miR-139-5p modulates cortical neuronal migration by targeting Lis1 in a rat model of focal cortical dysplasia

Accumulating evidence has indicated that microRNAs (miRNAs or miRs) play important roles in the developing rat brain. In this study, we investigated the role of miRNAs in the brains of immature (20–80 days) rats with liquid nitrogen lesion-induced focal cortical dysplasia. miRNA microarray demonstrated that the expression of miR-139-5p was associated with cortical development. Bioinformatic analysis and luciferase assays revealed that the Lis1 gene is a likely target of miR-139-5p. It is known that Lis1 plays a role in cell proliferation and migration and can lead to cortical dysplasia when mutated. Our data demonstrated an inhibitory effect of miR-139-5p on the expression of Lis1 in PC12 cells 24 h following transfection with pre-miR-139-5p. However, when the PC12 cells were transfected with anti-miR-139-5p, an increase was observed in the expression of Lis1. Cell migration assay revealed that miR-139-5p significantly inhibited the migration of PC12 and HCN-2 cells treated with or without Lis1 protein. In addition, a rat model of focal cortical dysplasia was established, wherein miR-139-5p was administered and Lis1 expression was found to be markedly reduced. Moreover, the injured cortex showed a certain degree of recovery following the administration of miR-139-5p, demonstrating that the reduction in miR-139-5p was at least partially responsible for the upregulation of Lis1 in the rat brains. Our data suggest that miR-139-5p modulates cortical neuronal migration by targeting Lis1.


Introduction
Cell proliferation, neuronal migration and cortical organization represent three important stages of brain maturation (1). Cortical development is critical for brain maturation as cortical malformations are increasingly recognized as causes of severe epileptic syndrome, neuropsychological disorders and mental retardation (2,3). Moreover, the incorrect positioning of cortical neurons following cortical cell migration often leads to cortical dysplasia. Therefore, the identification of regulatory factors responsible for brain development and maturation is important for understanding the pathogenesis of cortical abnormalities.
Lis1 is a brain-specific gene that encodes for the noncatalytic subunit of platelet-activating factor acetylhydrolase isoform 1B (PAFAH1B), which inactivates platelet-activating factor (PAF) (4,5). Lis1 is known to regulate cell proliferation and migration during brain development through its interaction with proteins, such as dynein (6). Subjects with Miller-Dieker syndrome (MDS) or isolated lissencephaly sequence (ILS) have a hemizygous deletion or mutation of the Lis1 gene (7,8). ILS and MDS often result from haploinsufficiency at human chromosome 17p13.3, a chromosomal region that includes the Lis1 gene. The disruption of Lis1 in patients with ILS and MDS (9) suggests that mutations within Lis1 are responsible for defective neuronal migration. microRNAs (miRNAs or miRs) are a class of small, noncoding, regulatory RNA molecules (10). Over the past decade, research has identified important regulatory roles for miRNAs in cell development, differentiation, proliferation, apoptosis and metabolism, as well as in the pathogenesis of several diseases (11). Approximately 70% of all known miRNAs are expressed in the mammalian brain, and the levels of many miRNAs are dramatically altered during brain development (12). However, the roles of miRNAs in the regulation of mammalian brain development are still poorly defined (13).
Further knowledge of the molecular mechanisms underlying cortical neuronal migration may provide insight into improved therapeutic options for the treatment of malformations of cortical development. In this study, we examined miRNA expression profiles in immature rats with liquid nitrogen lesioninduced focal cortical dysplasia. Our aim was to identify the miRNAs that modulate cortical neuronal migration. We identified and characterized miR-139-5p, indicating that the loss of miR-139-5p regulates cortical neuronal migration through the modulation of Lis1 expression.

Materials and methods
miRNA microarray analysis. RNA labeling and hybridization to miRNA microarray chips were performed as previously described (14). Whole brain tissues from immature Sprague-Dawley (20-80 days) rats were pooled and total RNA was extracted using TRIzol (Invitrogen, Shanghai, China). Briefly, 50 mg of total RNA were purified using the mirVana miRNA isolation kit (Ambion, Austin, TX, USA) resulting in a small, enriched RNA fraction. Purified RNA was labeled with Cy3 and hybridization was carried out using a miRNA microarray chip (CapitalBio Corp., Beijing, China) containing 381 probes in triplicate.
Western blot analysis for Lis1. The cells were rinsed once with phosphate-buffered saline (PBS) then lysed in lysis buffer [50 mmol/l Tris-HCl (pH 7.5), 5 mmol/l EDTA, 1% (V/V) Triton X-100 and 0.15 mol/l NaCl] on ice for 10 min. Insoluble components were removed by centrifugation (12,000 rpm for 5 min), and the protein concentration was measured, as previously described (19). Cellular proteins (100 µg) were adjusted to a total concentration of 5 µg protein/µl. After boiling for 5 min in loading buffer, proteins were separated by 8% Trisglycine gels for Lis1. Western blot analysis was performed using mouse anti-rat Lis1 antibody (Abcam Biotech, Cambridge, MA, USA) and goat anti-mouse HRP-linked secondary antibody. Immunocomplexes were visualized using the LumiGLO ® chemiluminescent detection kit (Cell Signaling Technology, Boston, MA, USA) according to the manufacturer's instructions.
Luciferase targeting assay and Transwell assay. We cloned 400 bp of the Lis1 3' untranslated region (3'UTR) containing the 7-bp target site for miR-139-5p into the SpeI/HindIII sites of a luciferase gene in the pMIR-REPORT luciferase vector (Ambion, Shanghai, China). PCR analyses were performed using rTaq polymerase (Takara Bio Inc., Japan). The PC12 cells (5x10 4 ) were co-transfected with 150 ng of pMIR-REPORT, Lis1-3'UTR plasmid and 25 nM of either a stably-enhanced non-targeting small RNA oligonucleotide as a negative control (control), or miR-139-5p mimics (both from GenePharma Co., Ltd.) using Lipofectamine™ 2000 (Invitrogen, Shanghai, China), as previously described (20,21). Cells transfected with pre-scramble miRNA or or anti-scramble miRNA were also used as negative controls. The transfected cells were harvested after 24 h and then assayed using a Dual-Luciferase Reporter assay system (Promega, Madison, WI, USA). The results were obtained from three separate experiments with each one conducted in triplicate.
A Transwell migration assay was performed as previously described (22). The cells (2x10 5 ) were seeded onto a 35-mm dish one day prior to transfection. The transfection protocol was conducted according to the cell culture and transfection protocols previously described (23). After 24 h of transfection, 5 ng/ml of Lis1 protein were added to the cell culture medium. The PC12 and HCN-2 cells that had migrated onto the membrane were fixed with methanol and stained with crystal violet after 24 h. Images of randomly selected fields of the fixed cells were acquired and the cells were counted. Experiments were repeated in triplicate.
Rat model of focal cortical dysplasia. Thirty-three days after birth, 30 Sprague-Dawley rats were obtained from the Laboratory Animal Center of Nanjing University (Nanjing, China). All animals were housed in a specific pathogenfree (SPF) facility and received human care according to the Chinese legal requirements. The experimental animal procedures were approved by the Nanjing Medical University Institutional Animal Care and Use Committee. All experiments were performed according to the guidelines of the European Community Council.
The rat model of liquid nitrogen lesion-induced focal cortical dysplasia was established as previously described (24). Twenty Sprague-Dawley rats with cortical dysplasia were randomly divided into two groups: the model group and the miR-139-5p administration group. In the model group, brain tissue was obtained on days 0, 20, 60 and 80. In the miR-139-5p administration group, miR-139-5p was injected into the rat brains using a rat brain locator at a 5 mg/kg dose on days 40 and 50, and brain tissue was obtained on day 60. The control (healthy; n=10) group was administered the same volume of PBS. On day 60, brain tissue was rapidly removed and prepared for hematoxylin and eosin (H&E) and Nissl staining.
H&E and Nissl staining. The rat brain tissues were rapidly fixed in 10% formaldehyde and embedded in paraffin. Sections (4-µm-thick) were mounted for H&E and Nissl staining. Brain tissue morphological characteristics and differences between the three experimental groups were observed under a microscope (Nikon, Tokyo, Japan).

Statistical analysis.
All experiments were performed in triplicate. Hierarchical cluster analysis was carried out using Gene Cluster software (Stanford University, Stanford, CA, USA). Comparisons between groups were made by a Student's t-test. A P-value <0.05 was considered to indicate a statistically significant difference, as previously described (25).

Cluster analysis for microarray data and qRT-PCR validation.
We examined the expression profiles of miRNAs in immature rats with liquid nitrogen lesion-induced focal cortical dysplasia. The dendrogram generated by cluster analysis revealed the separation of the model of dysplasia from the control samples on the basis of miRNA profiling (Fig. 1A). The relative changes in miRNA expression as shown by the qRT-PCR data were in agreement with the microarray data (Fig. 1B). These results indicate a downregulation in miR-139-5p expression during rat brain development.

miR-139-5p targets Lis1 mRNA as shown by luciferase assay.
Multiple algorithms (TargetScan, miRanda and PicTar) for predicting the putative targets of miR-139-5p identified several potential targets, such as Lis1, Capn8, Gmfb, Mapk1, Dclk1, Vim, Mgst1, Gnb1, Klf15 and Cdkn1b. Lis1 was selected for further examination due to its established role in cell proliferation and migration, as well as its role in brain development (26). Bioinformatic analysis for the target site of miR-139-5p in the Lis1 3'UTR revealed that mature miR-139-5p shares the same sequence in rats, mice and humans ( Fig. 2A). There is one 7 bp target site in the Lis1 3'UTR which is conserved in mammals. Wild-type or mutant Lis1 3'UTR was constructed in the luciferase reporter plasmid for conducting luciferase targeting assays (Fig. 2B) as it contains the 7-bp target site for miR-139-5p and the mutant 7-bp site. The luciferase activity of the wildtype plasmid was suppressed by approximately 42% at 24 h in the PC12 cells transfected with miR-139-5p mimics, while the activity of the mutant plasmid was not suppressed (Fig. 2B).
miR-139-5p inhibits cell migration. Transwell assays revealed that miR-139-5p significantly inhibited the migration of PC12 and HCN-2 cells treated with Lis1 protein (Fig. 4A). The migration ability of the PC12 and HCN-2 cells was enhanced only upon the addition of Lis1 protein; this enhanced migration was attenuated by transfection with miR-139-5p.
miR-139-5p modulates rat brain development by targeting Lis1. qRT-PCR (Fig. 5B) and western blot analysis (Fig. 5A) indicated that the mRNA and protein levels of Lis1 were higher at days 20-80 after the rat model of dysplasia was established compared with day 0 (healthy control group). Sixty days after the rat models were established, miR-139-5p was admin-istered, and this led to the marked downregulation of Lis1 mRNA (Fig. 5D) and protein levels (Fig. 5C). Additionally, it was found that miR-139-5p altered cell morphology in the rat brain, as indicated by H&E and Nissl staining (Fig. 6).

Discussion
Neuronal migration has been studied extensively for over 30 years in diverse mammalian species from mice to humans (27). Abnormal neuronal migration often leads to cortical dysplasia (28)(29)(30). Lis1 is known to regulate cell proliferation and migration during brain development (31,32) and its expression is known to be disrupted in patients with ILS and MDS, suggesting that a mutation of Lis1 leads to cortical dysplasia (30). The regulatory function of miRNAs in various developmental, migration and apoptotic pathways of diverse organisms is known. Therefore, the investigation of miRNAs that may regulate Lis1 expression could potentially lead to the development of novel therapeutic methods for the treatment of patients with cortical dysplasia.
In this study, we profiled the expression of 391 miRNAs in the brains of immature Sprague-Dawley (20-80 days) rats by miRNA microarray to identify the miRNAs responsible for cortical neuronal migration. Among the miRNAs found with significant changes in expression, we selected miR-139-5p to clarify its function in the brain. The screening standard required that the miRNA appear in all databases and have a 2 to 10-fold upregulation in expression, although multiple genes are predicted by several algorithms (TargetScan, miRanda and PicTar) as potential miR-139-5p targets. The microarray results revealed that the expression of miR-139-5p was decreased in the brains of Sprague-Dawley rats (on days 20-80), and that Lis1 is a target of miR-139-5p.
The regulatory function of miRNA in various developmental, differentiation, proliferation, migration and apoptotic pathways of diverse organisms is known (33)(34)(35). As expected, we found that miR-139-5p plays a role in rat brain development. Furthermore, miR-139-5p expression was decreased in Sprague-Dawley rats (on days 20-80) with focal cortical dysplasia induced by liquid nitrogen lesions. Due to the significant change in miR-139-5p expression during rat brain development, our data suggest that Lis1 is a target for miR-139-5p. As Lis1 is associated with neuronal migration, we speculate that miR-139-5p regulates rat cortical neuronal migration by modulating the expression of certain target genes. These targets, as predicted by multiple algorithms (36), include Lis1, Capn8, Gmfb, Mapk1, Dclk1, Vim, Mgst1, Gnb1, Klf15 and Cdkn1b (37).
Studies have previously demonstrated the upregulated expression of Lis1 by proteomic analysis of a freeze-lesion model of focal cortical dysplasia (38,39). Furthermore, Lis1 is a key gene in brain development due to its role in cell proliferation and migration (40,41). Notably, in our study, Lis1 expression was markedly upregulated at day 20 and its expression was maintained at these high levels until day 80 ( Fig. 5A and B). This is likely an important reflection of the critical role of Lis1 in promoting cell proliferation and migration. Cell migration assays revealed that miR-139-5p significantly inhibited the migration of PC12 and HCN-2 cells treated with or without Lis1 protein. At the same time, miR-139-5p was administered to the rats with focal cortical dysplasia; miR-139-5p administration markedly decreased the expression of Lis1. In addition, the injured cortex of these rat models showed a certain degree of recovery. Furthermore, we confirmed that miR-139-5p targets Lis1 and inhibits its expression, as confirmed by luciferase assay.
It can be concluded that the upregulation of Lis1 in the rat brain is at least partially caused by a reduction in miR-139-5p. As the upregulation of Lis1 can promote the occurrence of   certain developmental events in the rat brain, miR-139-5p modulates rat brain development thorugh the regulation of Lis1 expression. miR-139-5p may also contribute to rat brain development by affecting the expression of several other putative targets (30,42,43). Futher studies are required to validate other predicted targets involved in brain development. The data presented in our study suggest an important role of miR-139-5p in regulating cell migration, thus offering a novel target for the development of therapeutic agents against focal cortical dysplasia.