MiR‐199a‐5p enhances neuronal differentiation of neural stem cells and promotes neurogenesis by targeting Cav‐1 after cerebral ischemia

Abstract Aims MicroRNAs (miRs) are involved in endogenous neurogenesis, enhancing of which has been regarded as a potential therapeutic strategy for ischemic stroke treatment; however, whether miR‐199a‐5p mediates postischemic neurogenesis remains unclear. This study aims to investigate the proneurogenesis effects of miR‐199a‐5p and its possible mechanism after ischemic stroke. Methods Neural stem cells (NSCs) were transfected using Lipofectamine 3000 reagent, and the differentiation of NSCs was evaluated by immunofluorescence and Western blotting. Dual‐luciferase reporter assay was performed to verify the target gene of miR‐199a‐5p. MiR‐199a‐5p agomir/antagomir were injected intracerebroventricularly. The sensorimotor functions were evaluated by neurobehavioral tests, infarct volume was measured by toluidine blue staining, neurogenesis was detected by immunofluorescence assay, and the protein levels of neuronal nuclei (NeuN), glial fibrillary acidic protein (GFAP), caveolin‐1 (Cav‐1), vascular endothelial growth factor (VEGF), and brain‐derived neurotrophic factor (BDNF) were measured by Western blotting. Results MiR‐199a‐5p mimic enhanced neuronal differentiation and inhibited astrocyte differentiation of NSCs, while a miR‐199a‐5p inhibitor induced the opposite effects, which can be reversed by Cav‐1 siRNA. Cav‐1 was through the dual‐luciferase reporter assay confirmed as a target gene of miR‐199a‐5p. miR‐199a‐5p agomir in rat stroke models manifested multiple benefits, such as improving neurological deficits, reducing infarct volume, promoting neurogenesis, inhibiting Cav‐1, and increasing VEGF and BDNF, which was reversed by the miR‐199a‐5p antagomir. Conclusion MiR‐199a‐5p may target and inhibit Cav‐1 to enhance neurogenesis and thus promote functional recovery after cerebral ischemia. These findings indicate that miR‐199a‐5p is a promising target for the treatment of ischemic stroke.


| INTRODUC TI ON
Stroke as a common central nervous system (CNS) disease with high prevalence has been well established as a leading cause of disability and mortality. 1In particular, approximately 87% of cases can be further recognized as ischemic stroke. 2 Recanalization, such as thrombolysis and endovascular treatment (EVT), are common treatment strategies for ischemic stroke, 3 which, however, is limited by the narrow time range for therapeutic time window as well as by the risk of hemorrhagic transformation.Many patients thereby failed to receive such treatment in a timely manner. 4,5It has been observed that the regenerative processes following a stroke, which may be inefficient though, persist actively in the CNS from days to weeks. 6An optimization of such regenerative process through promoting brain tissue repair therefore seems to be a promising approach to overcome the narrow therapeutic time window of stroke. 6ural stem cells (NSCs) demonstrate such characteristic of self-renewal and pluripotent differentiation that they can generate neurons, astrocytes, and oligodendrocytes in the nervous system. 7,8In adult brains, NSCs are primarily located in two niches: the subventricular zone (SVZ) of the lateral ventricle and the subgranular zone (SGZ) of the dentate gyrus (DG) of the hippocampus. 9,10Numerous preclinical and clinical studies have confirmed the ischemic insults activated endogenous neurogenesis, involving proliferation, migration, differentiation of NSCs, and integration into impaired neural networks. 11The stroke-induced neurogenesis, however, is insufficient to restore full neurological function. 9,12e majority of injury-activated NSCs eventually differentiate into astrocytes.Transformation of the newly generated astrocytes into reactive ones in the injured brain contributes to the glial scar border and inhibits axon regeneration and neurogenesis. 13Exploring the molecular mechanisms of the stroke-induced neurogenesis may thus facilitate the development of new treatment strategies for superior functional recovery.
MicroRNAs (miRNAs, miRs), which by increasing evidence have been closely related to the neuronal differentiation of NSCs and adult neurogenesis, are small noncoding RNAs that combine to the 3'untranslated region (3'UTR) of specific mRNAs and act as their post-transcriptional suppressant. 14For one specific instance, miR-199a-5p has been detected in the CNS and participated in cerebral ischemic injury in rats. 15,16It has been found in NSCs through high-throughput sequencing approach a higher miR-199a-5p expression in ischemic SVZ than that in non-ischemic SVZ, suggesting that miR-199a-5p may mediate neurogenesis after cerebral ischemia. 17Caveolin-1 (Cav-1), 18 which hinders the differentiation of NSCs to neurons and oligodendrocytes and as well promotes the differentiation of NSCs to astrocytes, [19][20][21] is one of the main target genes of miR-199a-5p, together with silent information regulator 1(SIRT1), 22 hypoxia-inducible factor 1α (HIF-1α). 23However, whether miR-199a-5p promotes postischemic neurogenesis through targeted inhibition of Cav-1 remains unclear.
In this study, it is aimed to identify whether miR-199a-5p/Cav-1 pathway by enhancing endogenous neurogenesis promotes functional recovery after ischemic stroke.

| IdentificationofNSCs
Firstly, the cells were immunostained with Nestin, a marker of NSCs, and then identified by proliferation and differentiation.For proliferation, single NSCs were cultured in polyd-lysine-coated plates.Twenty-four hours later, the medium was changed with the 5-bromo-2′-deoxyuridine (BrdU) labeling solution (10 μM, Sigma-Aldrich) for 48 h, and the cells were then immunolabeled for Nestin and BrdU.For differentiation, the cells were cultured in DMEM/ F12 containing 1% fetal bovine serum (FBS, Gibco).After 7 days of differentiation, the cells were immunolabeled for neuronal, astrocytic, and oligodendrocytic markers (NeuN, GFAP, and Olig2, respectively).

| Experimentalanimals
Male SD rats (280-300 g, SPF grade) were obtained from the Sino-British SIPPR/BK Laboratory Animal (Shanghai, China).The rats were housed at 22 ± 2°C (a cycle of 12 h light/12 h dark).This study was authorized by the Experimental Animal Care and Ethics

| Establishmentofthemiddlecerebralartery occlusion (MCAO) model
The MCAO model was constructed as described previously. 26In brief, rats were anesthetized under isoflurane.After the right common (CCA) and external carotid artery (ECA) were isolated, the internal carotid artery (ICA) was exposed.A small opening was cut at the distal end of the ECA, and then a nylon monofilament suture with a tip diameter of 0.38 mm was advanced 18 ± 2 mm from the bifurcation of the carotid artery to block MCA origin.After 90 min of ischemia, the suture was withdrawn to restore the blood supply.Rats of the sham-operation group underwent similar operations, but the nylon suture was inserted with a depth of 5 mm.The temperature of rats was controlled at around 37°C by a heating lamp during the whole surgical procedure.

| Intracerebroventricularinjection
The intracerebroventricular injection was conducted following a previous report. 27In brief, the rats were anesthetized and set in a stereotaxic apparatus.The coordinates of intracerebroventricular injections were as follows: AP: −0.8 mm, L: −1.4 mm, V: −4.8 mm, approved by the stereotactic atlas of the rats.MiR-199a-5p agomir/ NC (RiboBio, 5 μL/2.5 nmoL, dissolved in aCSF) and miR-199a-5p antagomir/NC (Ribobio, 5 μL/10 nmoL, dissolved in aCSF) or sterile aCSF (5 μL) were then slowly injected into the lateral ventricle at a speed of 0.25 μL/min at day 5 following MCAO.Then, the needle was maintained for an additional 10 min, followed by slow retraction.

| Neurologicalfunctionevaluation
Neurological deficit assessments were detected by researchers who were blinded to the design of experiments.All rats were tested for the modified neurological severity score (mNSS) and the corner test to evaluate neurological disorders on days 1, 3, 7, and 14 following MCAO.The mNSS is an 18-point scoring system (0, normal; 18, maximal deficit). 28The corner test was applied to determine the sensorimotor function by counting the number of turns to the right of 10 trials. 29

| Detectionofthevolumeof cerebral infarction
Rats were sacrificed and were perfused with saline followed by 4% paraformaldehyde (PFA) on day 14 after ischemia.The brains were fixed and dehydrated for frozen sections slicing.A 20 μm coronal section of brain tissue was immersed in 1% toluidine blue (Sigma-Aldrich), followed to be dehydrated by gradient alcohol.The following equation measured the infarct volume percentage: volume of cerebral infarction (%) = (contralateral hemisphere volume − noninfarct ipsilateral hemisphere volume)/contralateral hemisphere volume × 100%.

| Immunofluorescencestaining
For immunofluorescence staining for cells, the cells were fixed with 4% paraformaldehyde (PFA) for 15 min.Cells were then blocked for 1 h (10% goat serum) after permeabilizing with 0.1% Triton X-100.For BrdU immunofluorescence, the cells were placed in HCl (2 moL/L) for 30 min, then neutralization with boric acids (0.1 moL/L) for 10 min, and then 3% H 2 O 2 .After that, cells were placed into primary antibodies of Nestin For immunofluorescence staining for brain slices, sections were post-fixed with 4% PFA for 0.5 h and incubated with 2 × salinesodium citrate buffer supplemented with 50% formamide for 2 h at 65°C and placed in HCl (2 moL/L) for 0.5 h at 37°C.After being washed 3 times, the sections were neutralized with boric acids (0.1 moL/L) and then permeabilized, followed by 3% H 2 O 2 at 37°C for 20 min.The tissue sections were then blocked for 1 h (5% goat serum), sequentially incubated with primary antibodies of BrdU (1:100), doublecortin (DCX, 1:100, Cell Signaling Technology), NeuN (1:500), and GFAP (1:100, Cell Signaling Technology) and then the corresponding secondary antibodies.The number of positive cells was counted from three sections per rat.Three fields were randomly selected from each section, and the average number of positive cells was calculated.All analysis was conducted with the examiner blinded to the identity of the samples being studied.

| Quantitativereversetranscriptionpolymerase chain reaction (RT-qPCR)
The total RNA was isolated with Trizol reagent (Invitrogen), and the reverse transcription reaction was conducted with Mir-XTM miRNA First-strand Synthesis Kit (TaKaRa) for miRNA and PrimeScript RT Master Mix (TaKaRa) for mRNA.The real-time quantitative PCR (qPCR) was performed with a TB Green Premix Ex Taq Kit II (TaKaRa) or TB Green Premix Ex Taq Kit (TaKaRa) on the Real-Time PCR instrument (Applied Biosystems® 7500, Thermo Fisher Scientific).
The results were calculated using the 2 −ΔΔCT method.The primers for RT-qPCR are listed in Table 1.

| Westernblotting
The total proteins were extracted using RIPA lysis buffer and measured with a BCA assay kit.Proteins were resolved by SDS-PAGE.

| Statisticalanalysis
All data are presented as mean ± SEM, and all statistical analyses were two-tailed with SPSS 22.0 Statistics.The Shapiro-Wilk test was used to analyze the normality of data.For normally distributed data, a one-way analysis of variance was conducted, followed by the Bonferroni test if there were more than two groups.Nonparametric Kruskal-Wallis H test was performed to analyze the results from the mNSS and the corner test.Differences in survival rate between the groups were analyzed using the log-rank (Mantel-Cox) test.p < 0.05 were considered statistically significant.

| IdentificationofNSCs
The primary NSCs separated from the cerebral cortex of fetal rats rapidly proliferated and gave rise to free-floating small clusters (neurospheres) after 48 h.The clusters continued to grow in size during expansion (Figure 1A).After 3 passages of expansion, the purity of NSCs was verified up to 99.12% by immunofluorescence staining of biomarker Nestin (a marker for NSCs) both in neurospheres and monolayer NSCs (Figure 1B).Meanwhile, the BrdU labeling was applied to examine the proliferation of NSCs.It was shown that approximately 20.83% of NSCs were dual-positive staining of BrdU/Nestin (Figure 1C).After 7 days of differentiation, 19.05% of NSCs were differentiated into NeuN + cells (a marker for neurons, Figure 1D), while 63.09% of NSCs into GFAP + cells (a marker for astrocytes, Figure 1D), and 17.86% of NSCs into Olig2 + cells (a marker for oligodendrocytes, Figure 1D).These results demonstrated that the cultured NSCs were capable of further proliferation and differentiation.

| miR-199a-5ppromotedneuronal differentiation of NSCs
To determine the function of miR-199a-5p on the differentiation of NSCs, we first measured the expression of miR-199a-5p by RT-qPCR at the 3rd, 7th, and 14th d during NSCs differentiation.The involvement of miR-199a-5p in the differentiation of NSCs can be indicated by its gradually rising expression over time during NSC differentiation (Figure 2A).Both gain-of-function and loss-offunction approaches indicated a dramatically enhanced expression of miR-199a-5p in the miR-199a-5p mimic group, whereas decreased expression of miR-199a-5p in the miR-199a-5p inhibitor group (Figure 2B,C).Furthermore, the percentage of NeuN + cells was significantly enhanced after the treatment of miR-199a-5p mimic, while those of GFAP-positive cells decreased (Figure 2D-F).In contrast, repressing miR-199a-5p by transfection with its specific inhibitor exerted the opposite effects (Figure 2D-F).The Western blotting results were similar to those obtained from the immunofluorescence staining (Figure 2G-I).Collectively, the above observations TA B L E 1 Primers used for the RT-qPCR.

| DISCUSS ION
That ischemic stroke often leads to long-term neurological deficits, with no specific therapeutic options available for late recovery currently has become a major concern. 30We demonstrate in the current study that miR-199a-5p by downregulating Cav-1 in vitro motivated neuronal differentiation and suppressed glial differentiation of NSCs.
Moreover, the in vivo study of miR-199a-5p showed in rats its enhancing endogenous neurogenesis and improving functional outcomes after focal cerebral ischemia.These data suggested that miR-199a-5p improve the recovery of neural function by promoting neuronal differentiation of NSCs after ischemic stroke.
miRNAs participation in the occurrence and progression of ischemic stroke in animals and patients have been confirmed by either miRNA expression profile or sequencing from multiple research. 31,32In particular, the effect of miR-199a-5p on ischemia-induced neural plasticity has been widely studied. 33,34Expression of miR-199a-5p was explicitly found in nerve tissue, with its biofunctions involving the remodeling of the neurovascular unit and synaptic plasticity. 33,34miR-199a-5p also preserves against hypoxia-induced cerebral injury, spinal cord injury, and myocardial injury. 16,35,36Nevertheless, the role of miR-199a-5p during the post-ischemic neurogenesis had not been determined.
NSCs as pluripotent stem cells with self-renewal ability can differentiate into neurons, astrocytes, and oligodendrocytes. 9Focal cerebral ischemia can motivate endogenous neurogenesis to improve functional recovery by promoting NSC proliferation, and differentiation into new neurons, as well as their migration to damaged brain areas. 11In our study, we verified the vital function of miR-199a-5p in regulating endogenous neurogenesis.DCX + neuroblasts are usually identified as migrating neuroblasts that already possess neuronal commitment. 9An increase of BrdU + /DCX + cells was observed following the administration of miR-199a-5p agomir, whereas the number of BrdU + /DCX + cells significantly decreased with the treatment of miR-199a-5p antagomir.Thus, these findings indicate that miR-199a-5p after stroke promotes the proliferation and recruitment of SVZ NSCs in rats.
Under pathophysiological conditions, most NSCs eventually differentiate into astrocytes which in the injured brain can be transformed into reactive astrocytes and promote the formation of glial scars, thereby inhibiting axon regeneration and hindering neurogenesis. 13,37In this study, we found that the expression of miR-199a-5p elevated at 14th d after cerebral ischemia.After transfected with miR-199a-5p mimic, NSCs enhanced neuronal differentiation and weakened astrocyte differentiation in vitro.However, this effect was reversed by the miR-199a-5p inhibitor.More importantly, miR-199a-5p promoted neuronal differentiation of NSCs in the periinfarct regions in the rat model of ischemic stroke, as confirmed by double labeling with BrdU and NeuN.Notably, the miR-199a-5p agomir did not affect the number of BrdU + /GFAP + cells in vivo.
Collectively, miR-199a-5p did change the differentiation fate of NSCs, leading to increased neurogenesis and decreased gliogenesis.
The predicted target genes of miR-199a-5p showed a corresponding binding site with Cav-1 at the 3′UTR.A dual-luciferase reporter assay apart from bioinformatic analysis was performed and further confirmed the relationship between Cav-1 and miR-199a-5p, which was consistent with the results of previous studies. 18,38The inhibited Cav-1 protein upon overexpression of miR-199a-5p and increased Cav-1 induced by miR-199a-5p repression suggested that Cav-1 in NSCs be a target gene of miR-199a-5p.Cav-1 refers to a small spherical invaginated plasma membrane of 50-100 nm.Li et al.
0][21] Studies have shown that Cav-1 inhibited the differentiation of NSCs into neurons by regulating VEGF and BDNF, [19][20][21] both of which were considered the most potent and specific angiogenesis and neurogenesis factors. 39,40In the present study, we found that the down-regulation of Cav-1 following the treatment of miR-199a-5p agomir was accompanied by the increased expression of VEGF and BDNF after ischemic stroke in rats.In contrast, the elevation of Cav-1 following the treatment of miR-199a-5p antagomir was simultaneously along with lower expression of VEGF and BDNF.These results suggest that the low expression of Cav-1 induced by miR-199a-5p contributes to the increased expression of BDNF and VEGF, eventually altering the differentiation fate of NSCs and promoting neurogenesis.
Although this study presented evidence that miR-199a-5p promoted neuronal differentiation of neural stem cells and endogenous neurogenesis after ischemic stroke, there were certain limitations.
First, previous reports showed the involvement of a large number of miRNAs in the pathophysiological processes of stroke, in the present study however, due to the major focus on exploring the role of miR-199a-5p, the remaining miRNAs that might also contribute to long-term outcomes of ischemic nerve repair were not included, but will further be taken into consideration in the following research.
Second, it is unsurprising that a single miRNA may modulate a series of transcripts, and various miRNAs can modulate the same mRNA as well.Multiple target genes of miR-199a-5p have been identified, such as CDKN1B and Sirtuin-1. 41,42Thus, we cannot rule out the possibility of the participation of potential targets other than Cav-1 in miR-199a-5p-mediated endogenous neurogenesis.

| CON CLUS IONS
Results of the present study supported a critical role of miR-199a-5p by inhibiting Cav-1 expression to promote the neuronal differentiation of NSCs and endogenous neurogenesis in rats after cerebral ischemia.These findings further suggest that miR-199a-5p/Cav-1 pathway be a novel therapeutic approach for the postischemic stroke functional recovery.
find out the effects of Cav-1 on NSCs differentiation, NSCs were transfected with different concentrations (20 nM, 50 nM, F I G U R E 1 Identification of NSCs.(A) Morphology of primary cultured NSCs passage 0 (upper panel) and day 3 of passage 3 (lower panel).Scale bar = 200 μm.(B) Immunofluorescence staining of NSC marker.Cells were positive for Nestin in neurospheres and monolayer NSCs cultured in PDL-coated well plates.Scale bar = 100 μm.(C) Representative immunofluorescence staining of BrdU (red) and Nestin (green) with nucleus (blue).Scale bar = 100 μm.(D) Representative immunofluorescence staining of NeuN (green), GFAP (red), and Olig2 (purple) to identify multipotency of the primary cultured NSCs.Scale bar = 100 μm.| 3973 JIN et al. and 100 nM) of Cy3-conjugated Cav-1 siRNA.The results showed a highest transfection efficiency of 100 nM Cav-1 siRNA (Figure4A).Dramatical inhibition of Cav-1 protein by Cav-1 siRNA was also seen from the western blot results (100 nM, Figure4B,C).Next, after cotransfection with miR-199a-5p inhibitor (100 nM) and Cav-1 siRNA (100 nM), immunofluorescence staining demonstrated a reduction in the ratio of NeuN + cells and enhancement in the ratio of GFAP + cells by the treatment of miR-199a-5p inhibitor (Figure4D-F).However, these effects were reversed as Cav-1 was concurrently silenced (Figure4D-F).In addition, the protein expression of NeuN and GFAP was further confirmed by Western blotting (Figure4G-I).These results indicated that miR-199a-5p promoted neuronal differentiation of NSCs by downregulating Cav-1.
miR-199a-5p antagomir facilitated the expression of Cav-1 at 14th d after ischemia (Figure7A-C).The above findings suggested that the miR-199a-5p boosted endogenous neurogenesis by negatively modulating the Cav-1 target gene.Subsequently, the expression of vascular endothelial growth factor (VEGF) and brain-derived neurotrophic factor (BDNF) were measured, which are key neurotrophic factors for neurogenesis after ischemic stroke.MiR-199a-5p agomir notably elevated the expression levels of VEGF and BDNF (Figure7B,D,E).In contrast, the administration of miR-199a-5p antagomir decreased their expressions compared with the MCAO group (Figure7B,D,E).

F I G U R E 6
miR-199a-5p promoted endogenous neurogenesis in the peri-infarct regions after ischemia in rats.(A-C) Representative images of BrdU/DCX, BrdU/NeuN, and BrdU/GFAP double immunofluorescence staining in the peri-infarct regions after ischemic stroke in rats.(D-F) Quantification of the number of BrdU + /DCX + , BrdU + /NeuN + and BrdU + /GFAP + cells in the peri-infarct regions.Scale bar = 50 μm, the data were presented as the mean ± SEM, n = 8, and **p < 0.01.

F I G U R E 7
miR-199a-5p promoted endogenous neurogenesis through inhibiting Cav-1 and increasing VEGF and BDNF expression.(A) The mRNA expression levels of Cav-1 were determined by RT-qPCR at 14th d after ischemia.n = 4. (B) Representative bands of Cav-1, VEGF, and BDNF expressions by western blot.(C-E) Semiquantitative analysis of the Cav-1, VEGF, and BDNF proteins.The data were presented as the mean ± SEM, n = 3. *p < 0.05, and **p < 0.01.