invasion and migration [13]. Recent genetic studies suggested that EphA8 was involved in regulating cell adhesion and apoptosis [14,15]. However, reports about the relationship of EphA8 and cell migration and invasion were still few. The process of metastasis consists of a series of steps. Among them, the epithelial–mesenchymal transition (EMT) is commonly characterized by the suppression of the cell–cell adhesion receptor E-cadherin and endows cells with more motile, invasive properties [16]. Accumulating evidence suggested that many miRNAs were involved in EMT [17,18]. Our previous studies have also suggested that miR-10a could promote cell migration and invasion by regulating EMT process in hepatoma [12].
http://dx.doi.org/10.1016/j.febslet.2015.02.005 0014-5793/Ó 2015 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved.
Y. Yan et al. / FEBS Letters 589 (2015) 756–765
In this study, we found that miR-10a promoted the migration and invasion of the human glioma cell lines U251 and U87. We identified EphA8 as a direct target of miR-10a. Furthermore, we found that miR-10a and EphA8 regulated the EMT process to influence the cell migration and invasion. Together, these data contribute to the characterization of the molecular mechanisms of glioma progression. 2. Materials and methods 2.1. Cell culture, transfection and RNA extraction Human glioma cell lines, including U251, U87, A172, U118 and LN18 were cultured at 37 °C with 5% CO2 in DMEM (Gibco, Gaithersburg, USA) supplemented with 10% fetal bovine serum (FBS). Transient transfection was performed using the Lipofectamine 2000 reagent (Invitrogen, Carlsbad, USA). Total RNA was extracted using the Trizol reagent (Invitrogen), and miRNAs were obtained using the mirVana miRNA isolation kit (Ambion, Austin, USA). 2.2. Clinical glioma samples Human glioma samples were collected from the neurosurgery department of Huan Hu Hospital of Tianjin in China from 20 adult patients, freshly resected during surgery. All samples were immediately sectioned from the resected glioma tissues, frozen in liquid nitrogen and stored at 80 °C. There were 8 low-grade (3 from WHO grades I and 5 from WHO grades II) and 12 high-grade tumors (5 from WHO grades III and 7 from WHO grades IV). None of patients had received chemotherapy, immunotherapy and radiotherapy prior to specimen collection. All patients gave written informed consent according to a study protocol that was approved by Tissue Committee and Research Ethics Board of Huan Hu Hospital of Tianjin. 2.3. Vector constructions The vector constructions used in this study was shown in Supplementary materials and methods.
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2.7. Migration and invasion assays For the transwell migration assay, 5 104 U251 or U87 cells were placed in the upper chamber of each insert (Corning, Cambridge, USA). For the invasion assay, 5 104 cells were placed on the upper chamber of each insert coated with 40 ll matrigel (Clontech, Mountain View, CA), which was diluted to 2 lg/ll with DMEM medium. Medium supplemented with 20% FBS (600 ll) was added to the lower chambers. After several hours of incubation (24 h for U251 and U87 in the migration assays; 48 h for U251 and U87 in the invasion assays), the upper surface of the membrane was wiped with a cotton tip and cells attached to the lower surface were stained for 20 min with crystal violet. Cells in five random fields of view at 100 magnification were counted and expressed as the average number of cells per field of view. 2.8. Statistical analysis Data are presented as the mean ± S.D. Statistical analyses were performed using a paired t-test to compare data. A P-value less than 0.05 were considered statistically significant. 3. Results 3.1. Upregulation of miR-10a in human glioma tissues and cell lines To determine whether miR-10a was involved in the tumorigenesis or development of glioma, we firstly examined the expression of miR-10a in clinical tissues and glioma cell lines. Among 20 paired clinical specimens, miR-10a was significantly upregulated in glioma tissues compared to the adjacent non-cancerous tissues. Meanwhile, the miR-10a expression was higher in grade III and IV gliomas compared to grade I and II tumors (Fig. 1A). qRT-PCR was performed on a panel of 5 human glioma cell lines and primary normal human astrocytes (NHA). Remarkable up-regulation of miR-10a could also be observed in glioma cell lines than that of NHA (Fig. 1B). These results suggested that miR10a could be closely related to human glioma and high level of miR-10a might be related to glioma oncogenesis and progression. 3.2. miR-10a promotes glioma cell migration and invasion in vitro
2.4. Bioinformatics analysis The target gene information of miR-10a was analyzed using miRanda, Targetscan, and PicTar. 2.5. EGFP reporter assay Glioma cells were transiently cotransfected with EGFP reporter plasmid and pcDNA3-pri-10a, pcDNA3, ASO-miR-10a and ASO-NC, respectively. The RFP expression vector, pDsRed2-N1, was used as the internal control. Intensities of EGFP and RFP fluorescence were detected with an F-4500 fluorescence spectrophotometer (Hitachi, Tokyo, Japan). 2.6. Quantitative real-time PCR (qRT-PCR) and Western blot analysis Target genes and controls were analyzed by qRT-PCR using SYBR Premix Ex TaqTM (TaKaRa, Dalian, China). Cell lysates were separated on 8% SDS denatured polyacrylamide gel, transferred to nitrocellulose membranes and blocked in phosphate-buffered saline/Tween-20 containing 5% non-fat milk. The membranes were incubated with antibodies. Details and antibodies were under Supplementary materials and methods.
To determine whether miR-10a had an effect on the malignant phenotype of glioma cells, we constructed a miR-10a expression plasmid (pcDNA3-pri-10a, pri-miR-10a) and validated the efficiency of pri-miR-10a and the antisense oligonucleotide of miR-10a (ASO-miR-10a) (Fig. S1). U251 and U87 cells were then transfected with them or their respective controls to explore their effects on the cancer cells. Since invasiveness is one of the pathophysiological features of glioma, we asked whether miR-10a over-expression was associated with the invasiveness of gliomas. Transwell migration and invasion assays showed that the migration (Fig. 2A) or invasion (Fig. 2B) capacities of U251 cells transfected with primiR-10a was increased by approximately 2.7- or 1.7-fold. ASOmiR-10a reduced these capacities by almost 76% or 69% when compared to the controls. The similar results were observed in U87 cells (Fig. 2C and D). These findings indicated that miR-10a promoted both the migration and invasion of glioma cells. 3.3. miR-10a targets the 30 -UTR of EphA8 transcripts and negatively regulates its expression miRNAs influence cell behavior by regulating the expression of target genes. To explore the molecular mechanism through which miR-10a exerts its function in glioma cells, we predicted and iden-
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Fig. 1. miR-10a was up-regulated in human glioma tissues and cell lines. (A) Real-time PCR analysis of miR-10a expression in glioma tissues from different grades and adjacent non-cancerous tissues. Box-plot lines represent medians and interquartile ranges of the normalized threshold values. (B) Real-time PCR analysis of miR-10a expression in several glioma cell lines. U6 was used as loading control. Differences between groups were assessed by one-way ANOVA with the LSD method, ⁄P < 0.05, ⁄⁄ P < 0.01.
tified the candidate target genes of miR-10a. Three algorithms, MiRanda, Targetscan and PicTar were used to predict the potential target gene(s). Considering the associated genes that could account for cell migration or invasion, Eph tyrosine kinase receptor 8, EphA8, was chosen for further study. Base-pairing complementation revealed that the 30 untranslated region (UTR) of EphA8 contains a putative binding region of miR-10a (Fig. 3A). To determine whether EphA8 is a direct target of miR-10a, a series of 30 UTR fragments-including full length 30 UTR, binding site 1 and binding site 2 were constructed and inserted into the region immediately downstream of the luciferase reporter gene. The pri-miR-10a or ASO-miR-10a constructs were then cotransfected with the EGFP reporter vector into U251 cells. Interestingly, EGFP intensities were reduced by almost 73% by miR-10a when the full-length wild-type 30 -UTR of EphA8 was present (Fig. 3B), and when miR-10a expression was blocked, the EGFP intensities were enhanced by approximately 2.2-fold (Fig. 3B). The repressive effect on EGFP activity was abrogated by a mutation in the seed region of the EphA8-30 UTR 1 fragment and partially attenuated by a mutation in the seed region of EphA8-30 UTR 2 fragment (Fig. 3C and D). Similar findings were observed in U87 cells (Fig. S2). These results indicated that these sites were necessary for miR-10a binding. To determine if miR-10a suppressed endogenous EphA8 expression, qRT-PCR and Western blot analysis was used and
demonstrated that over-expression of miR-10a dramatically suppressed the endogenous mRNA and protein levels of EphA8 by 64% and 45%, respectively, whereas the inhibition of miR-10a increased the expression of EphA48 by 2.2- and 1.8-fold in U251 cells (Fig. 3E and F). The similar results were detected in U87 cells (Fig. S2). 3.4. EphA8 is often down-regulated in glioma and suppresses glioma cell migration and invasion We have identified EphA8 as a direct target of miR-10a. To better understand the potential role of EphA8 in miR-10a-mediated migration and invasion, the mRNA level of EphA8 was also measured in 20 paired clinical specimens. The level of EphA8 was significantly down-regulated in glioma tissues when compared with adjacent non-cancerous tissues (Fig. 4A), and was strongly correlated with the up-regulation of miR-10a (Fig. 4B). Furthermore, the mRNA expression of EphA8 was also analyzed in glioma cells (Fig. 4C). These data suggested that EphA8 expression was reduced in glioma and was inversely correlated with miR-10a expression. In order to explore the biological functions of EphA8, we constructed the pSilencer/siR-EphA8 plasmid to knockdown endogenous EphA8 expression and Western blot analysis showed that EphA8 protein expression level was reduced by almost 75% or
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Fig. 2. miR-10a promotes glioma cell migration and invasion in vitro. (A) Transwell migration assay of human glioma cell line U251 cotransfected with either miR-10a or ASO-miR-10a and their control vectors. (B) Transwell invasion assay of U251 cells. (C and D) Transwell migration and invasion assays of human glioma cell line U87. Representative images are shown behind. Cells in five random fields of view at 100 magnification were counted and expressed as the average number of cells per field of view, ⁄⁄P < 0.01.
82% in U251 and U87 cells (Fig. 5A). As expected, knockdown of endogenous EphA8 resulted in a significant increase in cell migration by 3.1- and 3.5-fold (Fig. 5B), and an increase in invasion by 2.6- and 2.8-fold, respectively (Fig. 5C). These results suggested EphA8 could suppress cell migration and invasion of glioma cells. 3.5. Ectopic expression of EphA8 restores the influence of miR-10a in cell migration and invasion The data above indicated that miR-10a could negatively regulate the expression of EphA8 both at the mRNA and protein levels by directly binding to its 30 -UTR and knockdown of EphA8 could mimic the miR-10a over-expression. To further confirm that miR10a promoted the migration and invasion of glioma cells by regulating EphA8 directly, we perfumed a rescue experiment. U251 cells were cotransfected with miR-10a and pA3M1-EphA8, which encoded the entire EphA8 coding sequence but lacked the 30 -UTR. Western blot analysis was used to validate the EphA8 expression in the rescue experiment (Fig. 6A). As expected, the restoration of EphA8 inhibited the miR-10a-promoted migration and invasion (Fig. 6B). Similar data was detected in U87 cells (Fig. 6C). Taken together, these findings suggested that miR-10a promoted glioma cell migration and invasion by directly targeting EphA8. 3.6. miR-10a and EphA8 affect glioma cell migration and invasion by regulating of the EMT process The above observations suggested that miR-10a could promote glioma cell migration and invasion and exerted its function by directly regulating EphA8, we further investigated the mechanism by which miR-10a and EphA8 regulate the cell malignant
phenotype. We noticed a striking change in cellular shape due to the miR-10a inhibition or EphA8 over-expression, an initial fibroblast-like morphology was observed to switch to the cobblestone-like appearance of epithelial cells. To determine whether the typical molecular alternations of EMT occurred, the protein levels of epithelial (E-Cadherin) and mesenchymal (Vimentin and ICAM-1) markers were measured. As shown in Fig. 7A, EphA8 expression was increased by 1.5- or 2.8-fold after transfection of ASO-miR-10a or pA3M1-EphA8 plasmid in U251 cells. E-Cadherin protein expression was up-regulated by 1.1-fold, whereas Vimentin and ICAM-1 were down-regulated by 62% and 78% respectively when miR-10a was blocked (Fig. 7B) in U251 cells. In addition, similar results were observed when EphA8 was over-expressed (Fig. 7C). Furthermore, similar observations were obtained in U87 cells (Fig. S3). The above findings suggested that miR-10a and its target gene, EphA8, could affect glioma cell migration and invasion by regulating of EMT process. Above all, our findings indicate that miR-10a promotes, whereas EphA8 suppresses glioma cell migration and invasion in vitro. miR-10a exerts its function by directly repressing EphA8. miR10a and EphA8 affect glioma cell malignant phenotype by regulating of the EMT process. These observations proved a new miR10a/EphA8 regulation pathway of cell migration and invasion in glioma. 4. Discussion Diffusely infiltrating gliomas are one of the most devastating cancers because they often show locally aggressive behavior and cannot be cured by existing therapies [19]. Gliomas develop as a result of genetic alterations that accumulate throughout tumor progression [20]. Therefore, the elucidation of these molecular
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Fig. 3. miR-10a targets the 30 -UTR of EphA8 and down-regulates its expression. (A) The sequence of the predicted miR-10a binding site and the EphA8 30 -UTR segments containing the wild-type or mutant binding site are shown. The protein binding sequences for miR-10a within the EphA8 30 -UTR of human, chimpanzee, mouse, rat and guinea pig are also shown. Seed sequences are highlighted. (B) Relative EGFP activity was analyzed after the wild-type full-length EphA8-30 UTR plasmid was cotransfected with miR-10a or ASO-miR-10a in U251 cells. (C) EGFP intensity was analyzed after the mutant 30 -UTR-1 reporter plasmids was cotransfected with miR-10a or ASO-miR-10a. (D) EGFP intensity was analyzed after the mutant 30 -UTR-2 reporter plasmids was cotransfected with miR-10a or ASO-miR-10a. The histogram shows the mean ± S.D. of the normalized EGFP intensity. (E) Real-time PCR analysis was used to detect the mRNA level of EphA8 in transiently transfected U251 cells. Normalization was performed using b-actin. (F) Western blot analysis was used to detect the protein level of EphA8 in transiently transfected U251 cells. Normalization was performed using GAPDH, ⁄P < 0.05, ⁄⁄ P < 0.01.
mechanisms, in particular the ones associated with cellular migration and invasion is crucial for a better prediction of glioma patients outcome and response to therapies [21]. Recent studies have shown that miRNAs play a fundamental role in the migration and invasion of glioma. For example, miR-204 inhibits glioma cell migration and invasion by negatively regulating ezrin gene
expression [22]. MiR-18a regulates the proliferation, migration and invasion of human glioblastoma cell by targeting neogenin [23]. MiR-10a, which was studied in many research was involved in regulating the migration and invasion of many kinds of cancers, such as cervical and gastric cancers [24,25]. Our previous studies also suggested that miR-10a could promote cell migration and
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Fig. 4. EphA8 is often down-regulated in glioma tissues and cell lines. (A) Real-time PCR was used to detect the mRNA levels of EphA8 in clinical specimens. Box-plot lines represent medians and interquartile ranges of the normalized threshold values. The EphA8 abundance was normalized to b-actin. (B) Correlation between expression level of EphA8 and that of miR-10a in 20 matched tissues. Linear regression coefficient and statistical significance is indicated. (C) Real-time PCR was used to detect the mRNA levels of EphA8 in glioma cell lines, ⁄⁄P < 0.01.
invasion of hepatoma and cervical cancer cells [12,26]. However, effect of miR-10a on glioma has not been reported. In this study, miR-10a, a new regulator of glioma, was shown to promote migration and invasion of U251 and U87 glioma cells. To further explore the mechanism by which miR-10a exerts its function, we used bioinformatics analysis to predict and finally identified EphA8 as the direct target of miR-10a. This conclusion is based on several experimental evidences. First, miR-10a significantly decreases the expression of EphA8 both at the mRNA and protein levels in glioma cells. Second, the EGFP reporter assay showed that the fluorescence intensity of EGFP-EphA8-30 UTR was specifically responsive to miR-10a over-expression, however, mutation of the miR-10a binding site abolished the effect of miR-10a on the
regulation of EGFP fluorescence intensity. Third, EphA8 expression is down-regulated in HCC tissues, and this down-regulation is strongly correlated with the up-regulation of miR-10a. Fourth, knockdown of EphA8 phenocopies the effect of miR-10a expression, whereas restoration of EphA8 counteracts the function of miR-10a. These results indicate that miR-10a targets EphA8 and down-regulates its expression in glioma. Ephrin receptors comprise the largest known family of receptor tyrosine kinases (RTKs), with 16 Eph receptors and 9 ephrin ligands [27–29]. Eph receptors and ligands are divided into either A-class or B-class, and were initially implicated in cell positioning and migration during development, particularly of the nervous system. Until now, reports have suggested that many Eph receptors are involved
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Fig. 5. EphA8 suppresses the migration and invasion of glioma cells in vitro. (A) Western blot analysis of EphA8 protein level in U251 and U87 cells transfected with pSilencer/ shR-EphA8 or pSilencer plasmids. (B) Transwell migration analysis of glioma cells when EphA8 was inhibited. (C) Transwell invasion analysis of glioma cells when EphA8 was inhibited. Cells in five random fields of view at 100 magnification were counted and expressed as the average number of cells per field of view, ⁄⁄P < 0.01.
in regulating proliferation, migration, invasion and adhesion of glioma cells [30–34]. In this study, transwell assay demonstrated that knockdown of EphA8 could promote cell migration and invasion of glioma which was in accordance with miR-10a over-expression. Ectopic expression of EphA8 without the 30 UTR rescued the promotion of migration and invasion in U251 and U87 cells caused by miR-10a. As mentioned above, EMT is a process in which epithelial cells lose or modify their apical–basal polarity during embryonic development and tumor metastasis [35,36]. Our previous study
has suggested that miR-10a could affect cell malignant behavior via regulating the EMT process [12]. Barrios et al. [37] have indicated that one of the RTKs family members, EphA4, regulates the mesenchymal-to-epithelial transition (MET) of the paraxial mesoderm during somite morphogenesis. We speculated that miR-10a and EphA8 played roles in the EMT process in glioma. We examined the expression of the epithelial marker E-cadherin and the mesenchymal markers Vimentin and ICAM-1. Because miR-10a expression was increased whereas EphA8 was decreased in cancer tissues and glioma cells, we blocked miR-10a expression
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Fig. 6. Ectopic expression of EphA8 restores the influence of miR-10a in cell migration and invasion. (A) Western blot analysis of EphA8 expression in U251 and U87 cells with or without EphA8 restoration. (B) Restoration of EphA8 inhibits miR-10a-promoted glioma cell migration and invasion in U251 cells. (C) Restoration of EphA8 inhibits miR10a-promoted glioma cell migration and invasion in U87 cells. Cells in five random fields of view at 100 magnification were counted and expressed as the average number of cells per field of view, ⁄⁄P < 0.01.
by ASO-miR-10a and over-expressed EphA8 to detect the related molecular markers. As expected, inhibition of miR-10a or over-expression of EphA8 suppressed the EMT phenotype. In other words, miR-10a can increase, whereas EphA8 can suppress cell migration and invasion of glioma by mediating the EMT process.
In summary, we discovered that miR-10a can promote cell migration and invasion of glioma and exerts its function by directly targeting EphA8, thereby regulating EMT process. Our research thus provides a new insight into the mechanism of the pathogenesis of glioma and suggests miR-10a and EphA8 play important roles in progression of glioma.
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Fig. 7. miR-10a and EphA8 affect glioma cell migration and invasion via regulating of the EMT process. (A) Western blot analysis of EphA8 protein level when miR-10a expression was inhibited or EphA8 was over-expressed in U251 cells. (B) The protein expression levels of E-Cadherin, Vimentin and ICAM-1 when miR-10a was inhibited in U251 cells. (C) The protein expression levels of epithelial and mesenchymal markers mentioned above when EphA8 was over-expressed in U251 cells, ⁄⁄P < 0.01.
Acknowledgments We thank the Public Health College of Tianjin Medical University for their technical assistance in the fluorescence studies. This work was supported by the National Nature Science Foundation of China (No. 81301967). Appendix A. Supplementary data Supplementary data associated with this article can be found, in the online version, at http://dx.doi.org/10.1016/j.febslet.2015.02. 005.
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journal homepage: www.FEBSLetters.org
miR-10a controls glioma migration and invasion through regulating epithelial–mesenchymal transition via EphA8 Yan Yan a,⇑,1, Qin Wang a,1, Xiao-Ling Yan a,1, Yi Zhang b, Wei Li a, Fan Tang a, Xu Li a, Ping Yang a a b
Department of Clinical Laboratory, Tianjin Huan Hu Hospital, Tianjin Key Laboratory of Cerebral Vessels and Neural Degeneration, Tianjin, China Tianjin Life Science Research Center, Department of Microbiology, School of Basic Medical Sciences, Tianjin Medical University, Tianjin, China
a r t i c l e
i n f o
Article history: Received 4 January 2015 Revised 3 February 2015 Accepted 4 February 2015 Available online 12 February 2015 Edited by Tamas Dalmay Keywords: MicroRNAs Glioma Eph tyrosine kinase receptor A8 Migration Invasion Epithelial–mesenchymal transition
a b s t r a c t MicroRNAs (miRNAs) play a critical role in the development of cancers. However, the role of miRNAs in glioma is still poorly understood. In this study, we demonstrate that microRNA-10a (miR-10a) promotes cell migration and invasion by negatively regulating the expression of Eph tyrosine kinase receptor A8 (EphA8). Ectopic expression of EphA8 counteracts the promotion of migration and invasion induced by miR-10a. We further demonstrate that miR-10a and EphA8 regulate epithelial–mesenchymal transition (EMT) to affect cell migration and invasion. Collectively, we unveil a branch of the miR-10a/EphA8 pathway that regulates the progression of glioma. Ó 2015 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved.
1. Introduction Malignant gliomas are the most common primary tumors of the central nervous system. They are characterized by high invasion, migration and proliferation abilities. Grade IV astrocy-tomas are known as glioblastoma multiforme (GBM). Despite aggressive surgery combined with radiation, chemotherapy, and biological therapy, the median survival is only 12–15 months for patients with GBM [1]. Many factors influence the effectiveness of glioma therapies, including rapid and invasive tumor growth. Hence, there is a recognized need for new approaches based on increased understanding of the biological and molecular nature of these tumors. MicroRNAs (miRNAs) are endogenous, small, non-coding RNAs that regulate gene expression by antisense complementarity to specific mRNA. MiRNAs have been demonstrated to play critical roles in the biologic processes such as cell migration, invasion, proliferation, apoptosis, stress resistance, and cell identity [2–5]. Until
Abbreviations: miRNA, microRNA; miR-10a, microRNA-10a; EphA8, Eph tyrosine kinase receptor A8; EGFP, enhanced green fluorescence protein; UTR, untranslated region; qRT-PCR, quantitative real-time polymerase chain reaction; EMT, epithelial–mesenchymal transition ⇑ Corresponding author. at: No. 122 Qi-Xiang-Tai Road, Tianjin 300060, China. E-mail address: [email protected] (Y. Yan). 1 These authors contributed equally to this work.
now, many miRNAs have been reported to regulate glioma development, including miR-335 [6], miR-218 [7], miR-15b, miR-152 [8], miR-92b [9], miR-145 [10], and miR-124 [11], et al. However, the effect of miR-10a on glioma has not been reported. Our previous studies have demonstrated that miR-10a was involved in metastatic process in hepatoma cells [12]. Thus, we investigated whether miR-10a also contributed to the metastatic behavior of glioma cells. Ephrin receptors comprise the largest known family of receptor tyrosine kinases (RTKs), which interact with their ligands, ephrins. Recently, compelling evidence suggested that expression of ephrin ligands and receptors is often reduced in advanced-stage tumors and regulates tumor growth, metastasis and angiogenesis by altering cell proliferation, motility, invasion and migration [13]. Recent genetic studies suggested that EphA8 was involved in regulating cell adhesion and apoptosis [14,15]. However, reports about the relationship of EphA8 and cell migration and invasion were still few. The process of metastasis consists of a series of steps. Among them, the epithelial–mesenchymal transition (EMT) is commonly characterized by the suppression of the cell–cell adhesion receptor E-cadherin and endows cells with more motile, invasive properties [16]. Accumulating evidence suggested that many miRNAs were involved in EMT [17,18]. Our previous studies have also suggested that miR-10a could promote cell migration and invasion by regulating EMT process in hepatoma [12].
http://dx.doi.org/10.1016/j.febslet.2015.02.005 0014-5793/Ó 2015 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved.
Y. Yan et al. / FEBS Letters 589 (2015) 756–765
In this study, we found that miR-10a promoted the migration and invasion of the human glioma cell lines U251 and U87. We identified EphA8 as a direct target of miR-10a. Furthermore, we found that miR-10a and EphA8 regulated the EMT process to influence the cell migration and invasion. Together, these data contribute to the characterization of the molecular mechanisms of glioma progression. 2. Materials and methods 2.1. Cell culture, transfection and RNA extraction Human glioma cell lines, including U251, U87, A172, U118 and LN18 were cultured at 37 °C with 5% CO2 in DMEM (Gibco, Gaithersburg, USA) supplemented with 10% fetal bovine serum (FBS). Transient transfection was performed using the Lipofectamine 2000 reagent (Invitrogen, Carlsbad, USA). Total RNA was extracted using the Trizol reagent (Invitrogen), and miRNAs were obtained using the mirVana miRNA isolation kit (Ambion, Austin, USA). 2.2. Clinical glioma samples Human glioma samples were collected from the neurosurgery department of Huan Hu Hospital of Tianjin in China from 20 adult patients, freshly resected during surgery. All samples were immediately sectioned from the resected glioma tissues, frozen in liquid nitrogen and stored at 80 °C. There were 8 low-grade (3 from WHO grades I and 5 from WHO grades II) and 12 high-grade tumors (5 from WHO grades III and 7 from WHO grades IV). None of patients had received chemotherapy, immunotherapy and radiotherapy prior to specimen collection. All patients gave written informed consent according to a study protocol that was approved by Tissue Committee and Research Ethics Board of Huan Hu Hospital of Tianjin. 2.3. Vector constructions The vector constructions used in this study was shown in Supplementary materials and methods.
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2.7. Migration and invasion assays For the transwell migration assay, 5 104 U251 or U87 cells were placed in the upper chamber of each insert (Corning, Cambridge, USA). For the invasion assay, 5 104 cells were placed on the upper chamber of each insert coated with 40 ll matrigel (Clontech, Mountain View, CA), which was diluted to 2 lg/ll with DMEM medium. Medium supplemented with 20% FBS (600 ll) was added to the lower chambers. After several hours of incubation (24 h for U251 and U87 in the migration assays; 48 h for U251 and U87 in the invasion assays), the upper surface of the membrane was wiped with a cotton tip and cells attached to the lower surface were stained for 20 min with crystal violet. Cells in five random fields of view at 100 magnification were counted and expressed as the average number of cells per field of view. 2.8. Statistical analysis Data are presented as the mean ± S.D. Statistical analyses were performed using a paired t-test to compare data. A P-value less than 0.05 were considered statistically significant. 3. Results 3.1. Upregulation of miR-10a in human glioma tissues and cell lines To determine whether miR-10a was involved in the tumorigenesis or development of glioma, we firstly examined the expression of miR-10a in clinical tissues and glioma cell lines. Among 20 paired clinical specimens, miR-10a was significantly upregulated in glioma tissues compared to the adjacent non-cancerous tissues. Meanwhile, the miR-10a expression was higher in grade III and IV gliomas compared to grade I and II tumors (Fig. 1A). qRT-PCR was performed on a panel of 5 human glioma cell lines and primary normal human astrocytes (NHA). Remarkable up-regulation of miR-10a could also be observed in glioma cell lines than that of NHA (Fig. 1B). These results suggested that miR10a could be closely related to human glioma and high level of miR-10a might be related to glioma oncogenesis and progression. 3.2. miR-10a promotes glioma cell migration and invasion in vitro
2.4. Bioinformatics analysis The target gene information of miR-10a was analyzed using miRanda, Targetscan, and PicTar. 2.5. EGFP reporter assay Glioma cells were transiently cotransfected with EGFP reporter plasmid and pcDNA3-pri-10a, pcDNA3, ASO-miR-10a and ASO-NC, respectively. The RFP expression vector, pDsRed2-N1, was used as the internal control. Intensities of EGFP and RFP fluorescence were detected with an F-4500 fluorescence spectrophotometer (Hitachi, Tokyo, Japan). 2.6. Quantitative real-time PCR (qRT-PCR) and Western blot analysis Target genes and controls were analyzed by qRT-PCR using SYBR Premix Ex TaqTM (TaKaRa, Dalian, China). Cell lysates were separated on 8% SDS denatured polyacrylamide gel, transferred to nitrocellulose membranes and blocked in phosphate-buffered saline/Tween-20 containing 5% non-fat milk. The membranes were incubated with antibodies. Details and antibodies were under Supplementary materials and methods.
To determine whether miR-10a had an effect on the malignant phenotype of glioma cells, we constructed a miR-10a expression plasmid (pcDNA3-pri-10a, pri-miR-10a) and validated the efficiency of pri-miR-10a and the antisense oligonucleotide of miR-10a (ASO-miR-10a) (Fig. S1). U251 and U87 cells were then transfected with them or their respective controls to explore their effects on the cancer cells. Since invasiveness is one of the pathophysiological features of glioma, we asked whether miR-10a over-expression was associated with the invasiveness of gliomas. Transwell migration and invasion assays showed that the migration (Fig. 2A) or invasion (Fig. 2B) capacities of U251 cells transfected with primiR-10a was increased by approximately 2.7- or 1.7-fold. ASOmiR-10a reduced these capacities by almost 76% or 69% when compared to the controls. The similar results were observed in U87 cells (Fig. 2C and D). These findings indicated that miR-10a promoted both the migration and invasion of glioma cells. 3.3. miR-10a targets the 30 -UTR of EphA8 transcripts and negatively regulates its expression miRNAs influence cell behavior by regulating the expression of target genes. To explore the molecular mechanism through which miR-10a exerts its function in glioma cells, we predicted and iden-
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Fig. 1. miR-10a was up-regulated in human glioma tissues and cell lines. (A) Real-time PCR analysis of miR-10a expression in glioma tissues from different grades and adjacent non-cancerous tissues. Box-plot lines represent medians and interquartile ranges of the normalized threshold values. (B) Real-time PCR analysis of miR-10a expression in several glioma cell lines. U6 was used as loading control. Differences between groups were assessed by one-way ANOVA with the LSD method, ⁄P < 0.05, ⁄⁄ P < 0.01.
tified the candidate target genes of miR-10a. Three algorithms, MiRanda, Targetscan and PicTar were used to predict the potential target gene(s). Considering the associated genes that could account for cell migration or invasion, Eph tyrosine kinase receptor 8, EphA8, was chosen for further study. Base-pairing complementation revealed that the 30 untranslated region (UTR) of EphA8 contains a putative binding region of miR-10a (Fig. 3A). To determine whether EphA8 is a direct target of miR-10a, a series of 30 UTR fragments-including full length 30 UTR, binding site 1 and binding site 2 were constructed and inserted into the region immediately downstream of the luciferase reporter gene. The pri-miR-10a or ASO-miR-10a constructs were then cotransfected with the EGFP reporter vector into U251 cells. Interestingly, EGFP intensities were reduced by almost 73% by miR-10a when the full-length wild-type 30 -UTR of EphA8 was present (Fig. 3B), and when miR-10a expression was blocked, the EGFP intensities were enhanced by approximately 2.2-fold (Fig. 3B). The repressive effect on EGFP activity was abrogated by a mutation in the seed region of the EphA8-30 UTR 1 fragment and partially attenuated by a mutation in the seed region of EphA8-30 UTR 2 fragment (Fig. 3C and D). Similar findings were observed in U87 cells (Fig. S2). These results indicated that these sites were necessary for miR-10a binding. To determine if miR-10a suppressed endogenous EphA8 expression, qRT-PCR and Western blot analysis was used and
demonstrated that over-expression of miR-10a dramatically suppressed the endogenous mRNA and protein levels of EphA8 by 64% and 45%, respectively, whereas the inhibition of miR-10a increased the expression of EphA48 by 2.2- and 1.8-fold in U251 cells (Fig. 3E and F). The similar results were detected in U87 cells (Fig. S2). 3.4. EphA8 is often down-regulated in glioma and suppresses glioma cell migration and invasion We have identified EphA8 as a direct target of miR-10a. To better understand the potential role of EphA8 in miR-10a-mediated migration and invasion, the mRNA level of EphA8 was also measured in 20 paired clinical specimens. The level of EphA8 was significantly down-regulated in glioma tissues when compared with adjacent non-cancerous tissues (Fig. 4A), and was strongly correlated with the up-regulation of miR-10a (Fig. 4B). Furthermore, the mRNA expression of EphA8 was also analyzed in glioma cells (Fig. 4C). These data suggested that EphA8 expression was reduced in glioma and was inversely correlated with miR-10a expression. In order to explore the biological functions of EphA8, we constructed the pSilencer/siR-EphA8 plasmid to knockdown endogenous EphA8 expression and Western blot analysis showed that EphA8 protein expression level was reduced by almost 75% or
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Fig. 2. miR-10a promotes glioma cell migration and invasion in vitro. (A) Transwell migration assay of human glioma cell line U251 cotransfected with either miR-10a or ASO-miR-10a and their control vectors. (B) Transwell invasion assay of U251 cells. (C and D) Transwell migration and invasion assays of human glioma cell line U87. Representative images are shown behind. Cells in five random fields of view at 100 magnification were counted and expressed as the average number of cells per field of view, ⁄⁄P < 0.01.
82% in U251 and U87 cells (Fig. 5A). As expected, knockdown of endogenous EphA8 resulted in a significant increase in cell migration by 3.1- and 3.5-fold (Fig. 5B), and an increase in invasion by 2.6- and 2.8-fold, respectively (Fig. 5C). These results suggested EphA8 could suppress cell migration and invasion of glioma cells. 3.5. Ectopic expression of EphA8 restores the influence of miR-10a in cell migration and invasion The data above indicated that miR-10a could negatively regulate the expression of EphA8 both at the mRNA and protein levels by directly binding to its 30 -UTR and knockdown of EphA8 could mimic the miR-10a over-expression. To further confirm that miR10a promoted the migration and invasion of glioma cells by regulating EphA8 directly, we perfumed a rescue experiment. U251 cells were cotransfected with miR-10a and pA3M1-EphA8, which encoded the entire EphA8 coding sequence but lacked the 30 -UTR. Western blot analysis was used to validate the EphA8 expression in the rescue experiment (Fig. 6A). As expected, the restoration of EphA8 inhibited the miR-10a-promoted migration and invasion (Fig. 6B). Similar data was detected in U87 cells (Fig. 6C). Taken together, these findings suggested that miR-10a promoted glioma cell migration and invasion by directly targeting EphA8. 3.6. miR-10a and EphA8 affect glioma cell migration and invasion by regulating of the EMT process The above observations suggested that miR-10a could promote glioma cell migration and invasion and exerted its function by directly regulating EphA8, we further investigated the mechanism by which miR-10a and EphA8 regulate the cell malignant
phenotype. We noticed a striking change in cellular shape due to the miR-10a inhibition or EphA8 over-expression, an initial fibroblast-like morphology was observed to switch to the cobblestone-like appearance of epithelial cells. To determine whether the typical molecular alternations of EMT occurred, the protein levels of epithelial (E-Cadherin) and mesenchymal (Vimentin and ICAM-1) markers were measured. As shown in Fig. 7A, EphA8 expression was increased by 1.5- or 2.8-fold after transfection of ASO-miR-10a or pA3M1-EphA8 plasmid in U251 cells. E-Cadherin protein expression was up-regulated by 1.1-fold, whereas Vimentin and ICAM-1 were down-regulated by 62% and 78% respectively when miR-10a was blocked (Fig. 7B) in U251 cells. In addition, similar results were observed when EphA8 was over-expressed (Fig. 7C). Furthermore, similar observations were obtained in U87 cells (Fig. S3). The above findings suggested that miR-10a and its target gene, EphA8, could affect glioma cell migration and invasion by regulating of EMT process. Above all, our findings indicate that miR-10a promotes, whereas EphA8 suppresses glioma cell migration and invasion in vitro. miR-10a exerts its function by directly repressing EphA8. miR10a and EphA8 affect glioma cell malignant phenotype by regulating of the EMT process. These observations proved a new miR10a/EphA8 regulation pathway of cell migration and invasion in glioma. 4. Discussion Diffusely infiltrating gliomas are one of the most devastating cancers because they often show locally aggressive behavior and cannot be cured by existing therapies [19]. Gliomas develop as a result of genetic alterations that accumulate throughout tumor progression [20]. Therefore, the elucidation of these molecular
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Fig. 3. miR-10a targets the 30 -UTR of EphA8 and down-regulates its expression. (A) The sequence of the predicted miR-10a binding site and the EphA8 30 -UTR segments containing the wild-type or mutant binding site are shown. The protein binding sequences for miR-10a within the EphA8 30 -UTR of human, chimpanzee, mouse, rat and guinea pig are also shown. Seed sequences are highlighted. (B) Relative EGFP activity was analyzed after the wild-type full-length EphA8-30 UTR plasmid was cotransfected with miR-10a or ASO-miR-10a in U251 cells. (C) EGFP intensity was analyzed after the mutant 30 -UTR-1 reporter plasmids was cotransfected with miR-10a or ASO-miR-10a. (D) EGFP intensity was analyzed after the mutant 30 -UTR-2 reporter plasmids was cotransfected with miR-10a or ASO-miR-10a. The histogram shows the mean ± S.D. of the normalized EGFP intensity. (E) Real-time PCR analysis was used to detect the mRNA level of EphA8 in transiently transfected U251 cells. Normalization was performed using b-actin. (F) Western blot analysis was used to detect the protein level of EphA8 in transiently transfected U251 cells. Normalization was performed using GAPDH, ⁄P < 0.05, ⁄⁄ P < 0.01.
mechanisms, in particular the ones associated with cellular migration and invasion is crucial for a better prediction of glioma patients outcome and response to therapies [21]. Recent studies have shown that miRNAs play a fundamental role in the migration and invasion of glioma. For example, miR-204 inhibits glioma cell migration and invasion by negatively regulating ezrin gene
expression [22]. MiR-18a regulates the proliferation, migration and invasion of human glioblastoma cell by targeting neogenin [23]. MiR-10a, which was studied in many research was involved in regulating the migration and invasion of many kinds of cancers, such as cervical and gastric cancers [24,25]. Our previous studies also suggested that miR-10a could promote cell migration and
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Fig. 4. EphA8 is often down-regulated in glioma tissues and cell lines. (A) Real-time PCR was used to detect the mRNA levels of EphA8 in clinical specimens. Box-plot lines represent medians and interquartile ranges of the normalized threshold values. The EphA8 abundance was normalized to b-actin. (B) Correlation between expression level of EphA8 and that of miR-10a in 20 matched tissues. Linear regression coefficient and statistical significance is indicated. (C) Real-time PCR was used to detect the mRNA levels of EphA8 in glioma cell lines, ⁄⁄P < 0.01.
invasion of hepatoma and cervical cancer cells [12,26]. However, effect of miR-10a on glioma has not been reported. In this study, miR-10a, a new regulator of glioma, was shown to promote migration and invasion of U251 and U87 glioma cells. To further explore the mechanism by which miR-10a exerts its function, we used bioinformatics analysis to predict and finally identified EphA8 as the direct target of miR-10a. This conclusion is based on several experimental evidences. First, miR-10a significantly decreases the expression of EphA8 both at the mRNA and protein levels in glioma cells. Second, the EGFP reporter assay showed that the fluorescence intensity of EGFP-EphA8-30 UTR was specifically responsive to miR-10a over-expression, however, mutation of the miR-10a binding site abolished the effect of miR-10a on the
regulation of EGFP fluorescence intensity. Third, EphA8 expression is down-regulated in HCC tissues, and this down-regulation is strongly correlated with the up-regulation of miR-10a. Fourth, knockdown of EphA8 phenocopies the effect of miR-10a expression, whereas restoration of EphA8 counteracts the function of miR-10a. These results indicate that miR-10a targets EphA8 and down-regulates its expression in glioma. Ephrin receptors comprise the largest known family of receptor tyrosine kinases (RTKs), with 16 Eph receptors and 9 ephrin ligands [27–29]. Eph receptors and ligands are divided into either A-class or B-class, and were initially implicated in cell positioning and migration during development, particularly of the nervous system. Until now, reports have suggested that many Eph receptors are involved
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Fig. 5. EphA8 suppresses the migration and invasion of glioma cells in vitro. (A) Western blot analysis of EphA8 protein level in U251 and U87 cells transfected with pSilencer/ shR-EphA8 or pSilencer plasmids. (B) Transwell migration analysis of glioma cells when EphA8 was inhibited. (C) Transwell invasion analysis of glioma cells when EphA8 was inhibited. Cells in five random fields of view at 100 magnification were counted and expressed as the average number of cells per field of view, ⁄⁄P < 0.01.
in regulating proliferation, migration, invasion and adhesion of glioma cells [30–34]. In this study, transwell assay demonstrated that knockdown of EphA8 could promote cell migration and invasion of glioma which was in accordance with miR-10a over-expression. Ectopic expression of EphA8 without the 30 UTR rescued the promotion of migration and invasion in U251 and U87 cells caused by miR-10a. As mentioned above, EMT is a process in which epithelial cells lose or modify their apical–basal polarity during embryonic development and tumor metastasis [35,36]. Our previous study
has suggested that miR-10a could affect cell malignant behavior via regulating the EMT process [12]. Barrios et al. [37] have indicated that one of the RTKs family members, EphA4, regulates the mesenchymal-to-epithelial transition (MET) of the paraxial mesoderm during somite morphogenesis. We speculated that miR-10a and EphA8 played roles in the EMT process in glioma. We examined the expression of the epithelial marker E-cadherin and the mesenchymal markers Vimentin and ICAM-1. Because miR-10a expression was increased whereas EphA8 was decreased in cancer tissues and glioma cells, we blocked miR-10a expression
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Fig. 6. Ectopic expression of EphA8 restores the influence of miR-10a in cell migration and invasion. (A) Western blot analysis of EphA8 expression in U251 and U87 cells with or without EphA8 restoration. (B) Restoration of EphA8 inhibits miR-10a-promoted glioma cell migration and invasion in U251 cells. (C) Restoration of EphA8 inhibits miR10a-promoted glioma cell migration and invasion in U87 cells. Cells in five random fields of view at 100 magnification were counted and expressed as the average number of cells per field of view, ⁄⁄P < 0.01.
by ASO-miR-10a and over-expressed EphA8 to detect the related molecular markers. As expected, inhibition of miR-10a or over-expression of EphA8 suppressed the EMT phenotype. In other words, miR-10a can increase, whereas EphA8 can suppress cell migration and invasion of glioma by mediating the EMT process.
In summary, we discovered that miR-10a can promote cell migration and invasion of glioma and exerts its function by directly targeting EphA8, thereby regulating EMT process. Our research thus provides a new insight into the mechanism of the pathogenesis of glioma and suggests miR-10a and EphA8 play important roles in progression of glioma.
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Fig. 7. miR-10a and EphA8 affect glioma cell migration and invasion via regulating of the EMT process. (A) Western blot analysis of EphA8 protein level when miR-10a expression was inhibited or EphA8 was over-expressed in U251 cells. (B) The protein expression levels of E-Cadherin, Vimentin and ICAM-1 when miR-10a was inhibited in U251 cells. (C) The protein expression levels of epithelial and mesenchymal markers mentioned above when EphA8 was over-expressed in U251 cells, ⁄⁄P < 0.01.
Acknowledgments We thank the Public Health College of Tianjin Medical University for their technical assistance in the fluorescence studies. This work was supported by the National Nature Science Foundation of China (No. 81301967). Appendix A. Supplementary data Supplementary data associated with this article can be found, in the online version, at http://dx.doi.org/10.1016/j.febslet.2015.02. 005.
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