Microrna-222 Promotes Tumorigenesis Via Targeting DKK2 and Activating the Wnt/B-Catenin Signaling Pathway

FEBS Letters 587 (2013) 1742–1748

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MicroRNA-222 promotes tumorigenesis via targeting DKK2 and activating the Wnt/b-catenin signaling pathway

Qifeng Li a, Ke Shen b, Yang Zhao a, Xiaoguang He a, Chenkai Ma a, Lin Wang a, Baocheng Wang a, ⇑ Jianwen Liu b, Jie Ma a, a Department of Pediatric Neurosurgery, Xinhua Hospital, Shanghai Jiaotong University, School of Medicine, Shanghai 200092, PR China b State Key Laboratory of Bioreactor Engineering & Shanghai Key Laboratory of Chemical Biology, School of Pharmacy, East China University of Science and Technology, #268, 130 Meilong Road, Shanghai 200237, PR China article info abstract

Article history: MiR-222 in glioma can regulate cell cycle progression and apoptosis. However, the relationship Received 29 March 2013 between miR-222 and Wnt/b-catenin signaling pathway in glioma remains unknown. Here, we Accepted 3 April 2013 found that the Dickkopf-2 gene (DKK2) was a direct target of miR-222 by target prediction analysis Available online 12 April 2013 and dual luciferase reporter assay. RNA interference silencing of DKK2 proved that miR-222 overexpression led to constitutive activation of b-catenin through inhibition of DKK2 expression in glioma Edited by Tamas Dalmay cells. Furthermore, miR-222 siRNA signiﬁcantly inhibited tumorigenesis in vivo. Finally, Western blot analysis showed that miR-222 could regulate the expression of b-catenin and the downstream Keywords: genes of Wnt/b-catenin signaling pathway. Taken together, our ﬁndings reveal a new regulatory miR-222 DKK2 mechanism of miR-222 and suggest that miR-222 might be a potential target in glioma therapy. Wnt/b-catenin signaling pathway Ó 2013 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved. Glioma

1. Introduction be a promising strategy for cancer therapeutics. It has been demonstrated that abnormally high activation of the Wnt/b-catenin path- Gliomas are the most common form of malignancy in the central way is required for the initiation and progression of glioma[11]; nervous system [1]. Despite therapeutic advances, the median sur- however, until now, functional analysis of miR-222 and the Wnt/ vival time for high-grade gliomas is only 15 months [2]. Previous b-catenin pathway has not been well-documented. studies have suggested that the formation of gliomas is related to In the present study, we investigated the potential involvement the rates of cell proliferation and apoptosis. Therefore, understand- of miR-222 in glioma. We examined the expression level of miR- ing the main regulatory mechanism of this malignancy is the key to 222 in glioma cells, and tested its effects on cell growth, apoptosis, developing novel and effective therapeutic strategies for gliomas. and colony formation in vitro and tumorigenesis in vivo. Further- MicroRNAs (miRNAs) are non-coding RNA molecules that regu- more, we explored the underlying role of miR-222 in glioma. Final- late the expression of a wide variety of genes through sequence-spe- ly, we also analyzed the possible relationship between miR-222 ciﬁc binding to their mRNA targets, resulting in translational and the genes downstream of the Wnt/b-catenin pathway in gli- inhibition [3]. They have pivotal roles in various physiological and oma development and progression. Our study will provide a better pathological processes, including proliferation and apoptosis [4]. understanding of glioma pathogenesis. An increasing body of evidence has suggested that the aberrant expression of miRNAs may lead to the development and progression 2. Materials and methods of malignancy [5,6]. In recent years, miR-222 has been reported to be overexpressed in human carcinomas, including thyroid papillary 2.1. Reagents tumors and glioblastoma [7,8]. Functional studies have demonstrated that miR-222 may target multiple signaling pathway and Antibodies against b-actin, Bcl2, Bax, b-catenin, c-myc, DKK2, genes, such as p27Kip1 and PUMA, to promote the development and c-Jun were obtained from Santa Cruz Biotechnology Inc., (CA, and progression of malignancy [9,10]. Thus, miR-222 can be classi- USA). A dual luciferase reporter assay system was obtained from ﬁed as an oncomir, and the decreased expression of miR-222 might Promega Corporation (WI, USA). PGL3-promoter, PGL3-basic, and PRL-TK vectors were also purchased from Promega. All other ⇑ Corresponding author. Fax: +86 021 25078999. chemicals were purchased from Sigma–Aldrich unless otherwise E-mail address: [email protected] (J. Ma). stated.

0014-5793/$36.00 Ó 2013 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.febslet.2013.04.002 Q. Li et al. / FEBS Letters 587 (2013) 1742–1748 1743

2.2. Cell lines and cultures CGATAACTGAAACCAAAGTCAATAGCTGC-30 (anti-sense). Then, it was inserted into the XbaI restriction site, immediately down- Glioma cell lines, including U251, U87, SHG44, A172, and a hu- stream of the luciferase gene in the pGL3-promoter-vector (Prome- man astrocyte cell line (HA) (Cell Bank of Chinese Academy of Sci- ga, Madison, WI). A mutant version with a deletion of 7 bp from a ence Shanghai China), were cultured in RPMI 1640 medium (Gibco site of perfect complementarity was also generated using the Quik- Industries, Inc., Carlsbad, CA) with 10% fetal bovine serum (Gibco Change II Site-Directed Mutagenesis Kit (Stratagene, LaJolla, CA). Industries, Inc). U87 and U87-miR-222-disrupted fluorescent Wild-type and mutant inserts were confirmed by sequencing. (EGFP)-labeled cells were developed (Neuron Biotech co., Ltd, Twenty-four hours after transfection, the cells were seeded into Shanghai, China) and are referred to as Control and GR232 for 96-well plates (8 103 viable cells/well) and allowed to adhere conciseness. overnight. Then, 200 ng of pGL3-DKK2-30–UTR or pGL3-mut- DKK2-30–UTR plus 80 ng pRL-SV40 (Promega Corporation, WI, 2.3. 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide USA) was transfected alone or in combination with control oligonu- (MTT) assay cleotide (final concentration of 100 nM) inhibitor (100 nM) or mimics (100 nM) using Lipofectamine 2000 (Invitrogen Corporation, CA, The rate of proliferation was assessed using the colorimetric MTT USA) according to the manufacturer’s protocol. Luciferase activity assay as described previously [12]. All cells were seeded into 96-well was measured 48 h post-transfection using the Dual Luciferase Re- culture plates (2 103 cells/well). The cells were transfected with a porter Assay System (Promega Corporation, WI, USA). Firefly lucif- miR-222 mimics, control oligonucleotide, or inhibitor (antisense oli- erase activity was normalized to renilla luciferase activity for gonucleotide-miR-222) and grown for 4 days. One plate was devel- each transfected well. Three independent experiments were per- oped immediately after the medium was changed, and other plates formed in triplicate. were developed every 24 h for 4 d. Assays were initiated by adding 20 ll of MTT substrate to each well, followed by an additional 3 h 2.7. Western blot analysis incubation. Finally, the media was removed, and 200 ll DMSO was added to each well. Plates were read at a wavelength of 570 nm in The U251 and U87 cells were seeded in six-well plates an Automated Microplate Reader (Multiskan Ex, Lab systems, FIN). (4 10 cells/well) in antibiotic-free media, followed by transfec- All experiments were biologically repeated in triplicate to confirm tion withcontrol oligonucleotide (100 nM), inhibitor (100 nM), or consistency of the results obtained. mimics (100 nM) using Lipofectamine 2000 (Invitrogen Corpora- tion, CA, USA). After 48 h of culture, the cells were harvested and 2.4. Quantitative reverse transcription polymerase chain reaction homogenized with lysis buffer (50 mM Tris–HCl, pH 8.0, 150 mM (qRT-PCR) NaCl, 0.1% SDS, 1% Non-idet P-40, 0.5% sodium deoxycholate, 0.02% sodium azide, 100 g/ml PMSF, 1 g/ml aprotinin). Total pro- The relative expression levels of miRNAs were determined. To- tein extract (40 lg) was separated on 12% SDS–PAGE gels and tal RNA was isolated using TRIZOLTM reagent (Promega Corpora- transferred to nitrocellulose membranes (Millipore, Bedford, tion, WI, USA) according to the supplier’s instructions. qRT-PCR MA). After blocking with 5% non-fat dry milk in TBS, the mem- was performed using One Step PrimeScriptÒ miRNA cDNA Synthe- brane was probed with primary monoclonal antibody specific sis Kit (TaKaRa Biotechnology Ltd, Shandong, China). Real-time PCR to DKK2 (1:100; Cell Signaling Technology) which was used as was performed using SYBR Green Supermix with an iCyclerÒther- an internal control for protein loading. The membrane was fur- mal cycler (Bio-Rad Laboratories Inc, CA, USA). Primers of all genes ther probed with horseradish peroxidase-conjugated rabbit are described in the Supplementary data. Data were collected and anti-mouse IgG (1:2000; Santa Cruz), and the protein bands analyzed by the comparative Ct (threshold cycle) method using were visualized using enhanced chemiluminescence (Amersham GADPH as the reference gene. Pharmacia Corp., Piscataway, NJ). b-Actin was used as a loading control. 2.5. Flow cytometric analysis of apoptosis 2.8. Subcutaneous and orthotopic xenografts The extent of apoptosis was measured through the Annexin V- FITC apoptosis detection kit (Invitrogen Corporation, CA, USA) For subcutaneous implantation, 5 106 Control cells (U87 according to the manufacturer’s instructions. The cells (6 104/ cells) and GR232 cells (miR-222-disrupted U87 cells) suspended well) were seeded in six-well plates in antibiotic-free media, fol- in 100 ll PBS were injected subcutaneously into the flank of lowed by transfection with control oligonucleotide (100 nM) or BALB/C nude mice (Cancer Institute of the Chinese Academy of mimics (100 nM) using Lipofectamine 2000 (Invitrogen Corpora- Medical Science). The mice were randomly divided into two tion, CA, USA). After 48 h, the cells were collected, washed with groups (five mice per group). Tumor growth in mice was de- cold PBS twice, and gently resuspended in 400 ll1⁄binding buffer. tected by the Kodak In-Vivo FX Pro system (Kodak, New York, After adding 5 ll of Annexin V-FITC, the cells were gently vortexed USA). Tumor volume of xenografts was detected every 5 days and incubated for 10 min at 4 °C in the dark. Then, the tube was over the course of the study with fluorescence signaling. Mice incubated with 10 ll of PI for another 5 min at 4 °C in the dark. Fi- were sacrificed and examined 5 weeks after the subcutaneous nally, the cells were analyzed using a FACScan flow cytometer implantation. (Becton Dickinson, NJ, USA). In Fig. 2C, cells in the lower right All experiments were carried out under the approval of the quadrant represent early apoptosis, and those in the upper right Administrative Panel on Laboratory Animal Care of the Xinhua quadrant are representative of late apoptotic cells. Hospital.

2.6. Luciferase activity assay 2.9. In vivo optical imaging

The 30 UTR of human DKK2 (GeneBank accession number Prior to in vivo imaging, the mice were anesthetized with Phe- NM_000633) cDNA, containing the putative target sites for miR- nobarbital sodium. Fluorescence imaging was obtained with an 222, was ampliﬁed by PCR using the following primers: 50-GAT- excitation wavelength of 490 nm and emission wavelength of CGCCGTGTAATTCTAGAGTTTCATTGCCCTCT-30 (sense) and 50-CGT 535 nm. Exposure times ranged from 1 to 2 min. 1744 Q. Li et al. / FEBS Letters 587 (2013) 1742–1748

Fig. 1. MiR-222 overexpression promoted cell viability and colony formation, but inhibited apoptosis of glioma cells. (A) Real-time PCR was performed to quantify miR-222 in four glioma cell lines (U251, U87, SHG44 and A172) and a human astrocyte cell line (HA). Quantification real-time PCR reverse transcription-PCR was done on cDNA which was generated from total RNA. All data were presented as representative of an average of three independent experiments. (B) U251 and U87 cells were transfected with a miR-222 mimics, control oligonucleotide, or inhibitor (antisense oligonucleotide-miR-222). Up-regulated miR-222 promoted colony formation, whereas down-regulated miR-222 restrained this activity. (C) MiR-222 mimics increased cell survival; conversely, a miR-222 inhibitor significantly decreased survival (n = 3; means ± S.E.M. ⁄⁄P < 0.01, ⁄⁄⁄P < 0.001 t-test). (D) Cell apoptosis was determined by flow cytometry. A significant increase in cell apoptosis was induced by the miR-222 inhibitor compared with the negative control, while the miR-222 mimics group showed a decrease in cell apoptosis. Data are representative of 1 of 3 similar experiments.

2.10. Statistical analysis 3. Results

Statistical analyses based on the two-tailed Student’s t-test 3.1. MiR-222 was overexpressed in glioma cells were performed using the Statistical Package for the Social Sci- ences software. Signiﬁcance was determined at the 95% conﬁdence The four glioma cell lines (U251, U87, SHG44 and A172) were interval. All data were expressed as the mean ± standard deviation compared with a human astrocyte cell line (HA) (Fig. 1A). The (S.D.) from a representative experiment. miR-222 expression in U251, U87, SHG44 and A172 cells was Q. Li et al. / FEBS Letters 587 (2013) 1742–1748 1745

Fig. 2. MiR-222 siRNA signiﬁcantly inhibited tumorigenesis in vivo. (A) Tumors were induced by subcutaneous implant of control cells (U87 line) and GR232 cells (miR-222- disrupted U87 line). Tumor growth in the mice was monitored by a live imaging system detecting the ﬂuorescent (EGFP) signal (P = 0.0079, Mann–Whitney test). (B) Tumor volume was measured every 5 days. Tumors were photographed following euthanasia. All data were presented as the mean ± S.D. and as an average of three measurements from a representative experiment.

clearly up-regulated by 50- to 150-fold. The U251 and U87 glioma miR-222 triggered apoptosis; in contrast, up-regulated miR-222 cell lines, which possessed the highest levels of miR-222 expres- decreased the number of cells undergoing apoptosis (Fig. 1D). sion among all tested glioma cell lines, were selected for further studies. 3.3. miR-222 siRNA signiﬁcantly inhibited tumorigenesis in vivo

3.2. MiR-222 overexpression promoted cell viability and colony To evaluate the effect of miR-222 in glioma, we constructed formation, but inhibited apoptosis of glioma cells EGFP-labeled U87 cells and miR-222-disrupted U87 cells (referred to as control and GR232, respectively). Tumors were generated Given that miR-222 is overexpressed in glioma and that it may from the control and GR232 cells by subcutaneous implantation. act as an oncomiR, we next evaluated the oncogenic function of Tumor volume was measured every 5 days. Tumor growth in the miR-222 in the U251 and U87 glioma cell lines. Up-regulated mice was monitored by a live imaging system detecting the fluo- miR-222 promoted colony formation, whereas down-regulated rescent (EGFP) signals. During the course of the study, miR-222- miR-222 restrained this activity (Fig. 1B). MTT assay showed that disrupted U87 cells demonstrated significantly inhibited tumor transfection with miR-222 mimics increased cell survival; con- growth (Fig. 2A). All data were presented as the mean ± S.D. and versely, cells transfected with miR-222 inhibitors showed a signif- as an average of three measurements from a representative exper- icant decrease in survival (Fig. 1C). Next, we used flow cytometry iment (P = 0.0079, Mann–Whitney test). Tumor volumes were also to test the role of miR-222 in apoptosis. Down-regulated measured weekly. Tumors were photographed at the 5th week. 1746 Q. Li et al. / FEBS Letters 587 (2013) 1742–1748

When miR-222 was inhibited in EGFP-labeled U87 cells, we found containing miR-222 binding sites was cloned into a luciferase re- that tumor growth was signiﬁcantly inhibited (Fig. 2B). porter system. The plasmid lacking the binding sites was used as a positive control of luciferase activity. The resulting reporter vector 3.4. DKK2 is a direct target gene of miR-222 was transfected into the U87 cells together with increasing concen- trations of miR-222. Results showed that miR-222 inhibited lucifer- Using the algorithm Target Scan (http://www.targetscan.org), ase activity in the DKK2 wild-type group. No variation in the we found that the inhibitory protein encoded by DKK2 is a target luciferase activity was observed when the cells were transfected of post-transcriptional repression by miR-222. MiR-222 has 7 with the plasmid containing a DKK2 30-UTR fragment without the nucleotides of the ‘‘seed’’ region which are complementary to the miR-222 binding sites (Fig. 3C). In Fig 3D we show the level of DKK2 30-UTR (Fig. 3A), indicating that DKK2 is the potential target up-regulation or inhibition of miR-222 achieved upon transfection of miR-222. Consequently, the down-regulation of miR-222 expres- with either the mimics or inhibitor. Hence, these results demon- sion in U251 and U87 cells was concurrent with the up-regulation strate that miR-222 directly inhibits DKK2 expression at the post- of DKK2 protein expression. To ascertain whether miR-222 regu- transcriptional level through its 30-UTR. lates DKK2, we employed a gene overexpression approach to examine the effect of miR-222 on the DKK2 protein. The U251 and U87 3.5. Relationship between miR-222 and the Wnt/b-catenin pathway cells were transfected with a miR-222 mimics or inhibitor. DKK2 protein expression, determined via Western blot, was down-regu- DKK2 is the inhibitor of the Wnt/b-catenin signaling pathway lated in miR-222 mimics-transfected cells relative to the control [13], so we hypothesized that miR-222 might play a role relevant cells. By contrast, DKK2 protein expression was up-regulated in to Wnt/b-catenin signaling. To test our hypothesis, we detected miR-222 inhibitor-transfected cells (Fig. 3B). To examine whether the b-catenin protein levels by Western blot in miR-222 mimics- miR-222 directly targets DKK2, a segment of the DKK2 30-UTR and inhibitor-transfected cells. The b-catenin protein level was decreased in miR-222 inhibitor-transfected cells, contrary to the increased b-catenin protein level in miR-222 mimics-transfected cells (Fig. 4A). These data show that miR-222 functions by targeting the inhibitory protein DKK2, which in turn blocks theWnt/b- catenin signaling pathway. Furthermore, we detected the genes downstream of the Wnt/b-catenin signaling pathway, including Bcl2, c-myc, and c-Jun. Expression of these genes was tested using Western blot in miR-222 mimics-, control-, and inhibitor-transfected cells. Results showed that down-regulated miR-222 could re- press the protein levels of Bcl2, c-myc, and p-c-Jun, but did not affect c-Jun (Fig. 4B). To determine whether miR-222 functions by targeting DKK2, we inhibited DKK2 expression by siRNA and tested the protein levels of b-catenin, Bcl2, c-myc, and p-c-Jun. We observed up-regulated expression of ß-catenin, Bcl2, and c- myc; in contrast, p-c-Jun expression was down-regulated, suggest- ing that it is independent of miR-222 inhibition (Fig. 4C). The protein expression in tumors was detected by Western blot. As shown in Fig. 4D, DKK2 expression increased in GR232 cells in vivo, which also conﬁrmed the in vitro results. These results indicated that miR-222 functioned as an oncomiR by targeting DKK2.

4. Discussion

In recent years, emerging evidence has demonstrated an impor- tant role for miRNAs in regulating human cancers [14]. At least 30% of protein-coding genes in the human genome are regulated by miRNAs [15]. Functional studies have directly documented the po- tent pro- and anti-tumorigenic activity of specific miRNAs both in vitro and in vivo [16]; therefore, miRNAs can be classified as oncomirs or tumor-suppressive miRNAs. These miRNAs are associated with tumor cell processes like proliferation, apoptosis, angio- genesis, migration, and metastasis [17]. Recently, it was reported that miR-222 is overexpressed in different kinds of human cancers, including glioblastoma [9,10]. Several studies have revealed that miR-222 may target multiple signaling pathways, such as the Fig. 3. DKK2 is a direct target gene of miR-222. (A) TargetScan predicts that miR- AKT pathway and EGFR signaling pathway, to promote the devel- 222 targets the 30-UTR in the DKK2 gene. (B) DKK2 protein levels were assessed 48 h opment and progression of malignancy [18,19]. However, there post-transfection with miR-222 mimics (100 nM), control (100 nM), or inhibitor has been no functional analysis of miR-222 and the Wnt/b-catenin (100 nM) in U251 and U87 cells. Scanning densitometry of the blots was used to quantify the Western blotting data. (n = 3; means ± S.E.M. ⁄⁄P < 0.01, ⁄⁄⁄P < 0.001 t- pathway in glioma. test). (C) The luciferase activity in U87 cells transfected with the vector containing In the present study, we found that miR-222 was overexpressed the miR-222 binding sequence in the DKK2 30-UTR was inhibited by miR-222 in glioma cells (Fig. 1A). Ectopic expression of miR-222 in vitro sig- transfection. No change in luciferase activity was observed when the cells were nificantly promoted the proliferation and colony formation of gli- transfected with a plasmid containing a DKK2 30-UTR fragment without the miR- oma cells, while inhibiting their apoptosis (Fig. 1B–D). To 222 binding sites (n = 3; means ± S.E.M. ⁄⁄P < 0.01, ⁄⁄⁄P < 0.001 t-test). (D) MiR-222 expression after infection with miR-222 mimics or inhibitor (n = 3; means ± S.E.M. evaluate the carcinogenesis of miR-222 in vivo, we injected miR- ⁄⁄P < 0.01, ⁄⁄⁄P < 0.001 t-test). 222-disrupted U87 cells and normal U87 cells into nude mice. Q. Li et al. / FEBS Letters 587 (2013) 1742–1748 1747

Fig. 4. Mir-222 regulated b-catenin expression and the downstream genes of the Wnt/b-catenin signaling pathway at the protein level. U251 and U87 cells were transfected with miR-222 mimics (100 nM), control (100 nM), or inhibitor (100 nM). The protein levels were assessed 48 h post-transfection. Scanning densitometry of the blots was also performed to quantify the Western blotting data. ⁄⁄P < 0.01, ⁄⁄⁄P < 0.001. (A) Transfection with a Mir-222 mimics up-regulated b-catenin expression, while transfection with inhibitor down-regulated its expression. (B) The Bcl2, c-myc, and p-c-Jun protein expression was down-regulated when transfected with miR-222 inhibitor, and the expression was up-regulated post-transfection with miR-222 mimics. (C) Inhibition of DKK2 by siRNA clearly affected the associated protein levels. When DKK2 was inhibited by siRNA, the expression of b-catenin, Bcl2, and c-myc was up-regulated. In contrast, p-c-Jun expression was down-regulated and appeared to be independent of miR-222. (D) In vivo DKK2 expression was tested by Western blot analysis (P = 0.0079, Mann–Whitney test).

The results demonstrated that miR-222-disrupted U87 cells are for cancer therapeutics. One established characteristic of significantly less tumorigenic than the normal U87 cells in vivo microRNAs is the ability of a single microRNA to directly target (P = 0.0079) (Fig. 2). These findings demonstrated that miR-222 multiple downstream genes [20]. In previous studies, it has been can be classified as an oncomir and might be a promising target reported that miR-222 can target PUMA to induce cell survival in 1748 Q. Li et al. / FEBS Letters 587 (2013) 1742–1748 glioblastoma [10]. Another report demonstrated that miR-222 References could target RECK to promote cancer cell proliferation in gastric cancer [21]. These previously reported target genes may also par- [1] Wen, P.Y. and Kesari, S. (2008) Malignant gliomas in adults. N. Engl. J. Med. 359 (5), 492–507. tially explain the relationship between miR-222 and the genes de- [2] Gabayan, A.J., Green, S.B., Sanan, A., et al. 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