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MicroRNA-10b induces glioma cell invasion by modulating MMP-14 and uPAR expression via HOXD10
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available at www.sciencedirect.com
www.elsevier.com/locate/brainres
Research Report
MicroRNA-10b induces glioma cell invasion by modulating MMP-14 and uPAR expression via HOXD10 Lihua Suna,b,1 , Wei Yana,1 , Yingyi Wanga,1 , Guan Sunc,1 , Hui Luoa , Junxia Zhanga , Xiefeng Wanga , Yongping Youa , Zhengxiang Yangb,⁎, Ning Liua,⁎ a
Department of Neurosurgery, the First Affiliated Hospital of Nanjing Medical University, Nanjing 210029, PR China Department of Neurosurgery, the Affiliated Wuxi People's Hospital of Nanjing Medical University, Wuxi, 214023, PR China c Department of Neurosurgery, Fourth Affiliated Hospital of Nantong University, First Hospital of Yancheng, Yancheng, 224005, PR China b
A R T I C LE I N FO
AB S T R A C T
Article history:
MicroRNAs are small endogenous noncoding RNAs, which modulate target gene expression
Accepted 3 March 2011
by binding with target mRNA sequences in the 3′untranslated region (UTR) with an
Available online 16 March 2011
imperfect complementarity that inhibits the mRNA translation. Many microRNAs have been reported to function as tumor oncogenes or anti-oncogenes. Recently, more and more
Keywords:
microRNAs have been reported to contribute to a tumor's invasive potential. Here, we show
Glioma
that microRNA-10b (miR-10b) was over-expressed in glioma samples and directly associated
MicroRNA
with the glioma's pathological grade and malignancy. We also found that miR-10b induced
Invasion
glioma cell invasion by modulating tumor invasion factors MMP-14 and uPAR expression via
HOXD10
the direct target HOXD10. The miR-10b/HOXD10/MMP-14/uPAR signaling pathway might
MMP-14
contribute to the invasion of glioma. Accordingly, glioma cells lost their invasive ability
uPAR
when treated with specific antisense oligonucleotides (miR-10b inhibitors), suggesting that miR-10b could be used as a new bio-target to cure glioma. © 2011 Elsevier B.V. All rights reserved.
1.
Introduction
Glioma, the most common primary malignant neoplasm of the central nervous system (CNS), has a highly invasive malignant behavior and recurrence rate, and it remains one of the tumors most refractory to treatment. Recently, some studies reported that miRNAs play essential roles in tumor invasion and migration (Asangani et al., 2008; Hiyoshi et al., 2009; Huang et al., 2008; Ma et al., 2007; Sasayama et al., 2009; Shi et al., 2008; Weiss et al., 2009). Among these miRNAs, miR10b was first reported to induce breast cancer cell invasion and
metastasis, which was also found to be associated with tumor invasive potential in hepatic cancer (Ladeiro et al., 2008), pancreatic cancer (Bloomston et al., 2007), glioma (Sasayama et al., 2009), esophageal cancer (Tian et al., 2010) and neurofibromatosis (Chai et al., 2010). Ma et al. (2007) found that miR-10b could modulate invasion-associated protein Rhoc expression by directly target HOXD10 gene and Sasayama et al. (2009) reported that miR-10b level was associated with tumor invasive factors, uPAR and RhoC. But the exact function of miR-10b in glioma is still unknown. Under this condition, we first measured miR-10b levels in
⁎ Corresponding authors. N. Liu is to be contacted at Department of Neurosurgery, the First Affiliated Hospital of Nanjing Medical University, Nanjing 210029, PR China. Z. Yang, Department of Neurosurgery, the Affiliated Wuxi People's Hospital of Nanjing Medical University, Wuxi, 214023, PR China. Fax: +86 25 83716602. E-mail addresses: [email protected] (N. Liu), [email protected] (Z. Yang). 1 These authors contributed equally to this article. 0006-8993/$ – see front matter © 2011 Elsevier B.V. All rights reserved. doi:10.1016/j.brainres.2011.03.013
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different grade glioma samples and cell lines. Then, we used loss of function and recovery experiments to determine the function of miR-10b in glioma cell lines; furthermore, we also investigated that whether miR-10b direct binding to its target HOXD10. Additionally, tumor invasive factors MMP-14 and uPAR were also measured in our studies.
2.
Results
2.1. MiR-10b is over-expressed in glioma samples and cell lines, miR-10b expression is associated with glioma pathological grade and malignancy First, we used TaqMan®MicroRNA assay-based real-time RT– PCR to analyze miR-10b levels in 22 glioma samples and 4 well known glioma cell lines: U87, U251, LN229 and U373. Six normal brain tissues were considered as control. As shown in Fig. 1A, each glioma sample expressed higher miR-10b level than the normal controls. Furthermore, miR-10b expression in the glioma group was significantly higher than in the normal
brain group (p < 0.01) (Fig. 1B). Then, we divided all glioma samples into 3 groups according to their pathological diagnosis. Among them, 8 were diagnosed as WHO-II grade, 8 were WHO-III and 6 were WHO-IV. We found that miR-10b levels were all up-regulated in these 3 groups compared to the normal brain group (p < 0.05) and miR-10b expression was positively correlated with the tumor's grade. In WHO-IV, miR10b expression was higher than WHO-III and WHO-II (p < 0.01). However, there was no statistical difference between WHO-III and WHO-II (p = 0.093) (Fig. 1C). We also found that miR-10b expression in the 4 cell lines were all higher than in normal brain tissues (p < 0.01). As shown in Fig. 1D, the miR-10b level was the highest and lowest in the U87 cell line (94.79 fold compare to the normal control) and U251 cell line (18.40 fold compare to the normal control), respectively.
2.2. MiR-10b level is down-regulated/up-regulated by miR-10b inhibitors/mimics To determine whether miR-10b inhibitors/mimics could function in vitro, U87 and LN229 cell lines, which expressed
Fig. 1 – MiR-10b is over-expressed in glioma samples and cell lines. (A) The relative levels of miR-10b in 22 glioma samples were measured by using real-time quantitative RT–PCR. Six normal brain tissues were counted as control. (B) MiR-10b is up-regulated in the glioma group compared to the normal brain group. Student's t test was used to analyze the significant differences between the two groups. (C) Twenty-two glioma samples were divided into three groups according to the pathology diagnosis. One-way ANOVAs were used to analyze the significant differences among the groups. (D) The relative levels of miR-10b in the four glioma cell lines U251, U373, LN229 and U87. Student's t test was used to analyze the significant differences. Error bars mean standard deviation (SD) of triplicate independent experiments. *p < 0.05, **p < 0.01 compared to the control.
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higher levels of miR-10b, were selected and transfected with miR-10b inhibitors. The U251 cell line, which showed lower level of miR-10b, was chosen for transfection with miR-10b mimics. We analyzed miR-10b levels after transfection with the miR-10b inhibitors/mimics 48 h post transfection and found that miR-10b levels were significantly reduced by the miR-10b inhibitors (about 65.28% down-regulated compared to the control in the U87 cells and 54.17% in the LN229 cells) and elevated by the miR-10b mimics (about 20.36 fold compared to the control in the U251 cells) (Fig. 2).
2.3.
MiR-10b promotes glioma cell invasion
To investigate whether miR-10b could enhance glioma cell invasion, transwell assays were performed. First, we performed loss-of-function analysis and transfected miR-10b inhibitors into U87 and LN229 cell lines to reduce miR-10b expression. The results showed that the inhibition of miR-10b expression led to a 59.67% reduction in invasion compared to the control in U87 cells (Fig. 3A and D) and 43.29% in LN229 cells (Fig. 3B and E). Then, we used miR-10b mimics to up-regulate the level of miR10b in U251 cells, which led to a 1.82 fold increase in invasion (Fig. 3C and F). Additionally, both the miR-10b inhibitors and miR-10b mimics had no effect on cell apoptosis or cell cycle (Fig. 4).
This was in contrast to U251. In cell lines, the level of miR-10b was inversely related to the expression of HOXD10 protein (r = 0.94, p < 0.05) indicating that miR-10b might suppress HOXD10 gene expression (Fig. 5A and B). To test this hypothesis, we used miR-10b inhibitors to silence miR-10b expression in U87 and LN229 cells and found that HOXD10 protein levels were significantly increased by miR-10b inhibition compared to the blank control and scramble control. We also used miR-10b mimics to up-regulate miR-10b expression in U251 cells, and as a result, we found that HOXD10 protein expression was decreased by the miR-10b mimics (Fig. 5C and E). Tumor invasive factors MMP-14 and uPAR have been reported to be directly regulated by HOXD10 (Myers et al., 2002). To further investigate the signaling pathway, we measured MMP-14 and uPAR levels after transfection with HOXD10 vectors in U87 and LN229 cells. Expression of both genes was decreased by the HOXD10 vectors (Fig. 5D), which indicated that MMP-14 and uPAR were regulated by HOXD10 in glioma cell lines. Then, we examined the levels of MMP-14 and uPAR after transfection with miR-10b inhibitors in the U87 and LN229 cells; the results showed that both of them were downregulated by the miR-10b inhibitors, whereas transfection with the miR-10b mimics in U251 cells could up-regulate MMP-14 and uPAR expression levels (Fig. 5E).
2.5. 2.4. MiR-10b modulates invasion factors MMP-14 and uPAR expression via the target HOXD10 gene HOXD10 has been reported to be regulated by miR-10b in human breast cancer (Ma et al., 2007) and esophageal cancer (Tian et al., 2010) cell lines. To investigate whether miR-10b can modulate HOXD10 expression in glioma cell lines, we first examined the levels of HOXD10 protein in the four glioma cell lines. Interestingly, U87 cells which expressed the highest level of miR-10b showed the lowest level of HOXD10 protein.
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HOXD10 is a direct target of miR-10b
To verify whether miR-10b directly targeted HOXD10 in glioma cell lines, luciferase reporter assays were conducted. We constructed p3′UTR-HOXD10 and p3′UTR-mut-HOXD10-with a substitution of four nucleotides within the miR-10b binding site (Fig. 6A). We then cotransfected luciferase reporters and miR-10b expressing vectors into U87 and U251 cells. We found that cotransfection with p3′UTR-HOXD10 reporters and miR10b expressing vectors in U87 cells led to a significant decrease (45% decreased, p < 0.01) in the luciferase activity compared to
Fig. 2 – MiR-10b is reduced/elevated by miR-10b inhibitors/mimics after transfection. (A) U87 cells were seeded into 6-well plates, cells of 70% confluence were transfected with miR-10b inhibitors. Relative levels of miR-10b were analyzed 48 h after transfection. (B) LN229 cells of 70% confluence were transfected with miR-10b inhibitors. After 48 h, the relative levels of miR-10b were measured. (C) U251 cells of 70% confluence were transfected with miR-10b mimics, and miR-10b expression was also analyzed 48 h later after transfection. Error bars mean standard deviation (SD) of triplicate independent experiments. *p < 0.05, **p < 0.01 compared to the blank control.
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Fig. 3 – MiR-10b contributed to glioma cell invasion. (A and D) U87 cells, which were transfected with miR-10b inhibitors for 48 h later showed lower invasive potential (the reduction of cell number was about 59.67% in invasion assays) compared to the blank control. (B and E) LN229 cells, which were also transfected with the miR-10b inhibitors, showed 43.29% reduction in the number of invasive cells compared to the blank control. (C and F) U251 cells, which were transfected with the miR-10b mimics for 48 h, showed a higher invasive potential (the increasing cell number is about 1.82 fold in invasion assays) compared to the blank control. Error bars mean standard deviation (SD) of triplicate independent experiments. **p < 0.01 compared to the blank control.
the blank control (but not p3′UTR-mut-HOXD10) (Fig. 6B). In U251 cells, a similar effect was also found (51% decrease compared to the blank control, p < 0.01) (Fig. 6C).
3.
Discussion
MiRNAs are a class of small noncoding RNAs. Recently, a growing number of miRNAs have been found to be either
activators or suppressors of tumor invasion and metastasis (Shi et al., 2008; Tavazoie et al., 2008; Zhang et al., 2010; Zhu et al., 2008). Ma et al. (2007) has showed that TWIST1, which is a metastasis-promoting transcription factor, could modulate miR-10b expression by binding to the upstream portion of the miR-10b hairpin region. Moreover, miR-10b inhibited the translation of mRNA encoding homeobox D10 (HOXD10), which further regulated RHOC, a pro-metastasis gene belonging to the Ras homolog gene family. Therefore, the TWIST1-
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Fig. 4 – MiR-10b has no effect on cell apoptosis or cell cycle in glioma cell line. (A) U87 cell apoptosis rate and cell cycle was analyzed on FACScan by flow cytometry 48 h later with miR-10b inhibitors treatment; no differences were detected among the blank control group, scramble group and miR-10b inhibitors group. Annexin V− and PI− cell was used as control, Annexin V+ and PI− cell was considered as apoptotic while Annexin V+ and PI+ cell was designated as necrotic. (B) U251 cells were transfected with miR-10b mimics, after treatment 48 h, cell apoptosis assay and cell cycle analysis were done, and there are no differences among the blank control group, scramble group and miR-10b mimics group. Each independent experiment was done three times.
miR-10b–HOXD10–RHOC pathway was crucial for breast cancer cell invasion and metastasis. They also found that miR-10b antagomirs—a class of chemically modified antimiRNA oligonucleotides—could suppress breast cancer metastasis. Administration of miR-10b antagomirs to mice bearing highly metastatic cells could not reduce primary mammary tumor growth, but it markedly suppressed the
formation of lung metastases (Ma et al., 2010). Tumor invasion and metastasis is a complex and multi-step process: thus, miR-10b may play roles in different steps via different targets. More and more direct targets of miR-10b have been found. For example, miR-10b could promote human esophageal cancer cell migration and invasion by inhibiting KLF4 mRNA translation (Tian et al., 2010). MiR-10b was also identified to target
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Fig. 5 – MiR-10b regulates invasion factors MMP-14 and uPAR expression via target HOXD10 gene. (A and B) The relative expression levels of HOXD10 protein in four glioma cell lines; HOXD10 protein level in U87 cell line was considered as the control. The relationship between miR-10b expression and HOXD10 protein level was examined by using Pearson's correlation analysis. Error bars represent standard deviation (SD) of triplicate independent experiments. (C) Western Blot showed that HOXD10 protein level was significantly up-regulated while uPAR and MMP-14 levels were down-regulated by HOXD10 expressing Vectors both in U87 and LN229 cells. (D) Western blot showed that miR-10b inhibitors could enhance HOXD10 protein expression but significantly reduce HOXD10 downstream genes MMP-14, uPAR expression in U87 and LN229 cells. While miR-10b mimics has the opposite function, which could promote MMP-14 and uPAR expressing via decreasing HOXD10 protein expression.
neurofibromatosis type 1 (NF1) mRNA to activate RAS signaling in neurofibromin and to affect malignant peripheral nerve sheath tumor (MPNST) cell migration and invasion (Chai et al., 2010). However, there exists an opposing view that miR-10b directly targets T lymphoma invasion and metastasis 1 (Tiam1) and inhibited Tiam1-mediated Rac activation to suppress the ability of breast carcinoma cells to migrate and invade (Moriarty et al., 2010). MiR-10b was first found to be down-regulated in breast cancer (Iorio et al., 2005) and further confirmed by others (Gee et al., 2008), but Ma et al. (2007) found that miR-10b was upregulated in metastatic breast cancer. Although there exists some debate on whether miR-10b was up-regulated in breast cancer, there is no conflict on whether miR-10b was over-
expressed in glioma. Ciafre et al. (2005) first reported that the most up-regulated miRNA in glioblastoma was miR-10b by using microarray analysis and Northern blot, which was further confirmed by Sasayama et al. (2009) by real-time RT–PCR. They also identified that the level of miR-10b was associated with glioma maligancy and correlated with the expression of tumor invasion factors uPAR and RhoC. However, the mechanism of miR-10b-induced glioma cell invasion was still unknown. Thus, we first measured the levels of miR-10b in 22 glioma samples, 6 normal brain and 4 glioma cell lines. The results showed that miR-10b was over-expressed in all glioma samples compared to normal brain control, which complied with studies on human pancreatic adenocarcinomas (Bloomston et al., 2007) and esophageal cancer (Tian et al., 2010). We also found that miR-
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Fig. 6 – MiR-10b directly targets the 3′UTR of HOXD10 mRNA in glioma cell line. (A) Upper panel: the predicted binding sites between miR-10b and HOXD10 3′UTR in humans. Lower panel: the plasmid with the full length of HOXD10 3′UTR (p3′UTR-HOXD10) and the plasmid with a mutant HOXD10 3′UTR (p3′UTR-mut-HOXD10) which carried a substitution of four nucleotides with the miR-10b binding site. (B) U87 cells were cotransfected with miR-10b-expressing/blank vectors and p3′UTR-HOXD10/p3′UTR-mut-HOXD10 reporters. After 48 h, luciferase activities were measured. (C) U251 cells were also cotransfected with vectors and reporters, and similar results were observed. Error bars depict standard deviation (SD) of triplicate independent experiments. **p < 0.01 compared to the blank.
10b expression in WHO-IV was much higher than the other grades. In glioma cell lines, miR-10b levels were all higher than the normal control. All of this evidence indicated that miR-10b might contribute to a glioma's malignancy. To further verify that miR-10b could induce glioma cell invasion, we used miR-10b inhibitors, which could decrease miR-10b expression, and miR-10b mimics, which could increase miR-10b levels. When transfected with the miR-10b inhibitors, U87 and LN229 cells lost their invasion potential while U251 cells' invasive ability was enhanced by the miR-10b mimics. uPA is a specific serine protease that can convert plasminogen to plasmin and directly bind to its specific receptor uPAR, thereby promoting cell-surface plasmin activation and localization of degradation of the extracellular matrix to the migrating tumor cell surface. uPAR has been shown to be over-expressed in glioma (Salajegheh et al., 2005; Yamamoto et al., 1994), and high levels of uPA and uPAR were correlated with an invasive phenotype of glioma cells (Mohanam et al., 1993). Increased signaling pathways inducing glioma cell invasion were found through uPA and uPAR (Bryan et al., 2008; Kunigal et al., 2006; Young et al., 2009), while down-regulation of uPA and uPAR inhibited glioma cell invasion (Gondi et al., 2004a; Gondi et al., 2004b; Gondi et al., 2004c). MMP-14 is a number of the membrane-type matrix metalloproteinase (MT-MMP) subfamily involved in the breakdown of extracellular matrix in physiological or pathological processes. MMP-14 was found to be over-
expressed in neuroblastoma and increased with an increase in the extent of invasive depth and distant metastasis (Dong et al., 2010). MMP-14 inhibitor could effectively inhibit cancer cell migration and invasion in vitro (Suojanen et al., 2009). Both uPAR and MMP-14 were reported to be regulated by HOXD10 (Myers et al., 2002), a sequence-specific transcription factor, whose expression is progressively reduced in epithelial cells as malignancy increases in both breast and endometrial tumors. Retroviral gene transfer to restore expression of HOXD10 significantly impaired cell migration (Carrio et al., 2005) suggesting that HOXD10 may play a role as a suppressor of tumor invasive growth. To explore the molecular mechanism of miR-10b's function in glioma cell lines, we transfected HOXD10 expressing vectors into U87 and LN229 cells. Western blot showed that uPAR and MMP-14 were inactivated by HOXD10 gene. We then transfected miR-10b inhibitors into U87 and LN229 cells and found that HOXD10 protein levels were elevated while uPAR and MMP-14 levels were decreased. The transfection of miR-10b mimics into U251 cells resulted in an opposite effect. Finally, luciferase reporter assays certified that the HOXD10 gene was a direct target of miR-10b. Thus, we thought that miR-10b induced glioma cell invasion by modulating MMP-14 and uPAR expression via directly target the HOXD10 gene. The TWIST1/miR-10b/HOXD10/MMP-14, uPAR signaling pathway might be essential for glioma cells invasion. However, in the same way that miR-10a targets many genes (Fang et al.,
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2010; Han et al., 2007; Orom et al., 2008; Tan et al., 2009; Weiss et al., 2009; Woltering and Durston, 2008), the other target genes of miR-10b in glioma and their function need further investigation. Our studies also showed that glioma cells lost their invasive potential by using miR-10b antisense inhibitors, providing a new bio-target for glioma treatment. Because the function of a particular miRNA is often tissue specific, each miRNA may target many mRNAs, and the target genes themselves may have differential or even opposite effects in different tumors; therefore, using miR-10b antisense inhibitors for anti-metastasis treatment still warrant further investigation.
4.
Statistical analysis
All experiments were done three times and statistical analysis was performed on SPSS Graduate Pack 13.0 statistical software. Descriptive statistics including mean and SD. Student's t test and one-way ANOVAs were used to analyze significant differences. Pearson's correlation analysis was used to examine the relationship between miR-10b expression and HOXD10 protein levels. p<0.05 was considered to be statistically significant.
5.
Experimental procedures
5.1.
Cell lines and cell culture
Human glioma cell lines, U87, LN229, U251 and U373 were purchased from the Chinese Academy of Sciences Cell Bank; all cell lines were maintained in a 37 °C, 5% CO2 incubator in DMEM supplemented with 10% fetal bovine serum (FBS).
5.2.
Glioma samples and normal brain tissues
After informed consent patients diagnosed with glioma was obtained, human glioma samples were collected from the department of neurosurgery, the first affiliated hospital of Nanjing Medical University. Among the 22 samples, 8 samples were diagnosed as WHO-II grade, 8 were WHO-III and 6 were WHO-IV by pathological diagnosis. Six normal brain tissues were obtained after informed consent from patients with severe traumatic brain injury who required post-trauma surgery. After resection, all samples were immediately frozen in liquid nitrogen until RNA extraction.
5.3.
RNA isolation
Total RNA was extracted from tissues and four glioma cell lines by using the TRIzol reagent (Invitrogen, Carlsbad, USA). The amount of RNA was quantified by absorbance reading at A260/ A280= 1.6–1.8 and stored at −80 °C until use.
5.4.
Real-time quantification PCR
To detect the relative levels of miR-10b in glioma samples and cell lines, TaqMan-based real-time reverse transcription– polymerase chain reaction (RT–PCR) assay was used. The
primers and probes of has-miR-10b (P/N 4395329) and RNU6B (P/N 4373381) endogenous control for TaqMan miRNA assays were purchased from Applied Biosystems. Real-time PCR was performed according to the manufacturer's instructions on the ABI 7300 HT Sequence Detection System (Applied Biosystems, CA). The relative level of miR-10b was normalized to the RUN6B, and the quantitative miR-10b expression date was calculated by using 2−△△Ct method.
5.5.
Cell transfection
2′-O-methyl (2′-O-Me) hsa-miR-10b inhibitors (miR-10b antisense oligonucleotides) and mimics (miR-10b sense oligonucleotides) were chemically synthesized by Shanghai GenePharma Company (Shanghai, China). The 2′-O-Me-has-miR-10b inhibitor sequence was 5′-CACAAAUUCGGUUCUACAGGGUA-3′, and the scramble sequence was 5′-CAGUACUUUUGUGUAGUACAA-3′. The 2′-O-Me-has-miR-10b mimic sequence was 5′-UACCCUGUAGAACCGAAUUUGUG-3′, 3′-CAAAUUCGGUUCUACAGGGUAUU-5′, and the scramble sequence was 5′UUCUCCGAACGUGUCACGUTT-3′, 5′-ACGUGACACGUUCGGAGAATT-3′. Cells of 70%–80% confluence were transfected with oligonucleotide using Lipofectamine 2000 reagent (Invitrogen, Carlsbad, USA). Transfection was performed according to the manufacturer's instructions, and the final oligonucleotide concentration was 10 nmol/L. Transfection medium was replaced 6 h later. MiR-10b expressing vectors and blank vectors, HOXD10 vectors and mut-HOXD10 vectors (Genesil, Wuhan, China) were constructed by Wuhan Genesil. Vectors were transfected into human glioma cell lines with FuGENE HD6 (Roche, Basel, Switzerland) according to the manufacturer's instructions and screened by the aminoglycoside G418.
5.6.
Invasion assay in vitro
For transwell assay, 5 × 104 cells were plated into 24-well Boyden chambers (Coring costar, MA, USA), with an 8-μm pore polycarbonate membrane, which was coated with 20 μg of Matrigel (BD Biosciences, San Jose, CA). Cells were in the upper chamber with 200 μl of serum-free medium, and medium containing 20% FBS was added to the lower chamber to serve as a chemoattractant. After 24 h, the cells were washed 3 times with PBS, non-invasive cells were removed from the upper well by cotton swabs, and the invasive cells were then fixed with 4% paraformaldehyde, stained with 0.1% crystal violet, and photographed (×200) in six independent fields for each well. The results were averaged through three independent experiments.
5.7.
Apoptosis assay
First, 1 × 106 cells were plated into 6-well plates, and, after transfection with each oligonucleotide for 48 h, the apoptosis assay was conducted using Annexin V-FITC double stained detection kit (BD Biosciences, San Jose, CA). Annexin V− and PI− cells were used as controls. Annexin V+ and PI− cells were considered as apoptotic, and Annexin V+ and PI+ cells were designated as necrotic.
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5.8.
Cell cycle analysis
After each treatment for 48 h, cells in the log phase of growth were collected, washed with PBS for 3 times, and then fixed with 75% ethanol at −20 °C for at least 1 h. After extensive washing with PBS, the cells were suspended in HBSS containing 50 μg/ml of RNaseA (Boehringer Mannheim, Indianapolis, IN) and 50 μg/ ml of PI (Sigma-Aldrich), incubated for 1 h at room temperature and then analyzed by FACScan (Becton Dickinson, San Jose, CA).
5.9.
Luciferase reporter assay
The full length 3′UTR region of the HOXD10 gene was subcloned into luciferase reporter vectors from human genomic DNA and the mutant HOXD10 vectors were constructed, which carried a substitution of four nucleotides at the miR-10b binding sites. Cells of 60%–70% confluence in 24well plates were cotransfected with luciferase reporter vectors and miR-10b expressing vectors, and a 1 ng pRLSV40 Renilla luciferase construct was used for normalization. After 48 h, luciferase activity was analyzed by the Dual-Luciferase Reporter Assay System according to the manufacturer's protocols (Promega, Madison, USA).
5.10.
Western blot
Forty-eight hours after transfection, total protein was collected using a Total Protein Extraction Kit (KeyGen, China) and quantified using a BCA Protein Assay Kit (KeyGen, China). Twenty micrograms of protein were added and subjected to SDS–PAGE on (SDS)–polyacrylamide gel electrophoresis using the discontinuous buffer system of Laemmli (Bio-Rad Laboratories, USA). The electrophoresed proteins were transferred to a polyvinylidene difluoride (PVDF) membrane and subjected to immunoblot analysis with a goat primary antibody against HOXD10 (Santa Cruz, USA, 1:200), mouse against uPAR (Santa Cruz, USA, 1:200), rabbit against MMP-14 (R&D systems, USA, 1:2000), followed by HRP-conjugated rabbit anti-goat secondary antibodies (Bioworld Technology, USA, 1:5000), goat antimouse secondary antibodies (MultiSciences Biotech, China, 1:5000) and goat anti rabbit secondary antibodies (MultiSciences Biotech, China, 1:5000). GAPDH (KangCheng, China, 1:2000) was used as control.
Acknowledgments This work is supported by the China Natural Science Foundation (proj. no. 30872657), Jiangsu Province's Medical Major Talent Foundation (proj. no. RC2007061) and Jiangsu Province's “333” Key Talent Foundation (proj. no. 0508RS08).
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and stimulates invasion, intravasation and metastasis in colorectal cancer. Oncogene 27, 2128–2136. Bloomston, M., et al., 2007. MicroRNA expression patterns to differentiate pancreatic adenocarcinoma from normal pancreas and chronic pancreatitis. JAMA 297, 1901–1908. Bryan, L., et al., 2008. Sphingosine-1-phosphate and interleukin-1 independently regulate plasminogen activator inhibitor-1 and urokinase-type plasminogen activator receptor expression in glioblastoma cells: implications for invasiveness. Mol. Cancer Res. 6, 1469–1477. Carrio, M., et al., 2005. Homeobox D10 induces phenotypic reversion of breast tumor cells in a three-dimensional culture model. Cancer Res. 65, 7177–7185. Chai, G., et al., 2010. MicroRNA-10b regulates tumorigenesis in neurofibromatosis type 1. Cancer Sci. Ciafre, S.A., et al., 2005. Extensive modulation of a set of microRNAs in primary glioblastoma. Biochem. Biophys. Res. Commun. 334, 1351–1358. Dong, Q., et al., 2010. Expression of the reversion-inducing cysteine-rich protein with Kazal motifs and matrix metalloproteinase-14 in neuroblastoma and the role in tumour metastasis. Int. J. Exp. Pathol. 91, 368–373. Fang, Y., et al., 2010. MicroRNA-10a regulation of proinflammatory phenotype in athero-susceptible endothelium in vivo and in vitro. Proc. Natl Acad. Sci. U.S.A. 107, 13450–13455. Gee, H.E., et al., 2008. MicroRNA-10b and breast cancer metastasis. Nature 455, E8–E9 author reply E9. Gondi, C.S., et al., 2004a. Downregulation of uPA, uPAR and MMP-9 using small, interfering, hairpin RNA (siRNA) inhibits glioma cell invasion, angiogenesis and tumor growth. Neuron Glia Biol. 1, 165–176. Gondi, C.S., et al., 2004b. RNAi-mediated inhibition of cathepsin B and uPAR leads to decreased cell invasion, angiogenesis and tumor growth in gliomas. Oncogene 23, 8486–8496. Gondi, C.S., et al., 2004c. Adenovirus-mediated expression of antisense urokinase plasminogen activator receptor and antisense cathepsin B inhibits tumor growth, invasion, and angiogenesis in gliomas. Cancer Res. 64, 4069–4077. Han, L., et al., 2007. DNA methylation regulates MicroRNA expression. Cancer Biol. Ther. 6, 1284–1288. Hiyoshi, Y., et al., 2009. MicroRNA-21 regulates the proliferation and invasion in esophageal squamous cell carcinoma. Clin. Cancer Res. 15, 1915–1922. Huang, Q., et al., 2008. The microRNAs miR-373 and miR-520c promote tumour invasion and metastasis. Nat. Cell Biol. 10, 202–210. Iorio, M.V., et al., 2005. MicroRNA gene expression deregulation in human breast cancer. Cancer Res. 65, 7065–7070. Kunigal, S., et al., 2006. SPARC-induced migration of glioblastoma cell lines via uPA–uPAR signaling and activation of small GTPase RhoA. Int. J. Oncol. 29, 1349–1357. Ladeiro, Y., et al., 2008. MicroRNA profiling in hepatocellular tumors is associated with clinical features and oncogene/tumor suppressor gene mutations. Hepatology 47, 1955–1963. Ma, L., et al., 2010. Therapeutic silencing of miR-10b inhibits metastasis in a mouse mammary tumor model. Nat. Biotechnol. 28, 341–347. Ma, L., et al., 2007. Tumour invasion and metastasis initiated by microRNA-10b in breast cancer. Nature 449, 682–688. Mohanam, S., et al., 1993. Modulation of in vitro invasion of human glioblastoma cells by urokinase-type plasminogen activator receptor antibody. Cancer Res. 53, 4143–4147. Moriarty, C.H., et al., 2010. miR-10b targets Tiam1: implications for Rac activation and carcinoma migration. J. Biol. Chem. 285, 20541–20546. Myers, C., et al., 2002. Sustained expression of homeobox D10 inhibits angiogenesis. Am. J. Pathol. 161, 2099–2109.
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Orom, U.A., et al., 2008. MicroRNA-10a binds the 5′UTR of ribosomal protein mRNAs and enhances their translation. Mol. Cell 30, 460–471. Salajegheh, M., et al., 2005. Expression of urokinase-type plasminogen activator receptor (uPAR) in primary central nervous system neoplasms. Appl. Immunohistochem. Mol. Morphol. 13, 184–189. Sasayama, T., et al., 2009. MicroRNA-10b is overexpressed in malignant glioma and associated with tumor invasive factors, uPAR and RhoC. Int. J. Cancer 125, 1407–1413. Shi, L., et al., 2008. hsa-mir-181a and hsa-mir-181b function as tumor suppressors in human glioma cells. Brain Res. 1236, 185–193. Suojanen, J., et al., 2009. A novel and selective membrane type-1 matrix metalloproteinase (MT1-MMP) inhibitor reduces cancer cell motility and tumor growth. Cancer Biol. Ther. 8, 2362–2370. Tan, Y., et al., 2009. Transcriptional inhibiton of Hoxd4 expression by miRNA-10a in human breast cancer cells. BMC Mol. Biol. 10, 12. Tavazoie, S.F., et al., 2008. Endogenous human microRNAs that suppress breast cancer metastasis. Nature 451, 147–152.
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Research Report
MicroRNA-10b induces glioma cell invasion by modulating MMP-14 and uPAR expression via HOXD10 Lihua Suna,b,1 , Wei Yana,1 , Yingyi Wanga,1 , Guan Sunc,1 , Hui Luoa , Junxia Zhanga , Xiefeng Wanga , Yongping Youa , Zhengxiang Yangb,⁎, Ning Liua,⁎ a
Department of Neurosurgery, the First Affiliated Hospital of Nanjing Medical University, Nanjing 210029, PR China Department of Neurosurgery, the Affiliated Wuxi People's Hospital of Nanjing Medical University, Wuxi, 214023, PR China c Department of Neurosurgery, Fourth Affiliated Hospital of Nantong University, First Hospital of Yancheng, Yancheng, 224005, PR China b
A R T I C LE I N FO
AB S T R A C T
Article history:
MicroRNAs are small endogenous noncoding RNAs, which modulate target gene expression
Accepted 3 March 2011
by binding with target mRNA sequences in the 3′untranslated region (UTR) with an
Available online 16 March 2011
imperfect complementarity that inhibits the mRNA translation. Many microRNAs have been reported to function as tumor oncogenes or anti-oncogenes. Recently, more and more
Keywords:
microRNAs have been reported to contribute to a tumor's invasive potential. Here, we show
Glioma
that microRNA-10b (miR-10b) was over-expressed in glioma samples and directly associated
MicroRNA
with the glioma's pathological grade and malignancy. We also found that miR-10b induced
Invasion
glioma cell invasion by modulating tumor invasion factors MMP-14 and uPAR expression via
HOXD10
the direct target HOXD10. The miR-10b/HOXD10/MMP-14/uPAR signaling pathway might
MMP-14
contribute to the invasion of glioma. Accordingly, glioma cells lost their invasive ability
uPAR
when treated with specific antisense oligonucleotides (miR-10b inhibitors), suggesting that miR-10b could be used as a new bio-target to cure glioma. © 2011 Elsevier B.V. All rights reserved.
1.
Introduction
Glioma, the most common primary malignant neoplasm of the central nervous system (CNS), has a highly invasive malignant behavior and recurrence rate, and it remains one of the tumors most refractory to treatment. Recently, some studies reported that miRNAs play essential roles in tumor invasion and migration (Asangani et al., 2008; Hiyoshi et al., 2009; Huang et al., 2008; Ma et al., 2007; Sasayama et al., 2009; Shi et al., 2008; Weiss et al., 2009). Among these miRNAs, miR10b was first reported to induce breast cancer cell invasion and
metastasis, which was also found to be associated with tumor invasive potential in hepatic cancer (Ladeiro et al., 2008), pancreatic cancer (Bloomston et al., 2007), glioma (Sasayama et al., 2009), esophageal cancer (Tian et al., 2010) and neurofibromatosis (Chai et al., 2010). Ma et al. (2007) found that miR-10b could modulate invasion-associated protein Rhoc expression by directly target HOXD10 gene and Sasayama et al. (2009) reported that miR-10b level was associated with tumor invasive factors, uPAR and RhoC. But the exact function of miR-10b in glioma is still unknown. Under this condition, we first measured miR-10b levels in
⁎ Corresponding authors. N. Liu is to be contacted at Department of Neurosurgery, the First Affiliated Hospital of Nanjing Medical University, Nanjing 210029, PR China. Z. Yang, Department of Neurosurgery, the Affiliated Wuxi People's Hospital of Nanjing Medical University, Wuxi, 214023, PR China. Fax: +86 25 83716602. E-mail addresses: [email protected] (N. Liu), [email protected] (Z. Yang). 1 These authors contributed equally to this article. 0006-8993/$ – see front matter © 2011 Elsevier B.V. All rights reserved. doi:10.1016/j.brainres.2011.03.013
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different grade glioma samples and cell lines. Then, we used loss of function and recovery experiments to determine the function of miR-10b in glioma cell lines; furthermore, we also investigated that whether miR-10b direct binding to its target HOXD10. Additionally, tumor invasive factors MMP-14 and uPAR were also measured in our studies.
2.
Results
2.1. MiR-10b is over-expressed in glioma samples and cell lines, miR-10b expression is associated with glioma pathological grade and malignancy First, we used TaqMan®MicroRNA assay-based real-time RT– PCR to analyze miR-10b levels in 22 glioma samples and 4 well known glioma cell lines: U87, U251, LN229 and U373. Six normal brain tissues were considered as control. As shown in Fig. 1A, each glioma sample expressed higher miR-10b level than the normal controls. Furthermore, miR-10b expression in the glioma group was significantly higher than in the normal
brain group (p < 0.01) (Fig. 1B). Then, we divided all glioma samples into 3 groups according to their pathological diagnosis. Among them, 8 were diagnosed as WHO-II grade, 8 were WHO-III and 6 were WHO-IV. We found that miR-10b levels were all up-regulated in these 3 groups compared to the normal brain group (p < 0.05) and miR-10b expression was positively correlated with the tumor's grade. In WHO-IV, miR10b expression was higher than WHO-III and WHO-II (p < 0.01). However, there was no statistical difference between WHO-III and WHO-II (p = 0.093) (Fig. 1C). We also found that miR-10b expression in the 4 cell lines were all higher than in normal brain tissues (p < 0.01). As shown in Fig. 1D, the miR-10b level was the highest and lowest in the U87 cell line (94.79 fold compare to the normal control) and U251 cell line (18.40 fold compare to the normal control), respectively.
2.2. MiR-10b level is down-regulated/up-regulated by miR-10b inhibitors/mimics To determine whether miR-10b inhibitors/mimics could function in vitro, U87 and LN229 cell lines, which expressed
Fig. 1 – MiR-10b is over-expressed in glioma samples and cell lines. (A) The relative levels of miR-10b in 22 glioma samples were measured by using real-time quantitative RT–PCR. Six normal brain tissues were counted as control. (B) MiR-10b is up-regulated in the glioma group compared to the normal brain group. Student's t test was used to analyze the significant differences between the two groups. (C) Twenty-two glioma samples were divided into three groups according to the pathology diagnosis. One-way ANOVAs were used to analyze the significant differences among the groups. (D) The relative levels of miR-10b in the four glioma cell lines U251, U373, LN229 and U87. Student's t test was used to analyze the significant differences. Error bars mean standard deviation (SD) of triplicate independent experiments. *p < 0.05, **p < 0.01 compared to the control.
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higher levels of miR-10b, were selected and transfected with miR-10b inhibitors. The U251 cell line, which showed lower level of miR-10b, was chosen for transfection with miR-10b mimics. We analyzed miR-10b levels after transfection with the miR-10b inhibitors/mimics 48 h post transfection and found that miR-10b levels were significantly reduced by the miR-10b inhibitors (about 65.28% down-regulated compared to the control in the U87 cells and 54.17% in the LN229 cells) and elevated by the miR-10b mimics (about 20.36 fold compared to the control in the U251 cells) (Fig. 2).
2.3.
MiR-10b promotes glioma cell invasion
To investigate whether miR-10b could enhance glioma cell invasion, transwell assays were performed. First, we performed loss-of-function analysis and transfected miR-10b inhibitors into U87 and LN229 cell lines to reduce miR-10b expression. The results showed that the inhibition of miR-10b expression led to a 59.67% reduction in invasion compared to the control in U87 cells (Fig. 3A and D) and 43.29% in LN229 cells (Fig. 3B and E). Then, we used miR-10b mimics to up-regulate the level of miR10b in U251 cells, which led to a 1.82 fold increase in invasion (Fig. 3C and F). Additionally, both the miR-10b inhibitors and miR-10b mimics had no effect on cell apoptosis or cell cycle (Fig. 4).
This was in contrast to U251. In cell lines, the level of miR-10b was inversely related to the expression of HOXD10 protein (r = 0.94, p < 0.05) indicating that miR-10b might suppress HOXD10 gene expression (Fig. 5A and B). To test this hypothesis, we used miR-10b inhibitors to silence miR-10b expression in U87 and LN229 cells and found that HOXD10 protein levels were significantly increased by miR-10b inhibition compared to the blank control and scramble control. We also used miR-10b mimics to up-regulate miR-10b expression in U251 cells, and as a result, we found that HOXD10 protein expression was decreased by the miR-10b mimics (Fig. 5C and E). Tumor invasive factors MMP-14 and uPAR have been reported to be directly regulated by HOXD10 (Myers et al., 2002). To further investigate the signaling pathway, we measured MMP-14 and uPAR levels after transfection with HOXD10 vectors in U87 and LN229 cells. Expression of both genes was decreased by the HOXD10 vectors (Fig. 5D), which indicated that MMP-14 and uPAR were regulated by HOXD10 in glioma cell lines. Then, we examined the levels of MMP-14 and uPAR after transfection with miR-10b inhibitors in the U87 and LN229 cells; the results showed that both of them were downregulated by the miR-10b inhibitors, whereas transfection with the miR-10b mimics in U251 cells could up-regulate MMP-14 and uPAR expression levels (Fig. 5E).
2.5. 2.4. MiR-10b modulates invasion factors MMP-14 and uPAR expression via the target HOXD10 gene HOXD10 has been reported to be regulated by miR-10b in human breast cancer (Ma et al., 2007) and esophageal cancer (Tian et al., 2010) cell lines. To investigate whether miR-10b can modulate HOXD10 expression in glioma cell lines, we first examined the levels of HOXD10 protein in the four glioma cell lines. Interestingly, U87 cells which expressed the highest level of miR-10b showed the lowest level of HOXD10 protein.
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HOXD10 is a direct target of miR-10b
To verify whether miR-10b directly targeted HOXD10 in glioma cell lines, luciferase reporter assays were conducted. We constructed p3′UTR-HOXD10 and p3′UTR-mut-HOXD10-with a substitution of four nucleotides within the miR-10b binding site (Fig. 6A). We then cotransfected luciferase reporters and miR-10b expressing vectors into U87 and U251 cells. We found that cotransfection with p3′UTR-HOXD10 reporters and miR10b expressing vectors in U87 cells led to a significant decrease (45% decreased, p < 0.01) in the luciferase activity compared to
Fig. 2 – MiR-10b is reduced/elevated by miR-10b inhibitors/mimics after transfection. (A) U87 cells were seeded into 6-well plates, cells of 70% confluence were transfected with miR-10b inhibitors. Relative levels of miR-10b were analyzed 48 h after transfection. (B) LN229 cells of 70% confluence were transfected with miR-10b inhibitors. After 48 h, the relative levels of miR-10b were measured. (C) U251 cells of 70% confluence were transfected with miR-10b mimics, and miR-10b expression was also analyzed 48 h later after transfection. Error bars mean standard deviation (SD) of triplicate independent experiments. *p < 0.05, **p < 0.01 compared to the blank control.
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Fig. 3 – MiR-10b contributed to glioma cell invasion. (A and D) U87 cells, which were transfected with miR-10b inhibitors for 48 h later showed lower invasive potential (the reduction of cell number was about 59.67% in invasion assays) compared to the blank control. (B and E) LN229 cells, which were also transfected with the miR-10b inhibitors, showed 43.29% reduction in the number of invasive cells compared to the blank control. (C and F) U251 cells, which were transfected with the miR-10b mimics for 48 h, showed a higher invasive potential (the increasing cell number is about 1.82 fold in invasion assays) compared to the blank control. Error bars mean standard deviation (SD) of triplicate independent experiments. **p < 0.01 compared to the blank control.
the blank control (but not p3′UTR-mut-HOXD10) (Fig. 6B). In U251 cells, a similar effect was also found (51% decrease compared to the blank control, p < 0.01) (Fig. 6C).
3.
Discussion
MiRNAs are a class of small noncoding RNAs. Recently, a growing number of miRNAs have been found to be either
activators or suppressors of tumor invasion and metastasis (Shi et al., 2008; Tavazoie et al., 2008; Zhang et al., 2010; Zhu et al., 2008). Ma et al. (2007) has showed that TWIST1, which is a metastasis-promoting transcription factor, could modulate miR-10b expression by binding to the upstream portion of the miR-10b hairpin region. Moreover, miR-10b inhibited the translation of mRNA encoding homeobox D10 (HOXD10), which further regulated RHOC, a pro-metastasis gene belonging to the Ras homolog gene family. Therefore, the TWIST1-
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Fig. 4 – MiR-10b has no effect on cell apoptosis or cell cycle in glioma cell line. (A) U87 cell apoptosis rate and cell cycle was analyzed on FACScan by flow cytometry 48 h later with miR-10b inhibitors treatment; no differences were detected among the blank control group, scramble group and miR-10b inhibitors group. Annexin V− and PI− cell was used as control, Annexin V+ and PI− cell was considered as apoptotic while Annexin V+ and PI+ cell was designated as necrotic. (B) U251 cells were transfected with miR-10b mimics, after treatment 48 h, cell apoptosis assay and cell cycle analysis were done, and there are no differences among the blank control group, scramble group and miR-10b mimics group. Each independent experiment was done three times.
miR-10b–HOXD10–RHOC pathway was crucial for breast cancer cell invasion and metastasis. They also found that miR-10b antagomirs—a class of chemically modified antimiRNA oligonucleotides—could suppress breast cancer metastasis. Administration of miR-10b antagomirs to mice bearing highly metastatic cells could not reduce primary mammary tumor growth, but it markedly suppressed the
formation of lung metastases (Ma et al., 2010). Tumor invasion and metastasis is a complex and multi-step process: thus, miR-10b may play roles in different steps via different targets. More and more direct targets of miR-10b have been found. For example, miR-10b could promote human esophageal cancer cell migration and invasion by inhibiting KLF4 mRNA translation (Tian et al., 2010). MiR-10b was also identified to target
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Fig. 5 – MiR-10b regulates invasion factors MMP-14 and uPAR expression via target HOXD10 gene. (A and B) The relative expression levels of HOXD10 protein in four glioma cell lines; HOXD10 protein level in U87 cell line was considered as the control. The relationship between miR-10b expression and HOXD10 protein level was examined by using Pearson's correlation analysis. Error bars represent standard deviation (SD) of triplicate independent experiments. (C) Western Blot showed that HOXD10 protein level was significantly up-regulated while uPAR and MMP-14 levels were down-regulated by HOXD10 expressing Vectors both in U87 and LN229 cells. (D) Western blot showed that miR-10b inhibitors could enhance HOXD10 protein expression but significantly reduce HOXD10 downstream genes MMP-14, uPAR expression in U87 and LN229 cells. While miR-10b mimics has the opposite function, which could promote MMP-14 and uPAR expressing via decreasing HOXD10 protein expression.
neurofibromatosis type 1 (NF1) mRNA to activate RAS signaling in neurofibromin and to affect malignant peripheral nerve sheath tumor (MPNST) cell migration and invasion (Chai et al., 2010). However, there exists an opposing view that miR-10b directly targets T lymphoma invasion and metastasis 1 (Tiam1) and inhibited Tiam1-mediated Rac activation to suppress the ability of breast carcinoma cells to migrate and invade (Moriarty et al., 2010). MiR-10b was first found to be down-regulated in breast cancer (Iorio et al., 2005) and further confirmed by others (Gee et al., 2008), but Ma et al. (2007) found that miR-10b was upregulated in metastatic breast cancer. Although there exists some debate on whether miR-10b was up-regulated in breast cancer, there is no conflict on whether miR-10b was over-
expressed in glioma. Ciafre et al. (2005) first reported that the most up-regulated miRNA in glioblastoma was miR-10b by using microarray analysis and Northern blot, which was further confirmed by Sasayama et al. (2009) by real-time RT–PCR. They also identified that the level of miR-10b was associated with glioma maligancy and correlated with the expression of tumor invasion factors uPAR and RhoC. However, the mechanism of miR-10b-induced glioma cell invasion was still unknown. Thus, we first measured the levels of miR-10b in 22 glioma samples, 6 normal brain and 4 glioma cell lines. The results showed that miR-10b was over-expressed in all glioma samples compared to normal brain control, which complied with studies on human pancreatic adenocarcinomas (Bloomston et al., 2007) and esophageal cancer (Tian et al., 2010). We also found that miR-
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Fig. 6 – MiR-10b directly targets the 3′UTR of HOXD10 mRNA in glioma cell line. (A) Upper panel: the predicted binding sites between miR-10b and HOXD10 3′UTR in humans. Lower panel: the plasmid with the full length of HOXD10 3′UTR (p3′UTR-HOXD10) and the plasmid with a mutant HOXD10 3′UTR (p3′UTR-mut-HOXD10) which carried a substitution of four nucleotides with the miR-10b binding site. (B) U87 cells were cotransfected with miR-10b-expressing/blank vectors and p3′UTR-HOXD10/p3′UTR-mut-HOXD10 reporters. After 48 h, luciferase activities were measured. (C) U251 cells were also cotransfected with vectors and reporters, and similar results were observed. Error bars depict standard deviation (SD) of triplicate independent experiments. **p < 0.01 compared to the blank.
10b expression in WHO-IV was much higher than the other grades. In glioma cell lines, miR-10b levels were all higher than the normal control. All of this evidence indicated that miR-10b might contribute to a glioma's malignancy. To further verify that miR-10b could induce glioma cell invasion, we used miR-10b inhibitors, which could decrease miR-10b expression, and miR-10b mimics, which could increase miR-10b levels. When transfected with the miR-10b inhibitors, U87 and LN229 cells lost their invasion potential while U251 cells' invasive ability was enhanced by the miR-10b mimics. uPA is a specific serine protease that can convert plasminogen to plasmin and directly bind to its specific receptor uPAR, thereby promoting cell-surface plasmin activation and localization of degradation of the extracellular matrix to the migrating tumor cell surface. uPAR has been shown to be over-expressed in glioma (Salajegheh et al., 2005; Yamamoto et al., 1994), and high levels of uPA and uPAR were correlated with an invasive phenotype of glioma cells (Mohanam et al., 1993). Increased signaling pathways inducing glioma cell invasion were found through uPA and uPAR (Bryan et al., 2008; Kunigal et al., 2006; Young et al., 2009), while down-regulation of uPA and uPAR inhibited glioma cell invasion (Gondi et al., 2004a; Gondi et al., 2004b; Gondi et al., 2004c). MMP-14 is a number of the membrane-type matrix metalloproteinase (MT-MMP) subfamily involved in the breakdown of extracellular matrix in physiological or pathological processes. MMP-14 was found to be over-
expressed in neuroblastoma and increased with an increase in the extent of invasive depth and distant metastasis (Dong et al., 2010). MMP-14 inhibitor could effectively inhibit cancer cell migration and invasion in vitro (Suojanen et al., 2009). Both uPAR and MMP-14 were reported to be regulated by HOXD10 (Myers et al., 2002), a sequence-specific transcription factor, whose expression is progressively reduced in epithelial cells as malignancy increases in both breast and endometrial tumors. Retroviral gene transfer to restore expression of HOXD10 significantly impaired cell migration (Carrio et al., 2005) suggesting that HOXD10 may play a role as a suppressor of tumor invasive growth. To explore the molecular mechanism of miR-10b's function in glioma cell lines, we transfected HOXD10 expressing vectors into U87 and LN229 cells. Western blot showed that uPAR and MMP-14 were inactivated by HOXD10 gene. We then transfected miR-10b inhibitors into U87 and LN229 cells and found that HOXD10 protein levels were elevated while uPAR and MMP-14 levels were decreased. The transfection of miR-10b mimics into U251 cells resulted in an opposite effect. Finally, luciferase reporter assays certified that the HOXD10 gene was a direct target of miR-10b. Thus, we thought that miR-10b induced glioma cell invasion by modulating MMP-14 and uPAR expression via directly target the HOXD10 gene. The TWIST1/miR-10b/HOXD10/MMP-14, uPAR signaling pathway might be essential for glioma cells invasion. However, in the same way that miR-10a targets many genes (Fang et al.,
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2010; Han et al., 2007; Orom et al., 2008; Tan et al., 2009; Weiss et al., 2009; Woltering and Durston, 2008), the other target genes of miR-10b in glioma and their function need further investigation. Our studies also showed that glioma cells lost their invasive potential by using miR-10b antisense inhibitors, providing a new bio-target for glioma treatment. Because the function of a particular miRNA is often tissue specific, each miRNA may target many mRNAs, and the target genes themselves may have differential or even opposite effects in different tumors; therefore, using miR-10b antisense inhibitors for anti-metastasis treatment still warrant further investigation.
4.
Statistical analysis
All experiments were done three times and statistical analysis was performed on SPSS Graduate Pack 13.0 statistical software. Descriptive statistics including mean and SD. Student's t test and one-way ANOVAs were used to analyze significant differences. Pearson's correlation analysis was used to examine the relationship between miR-10b expression and HOXD10 protein levels. p<0.05 was considered to be statistically significant.
5.
Experimental procedures
5.1.
Cell lines and cell culture
Human glioma cell lines, U87, LN229, U251 and U373 were purchased from the Chinese Academy of Sciences Cell Bank; all cell lines were maintained in a 37 °C, 5% CO2 incubator in DMEM supplemented with 10% fetal bovine serum (FBS).
5.2.
Glioma samples and normal brain tissues
After informed consent patients diagnosed with glioma was obtained, human glioma samples were collected from the department of neurosurgery, the first affiliated hospital of Nanjing Medical University. Among the 22 samples, 8 samples were diagnosed as WHO-II grade, 8 were WHO-III and 6 were WHO-IV by pathological diagnosis. Six normal brain tissues were obtained after informed consent from patients with severe traumatic brain injury who required post-trauma surgery. After resection, all samples were immediately frozen in liquid nitrogen until RNA extraction.
5.3.
RNA isolation
Total RNA was extracted from tissues and four glioma cell lines by using the TRIzol reagent (Invitrogen, Carlsbad, USA). The amount of RNA was quantified by absorbance reading at A260/ A280= 1.6–1.8 and stored at −80 °C until use.
5.4.
Real-time quantification PCR
To detect the relative levels of miR-10b in glioma samples and cell lines, TaqMan-based real-time reverse transcription– polymerase chain reaction (RT–PCR) assay was used. The
primers and probes of has-miR-10b (P/N 4395329) and RNU6B (P/N 4373381) endogenous control for TaqMan miRNA assays were purchased from Applied Biosystems. Real-time PCR was performed according to the manufacturer's instructions on the ABI 7300 HT Sequence Detection System (Applied Biosystems, CA). The relative level of miR-10b was normalized to the RUN6B, and the quantitative miR-10b expression date was calculated by using 2−△△Ct method.
5.5.
Cell transfection
2′-O-methyl (2′-O-Me) hsa-miR-10b inhibitors (miR-10b antisense oligonucleotides) and mimics (miR-10b sense oligonucleotides) were chemically synthesized by Shanghai GenePharma Company (Shanghai, China). The 2′-O-Me-has-miR-10b inhibitor sequence was 5′-CACAAAUUCGGUUCUACAGGGUA-3′, and the scramble sequence was 5′-CAGUACUUUUGUGUAGUACAA-3′. The 2′-O-Me-has-miR-10b mimic sequence was 5′-UACCCUGUAGAACCGAAUUUGUG-3′, 3′-CAAAUUCGGUUCUACAGGGUAUU-5′, and the scramble sequence was 5′UUCUCCGAACGUGUCACGUTT-3′, 5′-ACGUGACACGUUCGGAGAATT-3′. Cells of 70%–80% confluence were transfected with oligonucleotide using Lipofectamine 2000 reagent (Invitrogen, Carlsbad, USA). Transfection was performed according to the manufacturer's instructions, and the final oligonucleotide concentration was 10 nmol/L. Transfection medium was replaced 6 h later. MiR-10b expressing vectors and blank vectors, HOXD10 vectors and mut-HOXD10 vectors (Genesil, Wuhan, China) were constructed by Wuhan Genesil. Vectors were transfected into human glioma cell lines with FuGENE HD6 (Roche, Basel, Switzerland) according to the manufacturer's instructions and screened by the aminoglycoside G418.
5.6.
Invasion assay in vitro
For transwell assay, 5 × 104 cells were plated into 24-well Boyden chambers (Coring costar, MA, USA), with an 8-μm pore polycarbonate membrane, which was coated with 20 μg of Matrigel (BD Biosciences, San Jose, CA). Cells were in the upper chamber with 200 μl of serum-free medium, and medium containing 20% FBS was added to the lower chamber to serve as a chemoattractant. After 24 h, the cells were washed 3 times with PBS, non-invasive cells were removed from the upper well by cotton swabs, and the invasive cells were then fixed with 4% paraformaldehyde, stained with 0.1% crystal violet, and photographed (×200) in six independent fields for each well. The results were averaged through three independent experiments.
5.7.
Apoptosis assay
First, 1 × 106 cells were plated into 6-well plates, and, after transfection with each oligonucleotide for 48 h, the apoptosis assay was conducted using Annexin V-FITC double stained detection kit (BD Biosciences, San Jose, CA). Annexin V− and PI− cells were used as controls. Annexin V+ and PI− cells were considered as apoptotic, and Annexin V+ and PI+ cells were designated as necrotic.
BR A IN RE S EA RCH 1 3 89 ( 20 1 1 ) 9 –1 8
5.8.
Cell cycle analysis
After each treatment for 48 h, cells in the log phase of growth were collected, washed with PBS for 3 times, and then fixed with 75% ethanol at −20 °C for at least 1 h. After extensive washing with PBS, the cells were suspended in HBSS containing 50 μg/ml of RNaseA (Boehringer Mannheim, Indianapolis, IN) and 50 μg/ ml of PI (Sigma-Aldrich), incubated for 1 h at room temperature and then analyzed by FACScan (Becton Dickinson, San Jose, CA).
5.9.
Luciferase reporter assay
The full length 3′UTR region of the HOXD10 gene was subcloned into luciferase reporter vectors from human genomic DNA and the mutant HOXD10 vectors were constructed, which carried a substitution of four nucleotides at the miR-10b binding sites. Cells of 60%–70% confluence in 24well plates were cotransfected with luciferase reporter vectors and miR-10b expressing vectors, and a 1 ng pRLSV40 Renilla luciferase construct was used for normalization. After 48 h, luciferase activity was analyzed by the Dual-Luciferase Reporter Assay System according to the manufacturer's protocols (Promega, Madison, USA).
5.10.
Western blot
Forty-eight hours after transfection, total protein was collected using a Total Protein Extraction Kit (KeyGen, China) and quantified using a BCA Protein Assay Kit (KeyGen, China). Twenty micrograms of protein were added and subjected to SDS–PAGE on (SDS)–polyacrylamide gel electrophoresis using the discontinuous buffer system of Laemmli (Bio-Rad Laboratories, USA). The electrophoresed proteins were transferred to a polyvinylidene difluoride (PVDF) membrane and subjected to immunoblot analysis with a goat primary antibody against HOXD10 (Santa Cruz, USA, 1:200), mouse against uPAR (Santa Cruz, USA, 1:200), rabbit against MMP-14 (R&D systems, USA, 1:2000), followed by HRP-conjugated rabbit anti-goat secondary antibodies (Bioworld Technology, USA, 1:5000), goat antimouse secondary antibodies (MultiSciences Biotech, China, 1:5000) and goat anti rabbit secondary antibodies (MultiSciences Biotech, China, 1:5000). GAPDH (KangCheng, China, 1:2000) was used as control.
Acknowledgments This work is supported by the China Natural Science Foundation (proj. no. 30872657), Jiangsu Province's Medical Major Talent Foundation (proj. no. RC2007061) and Jiangsu Province's “333” Key Talent Foundation (proj. no. 0508RS08).
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