MicroRNA-17-92 Cluster Mediates the Proliferation and Survival of Neural Progenitor Cells after Stroke*

Background: The role of miRNAs in mediating stroke-induced neurogenesis remains largely unknown. Results: The miR17-92 cluster regulated ischemia-induced neural progenitor cell proliferation, and activation of the Shh pathway up-regulated miR17-92 cluster expression. Conclusion: The miR17-92 cluster plays an important role in mediating adult neural progenitor cell proliferation. Significance: The present study provides molecular mechanisms underlying miR17-92 cluster in mediating stroke-induced neurogenesis. The role of microRNAs (miRNAs) in mediating adult neurogenesis after stroke has not been extensively studied. The present study investigated the function of the miR17-92 cluster in adult neural progenitor cells after experimental stroke. We found that stroke substantially up-regulated miR17-92 cluster expression in neural progenitor cells of the adult mouse. Overexpression of the miR17-92 cluster either in cultured ischemic neural progenitor cells or in the subventricular zone (SVZ) of ischemic animals significantly increased cell proliferation, whereas inhibition of individual members of the miR17-92 cluster, miR-18a and miR-19a, suppressed cell proliferation and increased cell death. The miR17-92 cluster mediated PTEN (phosphatase and tensin homolog) expression, which is a predicted target of the miR17-92 cluster. Addition of Sonic hedgehog (Shh) protein up-regulated miR17-92 expression and elevated c-Myc protein in ischemic neural progenitor cells, whereas blockade of the Shh signaling pathway down-regulated miR17-92 cluster expression and reduced c-Myc levels. Overexpression of c-Myc up-regulated miR17-92 cluster expression. Intraventricular infusion of Shh and a Shh receptor inhibitor, cyclopamine, to ischemic animals further elevated and suppressed, respectively, miR17-92 cluster expression in the SVZ. These data indicate that the miR17-92 cluster plays an important role in mediating neural progenitor cell function and that the Shh signaling pathway is involved in up-regulating miR17-92 cluster expression.

The role of microRNAs (miRNAs) in mediating adult neurogenesis after stroke has not been extensively studied. The present study investigated the function of the miR17-92 cluster in adult neural progenitor cells after experimental stroke. We found that stroke substantially up-regulated miR17-92 cluster expression in neural progenitor cells of the adult mouse. Overexpression of the miR17-92 cluster either in cultured ischemic neural progenitor cells or in the subventricular zone (SVZ) of ischemic animals significantly increased cell proliferation, whereas inhibition of individual members of the miR17-92 cluster, miR-18a and miR-19a, suppressed cell proliferation and increased cell death. The miR17-92 cluster mediated PTEN (phosphatase and tensin homolog) expression, which is a predicted target of the miR17-92 cluster. Addition of Sonic hedgehog (Shh) protein up-regulated miR17-92 expression and elevated c-Myc protein in ischemic neural progenitor cells, whereas blockade of the Shh signaling pathway down-regulated miR17-92 cluster expression and reduced c-Myc levels. Overexpression of c-Myc up-regulated miR17-92 cluster expression. Intraventricular infusion of Shh and a Shh receptor inhibitor, cyclopamine, to ischemic animals further elevated and suppressed, respectively, miR17-92 cluster expression in the SVZ. These data indicate that the miR17-92 cluster plays an important role in mediating neural progenitor cell function and that the Shh signaling pathway is involved in up-regulating miR17-92 cluster expression.
Stroke induces neurogenesis and neuroblasts generated in the SVZ 2 migrate to the ischemic boundary region, which par-ticipate in the brain repair process (1)(2)(3)(4). Molecular mechanisms underlying stroke-induced neurogenesis are not fully known. MicroRNAs (miRNAs), small noncoding RNAs, regulate neural stem cell function. Inhibition of miRNA biogenesis by ablation of Dicer, an endoribonuclease that cleaves doublestranded RNA and pre-miRNA into short double-stranded RNA, in neural stem cells during development results in reduction of neural stem cells, abnormal neuronal differentiation, and a thin cortical wall of the mouse brain (5). Multiple miR-NAs including miR-124a mediate adult neurogenesis (6 -13). Using miRNA array, we previously found that stroke robustly changes miRNA profiles in neural progenitor cells. Of interest, is that members of the miR17-92 cluster are increased in ischemic SVZ neural progenitor cells on the array, suggesting that this cluster could play a role in stroke-induced neurogenesis (11). The miR17-92 cluster comprises a cluster of six miRNAs on chromosome 13 that is transcribed as a single polycistronic unit (14). Despite the wealth of information about the biological effects of the miR17-92 cluster in cancer cells (15), the effect of this cluster on regulation of neural progenitor cells after stroke is unknown.
Sonic hedgehog (Shh), a secreted glycoprotein, is critical for embryonic tissue induction and patterning (16 -20). The Shh pathway plays an important role in adult neurogenesis under physiological conditions (21). Stroke up-regulates Shh expression, and blockage of the Shh signaling pathway suppresses ischemia -induced neural progenitor cell proliferation (19,21). Recent studies indicate that the Shh signaling pathway along with the miR17-92 cluster mediate the development of medulloblastomas (22,23). However, little is known about the effect of activation of the Shh signaling pathway on miR17-92 cluster expression and the biological function of the miR17-92 cluster in ischemic neural progenitor cells.
The present study investigated the effect of altering the miR17-92 cluster levels on the proliferation and differentiation of neural progenitor cells and the linkage between the Shh pathway and expression of the miR17-92 cluster. Using an animal model of focal cerebral ischemia and primary neural progenitor cells, we show that the miR17-92 cluster regulated ischemia-induced neural progenitor cell proliferation and that activation of the Shh pathway up-regulated miR17-92 cluster expression.

EXPERIMENTAL PROCEDURES
All experimental procedures were carried out in accordance with the NIH Guide for the Care and Use of Laboratory Animals and approved by the Institutional Animal Care and Use Committee of Henry Ford Hospital.
Model of Middle Cerebral Artery Occlusion (MCAO)-Male C57BL/6J mice (3-4 months) were employed in this study. The right MCA was permanently occluded by placement of an embolus at the origin of the right MCA as described previously (24). In this model, MCAO evokes a peak increase of neurogenesis 7 days after stroke (3,25). Therefore, all mice were sacrificed 7 days after MCAO.
Laser Capture Microdissection (LCM)-LCM was performed according to our published protocol (26,27). Briefly, frozen brain coronal sections (8 m) stored at Ϫ80°C were immediately immersed in acetone for 2 min of fixation and air-dried for 30s. After a brief rinse with 0.1% diethylpyrocarbonate-treated phosphate buffered saline, sections were stained with propidium iodide dye (1:5000 dilution, Sigma Aldrich) for 5 min and rinsed with phosphate-buffered saline twice. Sections were then air-dried under laminar flow for 10 min and immediately used for LCM. Dense SVZ cells on sections stained by propidium iodide were readily distinct from the ependymal cells that have cilia along the lateral wall of the lateral ventricle and from the adjacent striatal cells. In the non-ischemic mouse, the dorsal and ventral SVZ of the lateral wall was defined as a 20 -30-m-wide zone approximately of two to three cell bodies immediately adjacent to ependymal cells, whereas in the ischemic mouse, the SVZ was expanded to a 60 -80-m-wide zone (28). Propidium iodide-positive cells within the SVZ were dissected with a Leica AS LMD System (Leica Microsystems, Inc, Buffalo Grove, IL). The excised cells fell into a collection tube under gravity, ensuring contamination-free processing and minimizing sample damage. Eppendorf tubes containing 20 l of lysis buffer were stored at Ϫ80°C before miRNA isolation. Approximately 1,000 cells were isolated in the SVZ from each animal.
Implantation of an Osmotic Pump-An Alzet micro-osmotic pump was implanted in the animal brain for 7 days starting 1 day after MCAO. The intraventricular catheter with osmotic pump (1 l/hr, Alzet, Cupertino, CA) was placed into the right lateral ventricle (ipsilateral to ischemia). The catheter placement was 0.8 mm lateral and 0 mm caudal from the bregma and 2.2 mm deep. The pump was loaded with either 5 mol/liter recombinant mouse Shh (rmShh, catalog no. 461-SH-025, R&D Systems) or control vehicle (0.1% BSA in PBS).
To examine whether inhibition of the Shh pathway affects neural progenitor cell proliferation, cyclopamine (Millipore, Philadelphia, PA), a potent inhibitor of Smoothen (Smo), was intraventricularly infused at a dose of 0.11 g/h into the lateral ventricle of the ischemic hemisphere via the osmotic pump for 28 days starting 1 day after MCAO. Ischemic animals infused with vehicle were used as a control group. For labeling proliferating cells, BrdU (100 mg/kg) was administered (i.p.) daily for 7 consecutive days starting at 24 h after stroke. All animals were sacrificed 35 days after MCAO.
Plasmid DNA Transfection-The primary miR17-92 cluster (786 bp) was amplified by PCR using the following primers: forward, 5Ј-CGGAATTCGTCAGGATAATGTCAAAGTG-CTTACA and reverse, CGGGTACCACCAAACTCAACAG-GCCG, with EcoRI and KpnI at 5Ј-ends. pCAG-miR17-92 cluster plasmid was constructed by cloning a primary miR17-92 cluster fragment into the EcoRI and KpnI sites in the pCAG-GFP vector. The recombinant DNA protocol follows the National Institutes of Health guidelines. pCAG-miR17-92 plasmid (2 g) was introduced into neural progenitor cells by means of electroporation using a Nucleofector TM kit (Lonza, Walkersville, MD), an empty pCAG-GFP vector was used as a negative control. Briefly, DNA plasmids were mixed with 100 l of Nucleofector solution, and cell-DNA mixtures were transferred into a cuvette and electroporated using program A33. In addition, to examine the effect of c-Myc on miR17-92 cluster expression, neural progenitor cells were transfected with pCN-DA3.0 vector carrying c-Myc (Addgene, Cambridge, MA) or an empty vector. Total RNAs or proteins were extracted at 24 or 72 h after nucleofection for the following experiment.
miRNA and siRNA Electroporation in Vitro-miRNA hairpin inhibitors against miR-18a (mature sequence, UAAGGUG-CAUCUAGUGCAGAUAG), miR-19a (mature sequence, UGUGCAAAUCUAUGCAAAACUGA), and their mimics and cel-miR-67 (mature sequence, UCACAACCUCCUAGAAA-GAGUAGA) were purchased from Dharmacon (Chicago, IL). cel-miR-67 was used as the negative control for miRNA inhibitor and mimics. For attenuation of endogenous PTEN, SVZ neural progenitor cells were transfected with Silencer® Select siRNA-PTEN (s132220, Invitrogen), and a Silencer® Select Negative Control (Invitrogen, 4390843) was employed as a control. siRNA or miRNA oligonucleotides (200 pmol) were mixed with 100 l of nucleofector solution and were introduced into cultured neural progenitor cells using a Nucleofector TM kit as described above.
Quantification of Mature miRNAs by Real-time qRT-PCR-Total RNAs (10 ng) were reverse transcribed using a TaqMan® MicroRNA Reverse Transcription (RT) kit (Applied Biosystems, Carlsbad, CA). Each RT reaction contained 1ϫ stem-loop RT-specific primer, 1ϫ reaction buffer, 0.25 mM each of dNTPs, 3.33 units/l Multiscribe RT enzyme and 0.25 U/l RNase inhibitor. The 15-l reactions were incubated for 30 min at 16°C, 30 min at 42°C, and 5 min at 85°C and then held at 4°C. The PCR reaction was performed using a standard Taq-Man® PCR kit protocol (Applied Biosystems). Briefly, following the RT step, 1.33 l of the RT reaction were combined with 1 l of a TaqMan microRNA assay (20ϫ; forward primer, reverse primer, and probe) and 17.67 l of TaqMan® Universal PCR Master Mix, No AmpErase® UNG in a 20-l final volume. The reactions were incubated at 95°C for 10 min, followed by 40 cycles of 95°C for 15 s and 60°C for 1 min. The expression of miRNAs was normalized against the expression of U6 snRNA as an endogenous normalization control. All assays were performed in triplicate and were calculated on the basis of the ⌬⌬C t method. The n-fold change in miRNAs expression was determined according to the method of 2 Ϫ⌬⌬CT .
Neurosphere Assay-A neurosphere assay was employed to investigate the effect of miRNAs on SVZ neural progenitor cells. The assay has been widely used by us and others (7,11,19,26) as a valuable tool for investigating the biology of neural progenitor cells. The SVZ neural progenitor cells were transfected with miRNA mimics or inhibitors of miR-18a and miR-19a and incubated in growth medium in the presence of EGF and basic FGF for 24 h to allow the cells to recover. Then single cells at a density of 10 cells/l were plated directly onto laminin-coated glass coverslips and incubated for an additional 48 h to allow cell growth. Immediately after the termination of incubation, cell apoptosis was analyzed with TUNEL staining. To analyze cell proliferation, BrdU (30 g/ml, Sigma Aldrich), the thymidine analog that is incorporated into the DNA of dividing cells during S-phase, was added 18 h before the termination of incubation. BrdU-positive cells were measured (see below for quantification). To examine the SVZ cell differentiation, neurospheres were plated directly onto laminin-coated glass coverslips in DMEM-F-12 medium containing 2% FBS, which is referred to as differentiation medium. Every 4 days, one-half of the medium was replaced with fresh medium. Incubation was terminated 10 days after plating, and immunostaining for neuronal and astrocyte markers was performed for evaluation of cell differentiation.
Generation of Lentivirus and Injection of Lentivirus-Hightiter vesicular stomatitis virus-pseudotyped lentivirus were produced in 293TN cells by transient transfection of the transfer vector pre-miR17-92 cluster in pcopGFP lentivector (System Biosciences, Mountain View, CA) and pPACK packaging mixture, and concentrated by PEG-it virus precipitation solution as described in the manual. The titers of pseudoviral particles were determined by fluorescence real time PCR and were 1 ϫ 10 9 to 1 ϫ 10 10 transducing units/ml. Five l of vector concentrate was injected by a 33G Hamilton syringe (0.2 l/min) into the SVZ at stereotactic coordinates (mm from bregma): anterior-posterior ϭ 0, mediolateral ϭ ϩ0.8 cm, and dorsoventral ϭ 2.2 cm from the skull surface.
The number of BrdU-positive cells and total DAPI nuclei was counted under a 20ϫ objective (IX71; Olympus Optical, Tokyo, Japan), and the percentage of BrdU/DAPI was determined. For all measurements, we counted at least 500 cells from three wells/group (n ϭ 3 individual cultured cells).
ChIP-A ChIP assay was performed using the Magna ChIP TM A kit (Upstate, Charlottesville, VA). SVZ cells were cross-linked with 1% formaldehyde and sonicated to an average length of 200 -500 bp. The chromatin solutions were precleared with the addition of protein G beads for 2 h at 4°C. The precleared chromatins were incubated with Myc antibody (2 g, Santa Cruz Biotechnology, Inc., Santa Cruz, CA) and normal IgG serum as a negative control overnight. The antibody/ chromatin mixtures were precipitated with protein A/G magnetic beads, and magnetic beads were pelleted with a DynaMag TM -Spin (Invitrogen). The beads were sequentially washed with ChIP wash buffer. Cross-linkings were reversed by adding 4 l of 5 M NaCl and incubating at 65°C overnight. DNAs were purified by phenol/chloroform extraction and ethanol precipitation. The real-time PCR primers (forward primer, 5Ј-CCTTGTGCGACATGTGCTG, and reverse primer, 5Ј-GTTTCCCCAACTGCTGTGAT (202 bp) were used to amplify primary miR17-92 cluster promoter region flanking the Myc-binding site. Binding activities were calculated as percentage of pre-immunoprecipitated input DNA.
Statistical Analysis-The data are presented as mean Ϯ S.E. Independent sample t test was used for two-group comparisons from the non-MCAO and MCAO samples. One-way analysis of variance followed by a Student's Newman-Keuls test was performed for multiple sample analysis. A value of p Ͻ 0.05 was taken as significant.

RESULTS
The miR17-92 Cluster Up-regulated by Stroke Promotes the Proliferation and Survival of SVZ Neural Progenitor Cells-Using microarray, we previously demonstrated that stroke up-regulated miR17-92 expression in neural progenitor cells (11). To verify microarray findings, single neural progenitor cells in the SVZ were isolated from animals subjected to 7 days of MCAO by means of LCM (Fig. 1A), and levels of individual members of the miR17-92 cluster in these cells were measured by quantitative real-time RT-PCR. Compared with the cells isolated from non-ischemic SVZ, the cells from the ischemic SVZ had substantial increases in levels of miR-18a, miR-19a, and miR-19b (Fig. 1B), which is consistent with published microarray findings (11). To further confirm the in vivo findings, we examined miR17-92 cluster expression in cultured neural progenitor cells harvested from the SVZ of mice subjected to 7 days of stroke and found a significant elevation of individual members including miR-18a, miR-19a, miR-19b and miR-92a of the miR17-92 cluster (Fig. 1C). These data indicate that stroke up-regulates miR17-92 cluster expression in neural progenitor cells.
There are two miR17-92 cluster analogs, miR-106a-363 and miR-106b-25 (14). To examine whether stroke alters expression of these two clusters, levels of individual members of the two clusters were measured in ischemic neural progenitor cells using real-time RT-PCR. In contrast with the up-regulation of the miR17-92 cluster, we found that stroke substantially decreased the expression of miR-106a, miR-106b, miR-25, miR-93, and miR-363 (Fig. 1D). Another member of the miR-106a-363 cluster, miR-19b-2, was poorly expressed as indicated by a high C T value (ϳ35). We therefore, focused on the miR17-92 cluster for the following experiments.
Aforementioned data indicate that miR-18a and miR-19a were highly up-regulated among individual components of the miR17-92 cluster after stroke (Fig. 1B). Stroke increases neural progenitor cell proliferation and promotes progenitor cell differentiation into neuroblasts (1)(2)(3)(4). To examine whether upregulation of these two members affects cell proliferation and differentiation, neural progenitor cells harvested from the ischemic SVZ were transfected with miR-18a and miR-19a inhibitors, and in vitro cell proliferation and differentiation were measured. Quantitative RT-PCR analysis showed that transfection of miR-18a and miR-19a inhibitors almost completely abolished endogenous levels of miR-18a ( Fig. 2A) and miR-19a ( Fig. 2A), respectively, indicating the efficacy of ablation of endogenous miR-18a and miR-19a in ischemic neural progenitor cells. Analysis of the number of BrdU-positive cells showed that attenuation of endogenous miR-18a and miR-19a by the inhibitors substantially decreased the percentage of BrdU-positive cells (Fig. 2B). Blockade of endogenous miR-19a significantly increased the number of apoptotic cells, i.e. TUNEL-positive cells, whereas inhibition of miR-18a increased apoptotic cell death but did not reach a statistical significance (Fig. 2, C and D).
To examine whether forced increases in miR-18a and miR-19a levels further augment ischemia-induced cell proliferation, miR-18a and miR-19a mimics were delivered into ischemic neural progenitor cells. Compared with mimic controls, delivering the two mimics elevated miR-18a and miR-19a levels by 389-and 236-fold, respectively (Fig. 2E). Augmentation of miR-18a and miR-19a levels significantly increased the number of BrdU-positive cells (Fig. 2, F and G). In addition, ischemic progenitor cells were transfected with a pCAG-GFP vector carrying primary miR17-92 cluster or an empty vector (Fig. 3, A and  B). Overexpression of miR17-92 resulted in 4.3-and 6.5-fold increases in miR-18a and miR-19a levels, respectively, compared with levels in neural progenitor cells transfected with an  MAY 3, 2013 • VOLUME 288 • NUMBER 18 empty vector (Fig. 3C). Other members of the miR17-92 cluster, including miR-17, miR-19b, miR-20, and miR-92, were also up-regulated after transfection (Fig. 3C). Neural progenitor cells with overexpression of primary miR17-92 cluster exhibited a significant increase in proliferation measured by the BrdU-positive cells (Fig. 3, D and E). Overexpression of the miR17-92 cluster significantly reduced the number of glial fibrillary acidic protein (Fig. 3, F and G), but not Tuj1-and NG2-positive cells (Fig. 3G). The effect of the miR-18a and miR-19a mimics and overexpression of the miR17-92 cluster on apoptotic cells were not detected because only few TUNELpositive cells were found in control group (data not shown). To further examine the effect of overexpression of the miR17-92 cluster on neural progenitor cells in vivo, we intraventricularly injected the lentiviral vector carrying miR17-92 cluster precursor and GFP into the lateral ventricle of the ischemic hemisphere 1 day prior to stroke. Compared with ischemic animals treated with the lentiviral vector carrying only GFP, ischemic animals that received the lentiviral vector carrying miR17-92 cluster and GFP exhibited a substantial increase of neural progenitor cells measured by the number of GFP ϩ /SOX2 ϩ cells in the ischemic SVZ (Fig. 3, H and I), which is in line with in vitro data. Collectively, these in vitro and in vivo data suggest that either exogenous miR-18a and miR-19a or overexpression of the miR17-92 cluster enhance ischemia-increased neural progenitor cell proliferation.

miR17-92 Mediates Neurogenesis of SVZ Cells
Using Targetscan, miRanda, and Pictar, we found that PTEN is one of the predicted targets of miR-18a and miR19a (Fig. 4A). PTEN is well known to regulate cell proliferation and survival (29 -35). Accordingly, we examined whether elevation and attenuation of miR-18a and miR-19a alter PTEN protein levels in neural progenitor cells. Western blot analysis revealed that miR-18a and miR-19a mimics substantially reduced PTEN levels (Fig. 4B), whereas inhibitors of miR-18a and miR-19a elevated PTEN levels (Fig. 4C). These data suggest that the both miR-18a and miR19a target PTEN. Interestingly, although Fas ligand, an inducer of apoptosis (36), is not a putative target of miR-18a and miR-19a, we found that introduction of the mimics of miR-18a and miR-19a suppressed Fas ligand protein (Fig.  4B), whereas their inhibitors elevated Fas ligand levels in ischemic neural progenitor cells (Fig. 4C). To examine the effect of PTEN on neural progenitor cells, we transfected ischemic neural progenitor cells with a siRNA-PTEN or a scrambled control siRNA. Western blot analysis confirmed that siRNA-PTEN substantially decreased endogenous PTEN levels in ischemic neural progenitor cells 48 h after the transfection (Fig. 4D). Attenuation of PTEN by siRNA-PTEN considerably reduced cleaved caspase-3 proteins (Fig. 4D) and the number of TUNEL-positive cells (Fig. 4, E and F) and greatly increased the number of BrdU-positive cells (Fig. 4, G and H), compared with the cells transfected with the scrambled siRNA. These results

miR17-92 Mediates Neurogenesis of SVZ Cells
are consistent with the effect of miR-18a and miR-19a overexpression on the neural progenitor cells.
To examine the effect of Shh on miR17-92 cluster expression in neural progenitor cells, primary adult SVZ neural progenitor cells were treated with rmShh (100 ng/ml) or transfected with siRNA against Shh (siRNA-Shh) for 24 h. Transfection of siRNA-Shh reduced endogenous mRNA levels of Shh by 80% compared with cells transfected with control scrambled siRNA. Quantitative real-time RT-PCR analysis revealed that rmShh substantially increased levels of individual members of the miR17-92 cluster (Fig. 5A), whereas attenuation of endogenous Shh in ischemic neural progenitor cells by siRNA-Shh substantially decreased levels of miR-18a, miR-19a, and miR-92a compared with levels in ischemic neural progenitor cells transfected with scrambled siRNA (Fig. 5B). Shh binds to its transmembrane receptor Ptch that results in the activation of the transmembrane protein Smo and subsequently activates the Gli transcription factors (37)(38)(39). To examine whether the Shh signaling pathway regulates miR17-92 cluster expression, the ischemic neural progenitor cells were incubated with cyclopamine (5 M), a specific inhibitor of the Smo, in the presence of rmShh, and levels of individual members of the miR17-92 cluster were measured. Real-time RT-PCR analysis showed that cyclopamine suppressed rmShh-increased levels of miR-18a, miR-19a, miR-19b, and miR-92a (Fig. 5A). The effect of Shh on the miR17-92 cluster appears specific because rmShh treatment in the presence or absence of cyclopamine did not significantly affect the expression of miR-106a-363 and miR-106b-25 components on neural progenitor cells (Fig. 5D). To examine whether exogenous Shh regulates miR17-92 cluster expression in ischemic brain, rmShh or vehicle BSA was intraventricularly infused to ischemic mice for 7 consecutive days starting 1 day after stroke via an osmotic mini-pump. Single SVZ cells in these mice were isolated with LCM and levels of the miR17-92 cluster in SVZ cells were analyzed. Infusion of rmShh substantially increased levels of miR-18a, miR-19a, and miR-92a compared with treatment with BSA (Fig. 5C). In contrast, intraventricular infusion of cyclopamine suppressed ischemia-up-regulated miR-18a, miR-19a, miR-19b, and miR-92a in SVZ cells (Fig. 5E) and ischemia-increased BrdU-positive cells (Fig. 5, F and G) compared with the infusion of vehicle. These in vitro and in vivo data indicate that the Shh signaling pathway mediates miR17-92 cluster expression in neural progenitor cells.
Myc is a downstream target of Shh (40 -42). C-and N-Myc regulate miR17-92 cluster expression in HeLa cells (43), neuroblastoma (22,44,45), and cerebellar granule neuron precursors (22). Whether C-and N-Myc are involved in Shh-mediated miR17-92 cluster expression in adult neural progenitor cells is unknown. SVZ tissues were isolated from 7 day ischemic animals and ischemic animals treated with rmShh and protein levels of Myc were assayed by means of Western blot. We found that stroke significantly increased levels of c-Myc and N-Myc (Fig. 6, A and B), and intraventricular infusion of rmShh to ischemic mice further increased c-Myc protein levels (Fig. 6C). These in vivo data suggest that Shh targets Myc in neural progenitor cells. To confirm the in vivo observation, ischemic neural progenitor cells were treated with rmShh in the presence or absence of cyclopamine and Myc proteins were measured. Incubation of neural progenitor cells with rmShh considerably increased c-Myc levels, whereas cyclopamine abolished  MAY 3, 2013 • VOLUME 288 • NUMBER 18 rmShh-augmented c-Myc (Fig. 6D). These in vivo and in vitro data indicate that the Shh signaling pathway regulates c-Myc proteins in neural progenitor cells. We then examined the effect of c-Myc on miR17-92 cluster expression. Ischemic neural progenitor cells were transfected with pCNDA vector carrying c-Myc or an empty vector and expression of the miR17-92 cluster was measured. Western blot analysis confirmed that transfection of pCDNA-c-Myc plasmid increased protein levels of c-Myc (Fig. 6E). Elevation of c-Myc resulted in considerable increases in levels of individual members of the miR17-92 cluster (Fig. 6F). These data indicate that c-Myc activates transcription of the miR17-92 cluster in neural progenitor cells, which is consistent with published findings in fibroblasts (46). Using TRANSFAC software (BIOBASE Corp., Beverly, MA), we found that there are putative c-Myc binding sites in the promoter of primary miR17-92 cluster (Fig. 6G). To examine whether c-Myc binds to the miR17-92 cluster promoter, ChIP was performed. Proteins isolated from normal and ischemic SVZ neural progenitor cells were incubated with antibodies against c-Myc. The immune complexes were precipitated with magnetic protein A/G beads. The recovered DNA from the input, unbound, and bound fractions were determined by PCR, which amplified the c-Myc motif in the miR17-92 promoter sequences. A binding band was detected on ChIP (Fig. 6H), and stroke enhanced the binding of c-Myc to the promoter of miR17-92 (Fig. 6H), suggesting that Myc is involved in miR17-92-promoted neurogenesis. These results suggest that Shh could target c-Myc to regulate miR17-92 cluster expression in neural progenitor cells.

DISCUSSION
There are few reports on the biological functions of miRNAs in adult neural progenitor cells after stroke. In the present study, we found that stroke substantially up-regulated the miR17-92 cluster in SVZ neural progenitor cells. Furthermore, we demonstrate that the miR17-92 cluster up-regulated by stroke increased neural progenitor cell proliferation and sur-vival and that the Shh signaling pathway mediated miR17-92 cluster expression. Taken together, these data provide evidence for biological function of the miR17-92 cluster in ischemic neural progenitor cells.
The miR17-92 cluster comprises a cluster of seven miRNAs on chromosome 13 that are transcribed as a single polycistronic unit (47). During development, the miR17-92 cluster regulates neural progenitor cell proliferation and oligodendrogenesis (23,48). Clinical relevance of the miR17-92 cluster has recently been implicated in patients with Feingold syndrome, an autosomal dominant syndrome, and germ line deletion of the miR17-92 cluster in the patients causes microcephaly and skeletal abnormalities (49). We previously demonstrated that stroke alters miRNA profiles in SVZ neural progenitor cells (11). The present study showed that stroke robustly up-regulated members of the miR17-92 cluster, miR-18a, miR-19a, miR-19b, and miR-92a, in SVZ neural progenitor cells. Attenuation of endogenous miR-18a and miR-19a levels elevated by stroke suppressed neural progenitor cell proliferation and increased progenitor cell death, whereas addition of exogenous miR-18a and miR-19a further enhanced stroke-induced progenitor cell proliferation, indicating that these two miRNAs mediate stroke-induced neurogenesis by augmentation cell proliferation and survival. Others have reported that the miR17-92 cluster regulates oligodendrogenesis during development (48). Using conditional knock-out of the miR17-92 cluster in CNPase-expressing oligodendrocytes, Budde et al. (48) show that ablation of the miR17-92 cluster substantially reduces oligodendrocyte progenitor cell proliferation and survival at postnatal day 0. Our data showed that alteration of miR-18a and miR-19a levels inversely change PTEN protein levels and that the effect of attenuation of endogenous PTEN by siRNA-PTEN on ischemic neural progenitor cells mimics the effect of elevation of miR-18a and miR-19a on the progenitor cells, suggesting that PTEN is likely a target of miR-18a and miR-19a in mediating neural progenitor cell proliferation,

miR17-92 Mediates Neurogenesis of SVZ Cells
which is consistent with published studies showing that the miR17-92 cluster represses PTEN level on oligodendrocytes (48) and oncogenic cells (14,50,51). PTEN negatively regulates embryonic neural stem cell proliferation and survival (29 -35). Following a focal cerebral ischemia, PTEN null progenitors give rise to greater numbers of neuroblasts that migrate to periinfarct cortex (29). Interestingly, we also found that FasL levels were negatively regulated by miR-18a and miR-19a, although FasL is not a predicted target of these two miRNAs by the miRNA target algorithms. Studies in Pten ϩ/Ϫ mice and others have shown that Fas/FasL is involved in PTEN-mediated cell survival (52). Thus, the effect of miR-18a and miR-19a on FasL expression observed in the present study is likely indirectly affected by alteration of PTEN levels. Although PTEN is a featured target of miR18a and miR-19a, it is likely the miR17-92 cluster targets other potential genes in the neural progenitor cells, which coordinately regulate neurogenesis. In addition, contrary to the up-regulation of the miR17-92 cluster, we found that several components of its paralogs, miR-106b-25 and miR-106a-363 clusters, were decreased in ischemic neural progeni-  MAY 3, 2013 • VOLUME 288 • NUMBER 18 tor cells, suggesting different roles of miR-106b-25, miR-106a-363 clusters in stroke-induced neurogenesis.

miR17-92 Mediates Neurogenesis of SVZ Cells
The Shh signaling pathway mediates adult neurogenesis under physiological and stroke conditions (16 -19, 21). Our previous study demonstrated that the Shh pathway mediates carbamylated erythropoietin-enhanced proliferation of adult neural progenitor cells (19). The present study indicates that endogenous Shh in neural progenitor cells and exogenous Shh regulate miR17-19 cluster expression in neural progenitor cells and that administration of exogenous Shh to ischemic animals further up-regulated miR17-92 cluster in SVZ neural progenitor cells, whereas blockage of the Shh pathway suppressed ischemia-up-regulated miR17-92 cluster and ischemia-increased neural progenitor cell proliferation. These in vitro and in vivo data strongly suggest that the miR17-92 cluster likely mediates Shh-induced neural progenitor cell proliferation. Our findings are consistent with reports by others showing that the Shh signaling pathway up-regulates miR17-92 expression (22,23). Myc is an effector of the Shh signaling pathway, and Myc activates miR17-92 cluster expression by binding to the promoter of the primary miR17-92 cluster (40 -45). We found that Shh increased Myc levels, which was associated with up-regulation of the miR17-92 cluster. Collectively, these data suggest that the Shh signaling pathway up-regulates miR17-92 cluster expression at least in part through activation of Myc in neural progenitor cells.
In summary, the present study suggests that the miR17-92 cluster mediates adult neural progenitor cell proliferation and survival and that the Shh signaling pathway up-regulates miR17-92 cluster expression. Our data raise the possibility that augmentation of the miR17-92 cluster could amplify endogenous neurogenesis in ischemic brain.