Inhibition of MicroRNA-383 Ameliorates Injury After Focal Cerebral Ischemia via Targeting PPARγ Open Access

Globally, stroke is the second leading cause of death in the population aged > 60 years [1]. In China, it is the first leading cause of death. Moreover, morbidity and mortality from stroke has increased over the past two decades in China [2]. Stroke results from a complex of pathological mechanisms, including neuroinflammation [3,4,5,6,7,8]. Peroxisome proliferator-activated receptor gamma (PPARγ), as nuclear receptors and transcription factors, exerts the anti-inflammatory actions. PPARγ is expressed in neurons, microglia, astrocytes and oligodendrocytes [9]. Accumulating evidence indicates that a beneficial role of PPARγ in ischemic brain injury. For example, activation of PPARγ in Alzheimer's disease was shown to alleviate neuroinflammation via inducing expression of M2 macrophage markers which suggests that PPARγ agonist may influence chronic neuroinflammation [10]. Injection of PPARγ agonist was shown to reduce infarct volume, suggesting activation of PPARγ at an early stage after ischemia may represent a survival mechanism against ischemic brain injury [11]. These results indicate that PPARγ upregulation may protect ischemic brain injury in stroke. Identification of appropriate modalities to upregulate PPARγ expression may benefit treatment of ischemic brain injury. However, the underlying regulatory mechanisms of PPARγ expression in stroke are incompletely understood.

MicroRNA (miRNA) is endogenous ∼ 22 nucleotides non-coding RNAs, which regulates target gene expression at post-transcriptional level via transcription inhibition or mRNA degradation [12]. Numerous studies have demonstrated involvement of miRNAs in cancer, cardiovascular disease, and metabolic disorders [13]. Increasing evidences suggests a critical role of miRNAs as mediators in the pathophysiology of stroke. Notably, miRNAs have been shown to mediate angiogenesis, apoptosis, and oxidative stress in ischemic brain injury. For example, downregulation of miR-140-5p in the rat model of middle cerebral artery occlusion (MCAO) model was shown to regulate angiogenesis by targeting VEGFA. This suggests miR-140-5p as a novel biomarker for cerebral ischemia [14]. Stroke was shown to increase miR-146a expression, which in turn promoted oligodendrogenesis by targeting IPAK-1 gene [15]. Over-expression of miR-30a attenuates, down-regulation of miR-30a prevents ischemic brain infarction in stroke mice via regulating HSPA5 expression [16]. The increase in level of miR-23a-3p after reperfusion was shown to attenuate oxidative injury from cerebral ischemia reperfusion [17], which suggests that miRNA may promote neuronal recovery in the early stages after onset of stroke.

In our present study, we found that miR-383 was down-regulated in a rat model of middle cerebral artery occlusion (MCAO) model. Overexpression of miR-383 increase infarct size and aggravated neurological behavior in vivo. Likewise, inhibition of miR-383 had the opposite effect in vivo. Furthermore, miR-383 was shown to directly bind to the 3'UTR of PPARγ and regulate the expression of PPARγ both in vivo and in vitro.

Male Wistar rats (180-220 g) were obtained from the Animal Centre at the Second Affiliated Hospital of Harbin Medical University. Animals were housed in temperature-controlled room with constant humidity of 50 ± 5 % on a 12h: 12h light-dark cycle. Rat model of stroke was established by middle cerebral artery occlusion (MCAO) as described in our previous study. Briefly, rats were anesthetized by intraperitoneal administration of chloral hydrate (350 mg/kg), and the left common carotid artery was exposed. After isolation and clamping the artery and its branches, 3-0 nylon suture was carefully inserted into the internal carotid artery. The blood flow was restored by carefully removing the sutures after 90 min. Rats were allowed to recover from anesthesia after wound suture.

Rats with MCAO were anesthetized with chloral hydrate (350 mg/kg) by intraperitoneal administration, and perfused with ice-cold phosphate buffered saline (pH 7.4), followed by 4% paraformaldehyde. Whole brains were removed, post-fixed overnight in 4% paraformaldehyde, and, cryoprotected for 72 h using 30% sucrose at 4°C. Fifteen equidistant coronal sections (+3.7 mm from the bregma to -6.7 mm from the bregma) were stained with cresyl violet. Slice images were digitized, and the infarct area determined in each slice.

Post MCAO neurological evaluation was performed by a blinded observer using the grading system derived from Garcia [18]. The evaluation comprised of six tests: (1) spontaneous Activity; (2) symmetry in the Movement of Four Limbs; (3) forepaw outstretching; (4) climbing; (5) body proprioception; (6) response to vibrissae touch. Grade of 0 or 1 was considered as sever impairment.

Mixed culture both neurons and astrocytes were prepared as reported in previous study. Briefly, cerebral cortices were removed from postnatal day 0 rat pups. Cells were counted and plated onto 6-well plates at 2 × 10⁶ density which were pre-coated with poly-L-lysine (0.1 mg/mL). Cells were cultured in neurobasal medium (2 % B27 supplement, 0.5 mm L-glutamine, 100 U/mL penicillin, and 100 mg/mL streptomycin), and were kept at 37 °C in a humidified incubator with 5 % CO₂. Half of the culture medium was replaced every 3 days.

The transfection experiment was performed at Day 5 after initial plating. MiR-383 mimics or AMO-383 were transfected into mixed cortical cultures using Lipofectamine™ 3000 according to the manufacturer's instructions. The transfected cells were collected after 48 h for further experiments.

Proteins extracted from rat cerebral cortices and cultured primary neuron were lysed with RIPA buffer (50 mM Tris-HCl, pH7.4; 150 mM NaCl; 0.1% Tryton X-100; 0.25% Na-deoxycholate; 0.1 M EDTA and 1% SDS) containing protease inhibitor cocktail. The BCA kit was used to measure protein concentration in the samples. Proteins were separated on SDS-PAGE gel, transferred onto PVDF membranes. The membranes were blocked with 5% non-fat dry milk for 2 h. PPARγ or β-actin was incubated overnight with their primary antibodies at 4 °C. The secondary antibodies were incubated for 1 h. The images were captured on Odyssey Infrared Imaging System (LI-COR) and the bands were quantified with Odyssey v3.0 software.

Total RNA was isolated from rat cerebral cortices and cultured primary neuron with TRIZOL reagent (Invitrogen, USA) according to manufacturer's instructions. Total RNA (0.5µg) was reversed to cDNA. SYBR Green PCR Master mix was used to determine the level of miR-383. The procedure was as follows: 95 °C for 10 min, followed by 40 cycles with 95 °C for 15 sec, 60°C for 30 sec and 72°C for 30 sec. U6 was chosen as internal control.

The wild type and mutation type 3'UTR of PPARγ containing miRNA-binding site were synthesized by Sangon Biotech Co., Ltd. HEK293-T cells were plated at 50∼60% confluence and transfected with miR-383 mimics as well as the 3'UTR luciferase vector using Lipofectamine™ 3000 transfection reagent according to the manufacturer's instructions. Forty eight hours after transfection, luciferase assay was performed using dual luciferase reporter assay kit in compliance to the manufacturer's instructions.

All data are shown as mean ± SEM. Between-group differences were assessed by Student's t test; multigroup comparisons were performed by one-way Analysis of Variance (ANOVA). All statistical analyses were performed by SPSS 22 sorftware. P < 0.05 was considered as statistically significant.

To explore the role of miR-383 in rat model of MCAO, we first evaluated the level of miR-383 in cerebral region after focal cerebral ischemia. As shown in Fig. 1A, the level of miR-383 was significantly reduced compared to the sham group. The next question was whether miR-383 indeed is involved in controlling focal cerebral ischemia initiation and maintenance. MiR-383 agomir or miR-383 antogomir was intravenously injected for 3 days prior MCAO. We found that miR-383 agomir up-regulated, while miR-383 antogomir down-regulated the level of miR-383 in the rat model of MCAO (Fig. 1B). As shown in Fig. 2, compared with MCAO group, over-expression of miR-383 significantly induced the infarct volume after MACO. Further, inhibition of miR-383 expression after MACO markedly reduced the infarct volume compared to that in the MCAO group.

To further investigate the role of miR-383 in the rat model of MCAO, we observed the change of neurological outcomes through injection of miR-383 agomir and miR-383 antogomir. As shown in Fig. 3, compared with MCAO group, overexpression of miR-383 reduced the movement symmetry of four limbs, forepaw outstretching and body proprioception. However, inhibition of miR-383 had an opposite effect. The mean total neurological score in miR-383 antogomir group (9.41 ± 0.18) was significant elevated than that in MCAO group (8.09 ± 0.1).

In our previous study, we demonstrated increased PPARγ protein levels in the frontoparietal cortex and that activation of PPARγ by Ciglitazone had a neuroprotective effect; however, whether the effect of miR-383 in MCAO is mediated via regulation of PPARγ is not unclear. Therefore, we assessed the protein level of PPARγ in rats injected with miR-383 agomir or miR-383 antogomir. Western blotting analysis showed significant downregulation of PPARγ protein levels in the miR-383 agomir group, while a marked elevation owing to downregulation of miR-383 by transfection of miR-383 antogomir was observed (Fig. 4).

To further investigate the effect of miR-383 on PPARγ expression, we used Targetscan miRNA database for PPARγ gene prediction. We found that 3'-UTR of PPARγ indeed carries the binding sites for miR-383 (Fig. 5A). Therefore, in the next step, we established the relationship between miR-383 and PPARγ using both gain-and loss-of-function approaches. As illustrated in Fig. 5B, miR-383 significantly inhibited luciferase activities of PPARγ, while mutation at the binding sites reduced the repressive effects of miR-383. To observe the effect of miR-383 on PPARγ expression, we transfected the miR-383 mimics and AMO-383 into cortical cultures. Real-time PCR analysis showed that transfection of miR-383 mimics or AMO-383 was successful in cortical cultures (Fig. 5C). Compared with the NC group, over-expression of miR-383 could down-regulate the protein level of PPARγ. Conversely, known of miR-383 by AMO-383 elevated PPARγ protein levels (Fig. 5D), indicating that PPARγ is a potential target for miR-383.

In this present study, we found that the expression of miR-383 was decreased in rats following MCAO. MiR-383 could regulate its target gene PPARγ both in vivo and in vitro. Overexpression of miR-383 induced, inhibition of miR-383 reduced infract volume and neurological damages after MCAO surgery. Therefore, our study reveals novel insights into the molecular mechanism of PPARγ-mediated neuroprotection in stroke at the miRNA level. Further, our results indicate that the possibility of miR-383 as an endogenous miRNA agonist for stroke.

PPARγ is a nuclear transcription factor, which could regulate adipocyte differentiation, lipid metabolism, and insulin resistance. PPARγ agonist, such as Rosiglitazone or Pioglitazone, is usually used to treat hyperlipidemia or Type II diabetes. Recent studies have suggested a neuroprotective role of PPARγ in stroke. PPARγ agonist has been shown to be ideal candidates for central nervous system diseases owing to their effect in reducing the synthesis and secretion of pro-inflammatory cytokines. The anti-inflammatory effect of PPARγ agonist, Pioglitazone, was shown to attenuate ischemic brain injury in the rat model of permanent MCAO [19]. Further, pioglitazone may ameliorate ischemic injury by reducing oxidative stress and elevating nitric oxide level. In neurons, PPARγ agonists were shown to decrease the expression of COX-2, an enzyme that deteriorates the ischemic brain injury [20]. PPARγ ligands attenuated cell death in cerebellar granule cells [21]. Similarly, administration of PPARγ ligand in MCAO could upregulate PPARγ in the peri-infarct cortex. Our findings both in the present and previous studies also showed that PPARγ protein level was increased after 24h MCAO, which is consistent with other studies [22,23].

Increasing evidences have been indicated that miRNAs play the critical roles in response to cerebral ischemia. For example, miR-497 was shown to upregulate in the ischemic brain through binding with 3'UTR of Bcl-2. Inhibition of miR-497 could upregulate Bcl-2 expression and reduces the infarct volume [24]. MiR-140-5p regulated angiogenesis in response to MCAO by influencing cell proliferation, migration and tube formation by directly targeting VEGFA. MiR-107 directly binds to Dicer-1, influences angiogenesis and protects ischemia-induced injury after stroke [25]. Inhibition of miR-155 could reduce infarct size, improve brain microvasculature and ameliorate functional recovery in animal model [26]. Hamzei et al. found that injection of miR-124 at early stage after MCAO significantly increased neuronal survival and number of M2-like polarized microglia/macrophages [27]. Decreased levels of miR-383 in human malignant tumors were shown to inhibit growth, proliferation, migration, and invasion of U85 and U251 cells [28,29]. MiR-383 acts as an antineoplastic agent via regulation of cancer cell apoptosis [30]. In our present study, decreased expression of miR-383 was found at 24h after MCAO. As indicated by the results of western blotting and luciferase assay, miR-383 could directly regulate PPARγ expression, which suggests a neuroprotective effect of miR-383 due to it anti-inflammatory properties. Here, we found that injected miR-383 agomir to overexpression of miR-383 lead to induce the infarct volume and neurological damage; however, inhibition of miR-383 owing to miR-383 antogomir injection could decrease infarct volume and improve neurological damage in response to stroke.

Our study provided evidence that downregulation of miR-383 could upregulate PPARγ expression at an early stage after ischemia, and exert anti-inflammatory effect and neuroprotection function against ischemic brain injury, suggesting a novel way to prevent damage of stroke, and provide a potential therapeutic target for stroke treatment.

This work was supported in part by the Postdoctoral Science Foundation of the Second affiliated Hospital of Harbin Medical University (BS 2011-09).

The authors declare that no competing interest exists.