microRNA152-3p protects against ischemia/reperfusion-induced BBB destruction possibly targeting MAP3K2/JNK/c-Jun pathway

In the current study, we reported that overexpression of miR-152-3p effectively ameliorated neurological de�cit and protected blood-brain barrier(BBB) integrity in middle cerebral artery occlusion (MCAO) rats. In in vitro model, the level of miR-152-3p was signi�cantly decreased in bEnd.3 after oxygen–glucose deprivation/reperfusion (OGD/R) insult. miR-152-3p overexpressed bEnd.3 cell monolayers were protected from OGD/R-induced microvascular hyperpermeability. miR-152-3p-mediated protective effect was associated with lower apoptosis of endothelia by negatively modulating MAP3K2/JNK/c-Jun pathway.

Invitrogen), active caspase-3(1:1000, Invitrogen), caspase-3(1:2000, Invitrogen), β-actin (1:5000, Millipore Sigma) was used as a loading control. Afterwards, the membranes were washed and transferred into a buffer with appropriate secondary antibodies for 1h at room temperature. An ECL detection kit was used to visualize the protein bands. Image J (LOCI, Madison, WI, USA) software was used for quantitative analysis of western blot results.

RT-qPCR
The total RNA was extracted using TRIzol®. Total RNA was reverse transcribed into cDNA using RNA Reverse Transcription Kit (AB-4366596, Invitrogen). The expression of miR-152-3p was measured using SYBR® Premix Ex Taq™ II (Takara) (Applied Biosystems, Foster City, CA, USA). The reaction was performed as follows: 94°C for 2 min and 40 cycles of 94°C for 30s, 60°C for 20 s, and 72°C for 20s. U6 served as an internal reference. The data were analyzed based on the 2-ΔΔCt method [16]. According to GenBank, the primers were designed as follow (Table 1). The barrier integrity of Bend.3 and FITC-Dextran permeation were analyzed according to the protocols detailed in our previous study [17].

Flow cytometry
Apoptosis of bend.3 was measured by ow cytometry using an assay kit (C1062L, Beyotime, Shanghai, China) in accordance with the manufacturer's protocol. Brie y, Bend.3 were made into cell suspension (5 × 105 cells/mL) using PBS. Then 5 µl FITC-annexin V and 5 µl propidium iodide (PI) were added into suspension for 10 minutes at room temperature without light exposure. The cells were analyzed by ow cytometry using FACScan ow cytometry system (Becton Dickinson, San Diego, CA, USA).

TUNEL staining
To measure the cell apoptosis, TUNEL staining (Yisheng Biotechnology, shanghai, China) was performed according to the manufacturer's instructions. Consecutive 1 mm2 elds in the ischemic penumbra of six randomly rats were selected to take photographs by a uorescence microscope. The nal results were quanti ed as number of TUNEL-positive cells/ mm2 tissue.

Luciferase assay
The wild type of the 3′UTR of the MAP3K2 gene (containing the binding site with miR-152-3p) and mutant 3′UTR of the MAP3K2 gene were synthesized by Shanghai Beinuo Biological Technology and inserted into pmirGLO luciferase reporter vectors to generate MAP3K2 WT and MAP3K2 MUT. Luciferase assays were performed via co-transfection with pmiR-MAP3K2-WT or pmiR-MAP3K2-MAP3K2-Mut and miR-152-3p mimics using Lipofectamine 2000 (Invitrogen). After 48 h, relative luciferase activity was measured using the dual-luciferase reporter assay system (Promega).

Statistical analysis
All data were shown as mean ± standard error (SEM). Differences were evaluated using one-way ANOVA (Tukey's multiple comparison test) or Student's t-test (two groups). For statistical analyses, GraphPad Prism 8(GraphPad Software) was used. p < 0.05 was considered statistically signi cant.

Intracerebroventricular injection of agomir-miR-152-3p improved poststroke outcomes in MCAO rats
To explore the protective effect of miR-152-3p in vivo, the rats received an intracerebroventricular (i.c.v) injection of agomir-miR-152-3p prior to MCAO treatment. Based on qRT-PCR results, miR-152-3p level was signi cantly higher in rats injected with agomir-miR-152-3p compared with rats injected with agomir-NC ( Fig. 1A). As shown in Fig. 1B and 2C, TTC staining of brain sections showed that the sham group exhibited no damage, while at 24h post-MCAO, an infarcted area was obviously observed. The infarcted region was signi cantly reduced by agomir-miR-152-3p. Moreover, after MCAO, rats exhibited signi cant neurological de cits compared with sham group, while pre-treatment of agomir-miR-152-3p signi cantly reduced neurological de cit scores compared with MCAO group (Fig. 1D).

miR-152-3p blunted the BBB damage after MCAO/R
Evans blue (EB) extravasation were used to evaluate BBB disruption. We observed that EB leakage was increased in the MCAO + agomir-NC group compared to the sham group, while agomir-miR-152-3p pretreatment remarkably reduced MCAO-induced EB extravasation ( Fig. 2A). Tight junction proteins (TJPs) such as occludin and claudin-5 are strongly related with BBB stability [18]. Hence, the expressions of occludin and claudin-5, the major TJPs in the membrane of BMECs, were also assessed by western blot in the present study. The level of occludin and claudin-5 was signi cantly decreased after MCAO compared with the sham group, and this effect was signi cantly ameliorated by miR-152-3p ( Fig. 2B-D). Consistently, occludin/CD31 double immune-uorescence staining revealed that MCAO induced lower intensity of occludin and junctional discontinuity than sham group. The degradation of TJPs was reversed by miR-152-3p (Fig. 2E).
A monolayer of Bend.3 grown in the Transwell insert was employed as an in vitro blood-brain barrier model. In order to examine the neuro-protective effect of miR-152-3p during OGD/R, we rst investigated miR-152-3p expression in bEnd.3 under OGD/R condition. RT-qPCR result showed that miR-152-3p expression was markedly downregulated following OGD/R (Fig. 3A), indicating that miR-152-3p may be associated with BBB pathology after OGD/R. To investigate the effect of miR-152-3p on barrier function after OGD/R injury, gain-of-function experiment was performed through transfecting miR-152-3p mimics into bEnd.3. miR-152-3p expression was upregulated by miR-152-3p mimics con rmed by RT-qPCR (Fig. 3B). We observed that OGD/R caused signi cant increase in paracellular permeability to FITC-(4kDa) dextrans across bend.3 monolayers compared to the control group, while miR-152-3p overexpression reduced the permeability compared to OGD/R group (Fig. 3C). In line with the results of paracellular permeability, the TEER value in OGD/R-exposed endothelial monolayers was signi cantly decreased compared with control group. However, miR-152-3p protected endothelial monolayers from OGD-induced decrease in TEER (Fig. 3D).
3.4 miR-152-3p decreased bend.3 apoptosis after OGD/R Previous study indicated that apoptosis of BMEC during ischemic injury contributes to compromised formation of TJs and loss of microvascular integrity [19,20]. Results of ow cytometric analysis showed apoptosis cells were signi cantly increased after OGD/R compared with the control group (Fig. 4A-B).
Overexpression of miR-152-3p partially reversed the apoptosis of bend.3 in the OGD/R group. Furthermore, a large number of TUNEL-positive cells was observed in bend.3 in OGD/R group, while the number of TUNEL-positive cells was remarkably reduced in miR-152-3p group compared with the OGD/R group (Fig. 4C-D).

MAP3K2 is a miR-152-3p target gene
To uncover the mechanism underlying the protective effect of miR-152-3p during OGD/R, downstream targets of miR-152-3p bioinformatics were predicted by online software TargetScan (www.targetscan.org). Among these predicted targets, MAP3K2 attracted our interest, as an important regulator of apoptosis. The 3 -UTR of MAP3K2 mRNA possesses a conserved binding site for miR-152-3p (Fig. 5A). We further investigated the relationship between miR-152-3p and MAP3K2 in bend.3. The protein level of MAP3K2 in was decreased after overexpression of miR152-3p with miR152-3p mimics and increased after suppression of miR152-3p with miR152-3p inhibitor ( Fig. 5B-C). To further explore whether miR-152-3p could directly bind to the 3′-UTR region of MAP3K2 mRNA, the luciferase assay was perfored in the HEK293 cells, in which the 3′-UTR fragment of MAP3K2 mRNA was used to construct the psiCHECK vector. As expected, miR-152-3p mimics signi cantly decreased the luciferase activity of pMAP3K2-WT in HEK293cells, without any effect on the luciferase activity of pMAP3K2-MUT (Fig. 5D).
3.6 miR-152-3p inhibited apoptosis of Bend.3 and improved the integrity of the BBB by negatively regulating MAP3K2 The present study was also aimed to determine whether MAP3K2 was involved in the protective action of miR-152-3p. We treated bend.3 cells with miR-152-3p mimics, miR-152-3p mimics + oe-NC or miR-152-3p mimics + oe-MAK3P2 before OGD/R. Western blot showed that expression of MAP3K2 was downregulated by miR-152-3p mimics transfection. However, this effect was reversed by co-transfected with miR-152-3p mimics and oe-MAP3K2 (Fig. 6A-B). The results of ow cytometry indicated that transfection with oe-MAP3K2 partially reversed the anti-apoptotic effect of miR-152-3p after OGD/R (Fig. 6C-D). Similarly, the results of uorescence intensity of cleaved caspase-3 demonstrated that caspase-3 activation was signi cantly decreased by miR-152-3p mimics compared with OGD/R group, which was reversed by co-transfection with miR-152-3p mimics and oe-MAP3K2 group(6E-F).
3.7 miR-152-3p modulated JNK/c-Jun pathway through targeting MAP3K2 after OGD/R MAP3K2 has been found to be an activator of JNK/c-Jun pathway, which has been reported to promote apoptosis in endothelial cells. We speculated that MAP3K2 may regulate the JNK/c-Jun pathway to attenuated Bend.3 apoptosis after OGD/R. As illustrated in Fig. 8A-C, compared with NC group, OGD/R signi cantly increased phosphorylation of JNK and p-c-Jun, and up-regulated active caspase-3; while overexpression of miR-152-3p inhibited the p-JNK/JNK and p-c-Jun/c-Jun ratio signi cantly and decreased the active caspase-3 compared with OGD/R group. In co-transfected with miR-152-3p mimics and oe-MAP3K2 group, the level of p-JNK/JNK, p-c-Jun/Jun and activate caspase-3 were increased again ( Fig. 7A-D). These results suggested that miR-152-3p modulated JNK/c-Jun signaling pathway after OGD/R, and MAP3K2 mediated this effect.

Discussion
Recently, the bene cial effect of miR-152-3p during brain ischemia has been demonstrated, but its potential impact on ischemia-induced BBB breakdown had remained elusive. BBB disruption following acute ischemic stroke is the primary cause of brain edema and hemorrhagic transformation, which contributes to worse stroke outcome [21,22]. Researches have increasingly suggested that BBB protection is important for ischemic stroke therapy [23]. We found that miR-152-3p played a protective role against MCAO-induced BBB collapse in rats and OGD-stimulated endothelial barrier damage. Mechanistically, miR-152-3p inhibited bend.3 apoptosis through targeting MAP3K2.
In the previous investigations, the expression of serum exosome miR-152-3p in patients with who suffered acute ischemic stroke was signi cantly lower than in healthy controls [11]. In addition, miR-152-3p was found to be downregulated in the hippocampus of ischemia/ reperfusion [12]. These data indicate that miR-152-3p may be involved in the regulation of ischemic injury. As expected, pre-ischemic intracerebroventricular injection of agomir-miR-152-3p improved stroke outcomes by reducing infarct volume and improving neurological functions in our study. Importantly, miR-152-3p protected against BBB destruction following MCAO. The failure of BBB can be characterized by reduced levels of TJPs such as claudin-5 and occludin after ischemia [6,24]. In the current study, we also demonstrated that miR-152-3p attenuated MCAO-induced TJPs degradation and improved TJP integrity in occudin immuno uorescence localization.
BMECs play an important role in the function of BBB by establishing a highly selective barrier [25]. We discovered that miR-152-3p expression was downregulated in the OGD/R-treated Bend.3, implying that miR-152-3p may play a role in the pathological process of BBB. Previous studies have shown that ischemic stroke leads to Bend.3 apoptosis associated with BBB disruption[26-28]. Hence protecting endothelial cells can maintain the integrity of BBB. The function of miR-152-3p to modulate apoptosis in cancers has been well established [29,30]. Recent years, the anti-apoptotic effect of miR-152-3p in injury diseases have received increasing investment. miR-152-3p regulates TCF-4 pathway in OA rats to weaken chondrocyte apoptosis [31]. miR-152-3p overexpression suppressed angiotensin II-induced apoptosis [32].
In addition, miR-152 overexpression prevented hypoxia-induced cardiomyocyte apoptosis in vitro [33]. A recent study revealed that miR-152-3p overexpression protected neurons from OGD/R-induced apoptosis [34]. Adding to the existing ndings, we demonstrated that miR-152-3p elicited anti-apoptotic effect on Bend.3 under I/R condition, which was con rmed by TUNEL staining and ow cytometry.
Since miRNAs do not code for proteins, the protective effect of miR-152-3p on cerebral I/R injury is assumed to be exerted via the repression of speci c proteins. In the present study, we provided evidence that miR-152-3p could target MAP3K2 to exert its protection action by following experiments: 1) With the assistance of bioinformatics tools (http://www.targetscan. org/vert_72/), MAP3K2 was identi ed as the target of miR-152-3p; 2) suppression of miR-152-3p promoted MAP3K2 expression, whereas over- MAP3K2 is an upstream kinase in the mitogen-activated protein kinase (MAPK) signaling pathway, which preferentially regulates JNK and ERK5 pathways. Multiple lines of evidence have linked MAP3K2 to apoptosis [35,36]. It is noteworthy that MAK3P2 may induce distinct effects on apoptosis. The MAP2K5/ERK5 pathway improves EC viability and confers resistance to apoptosis [37,38]; conversely, JNK MAPKs serves a pro-apoptotic role in ECs under most conditions [39]. Targeted disruption of JNK pathway has been reported to be involved in anti-apoptotic mechanism, which reduces cerebral I/R injury [15,40]. c-Jun is the major downstream substrate of the JNK pathway. Under the action of JNK, phosphorylated c-Jun further promotes the expression of a variety of pro-apoptotic proteins [41]. In this study, we found that miR-152-3p positively modulated the phosphorylation state of JNK and c-Jun and decreased the expression of active caspase-3. This phenomenon was reversed by MAP3K2 overexpression. Therefore, the effects of miR-152-3p on endothelial apoptosis during OGD/R may be induced by MAP3K2 suppression through JNK/c-Jun pathway.

Conclusion
In summary, miR-152-3p attenuated BBB damage under brain ischemia. Mechanistically, miR-152-3p attenuated OGD/R-induced endothelial apoptosis by regulating the MAP3K2 mediated JNK/c-Jun pathway. The anti-apoptotic effect of miR-152-3p might serve as a potential therapeutic target for cerebral I/R injury. The data used to support the ndings of this study are included within the article.

Con icts of interest
The authors declare no con ict of interest.
A. The level of miR-152-3p in rat brain tissue was detected by RT-qPCR; B. Representative TTC-stained brain slices in rats of different groups. C. Quantitative results of the infarction ratio. D. modi ed Garcia scores at 24 hours after MCAO. Bars represent means ± SEM. n = 5 per group. **P < 0.01, compared with the indicated group.     Bars represent means ± SEM. n = 3 per group. *P < 0.05, compared with the indicated group; **P < 0.01, compared with the indicated group.