MiR-21 involve in ERK-mediated upregulation of MMP9 in the rat hippocampus following cerebral ischemia

Brain Research Bulletin 94 (2013) 56–62

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MiR-21 involve in ERK-mediated upregulation of MMP9 in the rat hippocampus following cerebral ischemia XiaHeng Deng a,b , Yun Zhong b , LiZe Gu b,c , Wei Shen a,∗ , Jun Guo b,c,∗ a b c

Department of Neurology, Puai Hospital of Tongji Medical College, Huazhong University of Science and Technology Wuhan 430033, PR China Laboratory Center for Basic Medical Sciences, Nanjing Medical University, Nanjing 210029, PR China Department of Biochemistry and Molecular Biology, Nanjing Medical University, Nanjing 210029, PR China

a r t i c l e

i n f o

Article history: Received 7 October 2012 Received in revised form 26 January 2013 Accepted 21 February 2013 Available online 7 March 2013 Keywords: Cerebral ischemia miRNA MMP9 ERK

a b s t r a c t Matrix metallinoprotease-9 (MMP9) plays a key role in the pathogenesis of post-ischemic blood brain barrier (BBB) disruption and the formation of lesions after cerebral ischemia. In this study we investigate the effect of brain-specific miRNAs on MMP-9 protein level in the rat hippocampus following cerebral ischemia and its underlying mechanism. Cerebral ischemia significantly upregulated miR-21 and -224 in the hippocampus; however, expression of miR-122 and -338-3p was not significantly affected by ischemia. Silencing of miR-21, but not -224, reduced MMP9 protein level after cerebral ischemia. Downregulation of extracellular signal-regulated kinase (ERK) signaling using the ERK inhibitor U0126 and the calcium-channel blocker ketamine inhibited the upregulation of miR-21 expression and MMP9 protein level after cerebral ischemia. The study suggests that cerebral ischemia up-regulates expression level of miR-21, which is involved in ERK-stimulated upregulation of MMP9 following cerebral ischemia via a calcium-dependent mechanism. © 2013 Elsevier Inc. All rights reserved.

1. Introduction Matrix metalloproteinase 9 (MMP9) is a member of the MMP protein family, which are a group of more than 20 zinc-dependent proteinases with proteolytic activity against extracellular matrix components including collagen and elastin, containing numerous proteins involved in angiogenesis, cell migration, growth and apoptosis (He et al., 2009). MMP9 is considered to be critical in the pathogenesis of post-ischemic blood–brain barrier (BBB) disruption by degrading the major components of the basement membrane which surrounds brain vessels, such as collagen IV, collagen V, collagen-based gelatin, laminin and fibronectin (Romanic et al., 1998; Liu et al., 2011). Thus, ischemia-induced upregulation of MMP9 may have a close association with the consequent brain injuries. Although MMP9 is upregulated after a stroke, the mechanisms which regulate MMP9 protein level post-infarct remain poorly understood. Extracellular signal-regulated kinase (ERK), a prototypical member of the mitogen-activated protein kinase (MAPK)

∗ Corresponding author at: (Wei Shen): Department of Neurology, Puai Hospital of Tongji Medical College, Huazhong University of Science and Technology, Wuhan 430033, PR China. (Jun Guo): Laboratory Center for Basic Medical Sciences, Nanjing Medical University, Nanjing 210029, PR China. Tel.: +86 25 86862729; fax: +86 25 86862728. E-mail addresses: [email protected] (W. Shen), [email protected] (J. Guo). 0361-9230/$ – see front matter © 2013 Elsevier Inc. All rights reserved. http://dx.doi.org/10.1016/j.brainresbull.2013.02.007

family, is thought to be necessary for the expression of MMP9 in response to ischemic stimuli (Yen et al., 2011). Expression of MMP9 can be induced by ERK signaling via the binding of transcription factor to ets-1 site in MMP9 promoter (Jiang et al., 2001; Tower et al., 2003). Calcium influx is also involved in cerebral ischemiainduced ERK activation through the N-methyl-d-aspartate (NMDA) receptor-coupled calcium channel (Sugino et al., 2000; Song et al., 2006; Zhao et al., 2008). MicroRNAs are a group of small endogenous non-coding RNAs, 20–25 nucleotides in length, which function as regulators of posttranscriptional gene expression (Yan et al., 2011). MicroRNAs are encoded by genes, and bind to their target mRNAs to regulate gene expression (Saugstad, 2010). Approximately 20–40% of miRNAs in rat primary cerebral neurons appear to be developmentally regulated (Kim et al., 2004). MicroRNAs are abundant in the hippocampus, and function as effectors of neuronal development and the sustainability of the neuronal phenotype. Current findings suggest that MMP9 protein level is closely linked to microRNAs MiR-122 can significantly promote MMP9-mediated hepatocarcinogenesis (Kutay et al., 2006), and miR-21 can upregulate MMP9 protein level in hypertrophic heart fibroblasts by stimulating MAP kinase signaling (Thilo et al., 2010; Thum et al., 2008). Other research has shown that miR-224 enhanced MMP9 function and promoted cellular migration and invasion (Li et al., 2010). Inhibition of miR-338-3p has been associated with MMP9-mediated migration and invasion of hepatocellular carcinoma cells (Huang et al., 2011). However, it remains to be clarified whether miR-122,

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-21, -224 or -338-3p regulate MMP9 following cerebral ischemia, or whether these microRNAs are involved in the calcium-dependent ERK pathway-mediated expression of MMP9. In the present study, we investigated the effect of miR-122, -21, -224 and -338-3p on MMP9 protein following cerebral ischemia, and the relationship of these miRNAs and MMP-9 with the calciumdependent ERK pathway following cerebral ischemia. This study demonstrates that miR-21 and -224, but not miR-122 or -338-3p, are significantly upregulated after ischemia. Antagomirs of miR21, but not -224, inhibited the ischemia-induced upregulation of MMP9 protein level, and expression of miR-21 could be stimulated via the ERK signaling cascade in a calcium-dependent manner. This study provides a new perspective of the regulation of MMP9 following cerebral ischemia. 2. Materials and methods 2.1. Ethics statement All procedures using animals in this study, including surgical procedures and postoperative care, were approved by the Institutional Animal Care and Use Committee (IACUC). Every effort was made to minimize the number of animals used and their suffering. 2.2. Animal model of ischemia Adult male Sprague-Dawley rats (Experimental Animal Center of Nanjing Medical University) weighing approximately 220–250 g were selected in the present study. They were housed in individual cages under room temperature (24 ◦ C or so) and a 12:12 h light/dark cycle, with free access to food and water. Transient global ischemia was induced by four-vessel occlusion, as previously described (Wu et al., 2008; Cao et al., 2011). Briefly, the animals were deprived of food overnight before surgery, and then the rats were anesthetized with 20% chloral hydrate (300 mg/kg, ip). Both vertebral arteries were occluded by electrocauterization, and the common carotid arteries were separated from connective tissue and nerves and marked with surgical thread. On the following day, 10-min 4-VO ischemia was induced by clasping both the common carotid arteries and the suture. To minimize variability, the following criteria were met: Rats which lost their righting reflex within 30 s, whose pupils were dilated and unresponsive to light, and whose body temperature was maintained at approximately 37 ◦ C were used for the subsequent experiments. Sham-operated animals received identical surgical procedures; however, the arteries were not occluded. 2.3. Experimental design, injection (icv or ip) and drug treatment The experimental animals were randomly divided into three or four groups in each drug (or antagomir) treatment as follows: (1) sham group; (2) ischemia/reperfusion (I/R) group (partly); (3) I/R + drug (or antagomir) group; (4) I/R + vehicle group. For intracerebroventricular injection, U0126 (5 ␮g/␮l, 4 ␮l in DMSO) or its vehicle was administered into the right cerebral ventricle (icv) using a micro-injector (coordinates: 15 mm lateral and 0.8 mm posterior of Bregma; 35 mm below the skull surface) 30 min before occlusion, as previously described (Zhao et al., 2008). Antagomirs (50 ␮mol/l, 20 ␮l) or its control was administered into the rat bilateral cerebral ventricle 24 h before occlusion. After injection, the micro-injector was kept in place for an additional 5 min, to reduce backflow of the liquid along the injection void. For intraperitoneal injection (ip), the selective NMDA receptor inhibitor ketamine (50 mg/kg) or its vehicle (normal saline) was administrated intraperitoneally 30 min before ischemia.

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2.4. Protein extraction and Western blot assay Animals were euthanized 6, 12 and 24 h after the 10 min ischemia by perfusion followed by decapitation. The hippocampus was dissected from the unilateral hemisphere on ice in a cold room. Cytoplasmic and membrane proteins were extracted from the hippocampus with sample buffer containing 1% mammalian protease inhibitor cocktail (Sigma) and stored at −80 ◦ C. The protein contents were determined by the Bradford assay (Kim et al., 2011). Denatured homogenates (approximately 25 ␮g protein) were separated by 10% SDS-PAGE and transferred onto nitrocellulose membranes. The membranes were probed with antibodies specific for MMP9 (92 kDa, Bioss Co, Beijing, China) and ␤-actin (Boster Biotechnology, WuHan, China), and detected using horseradish peroxidase (HRP)-conjugated IgG antibodies and enhanced chemiluminescence (Amersham Bioscience, Piscataway, NJ, USA). The bands were scanned and analyzed using the Jieda image analyzer (Jieda, Nanjing, China). 2.5. RNA extraction and quantitative real-time PCR (qRT-PCR) Total RNA of the samples mentioned above was extracted using TRIzol reagent (Invitrogen, Carlsbad, CA, USA). cDNA was reverse transcribed using the High Capacity cDNA Archive kit (Invitrogen). cDNA synthesis of miRNAs was performed using the miScript II RT Kit (Qiagen, Hilden, Germany) according to the manufacturer’s instructions. The sequences of primers specific to the published miR sequences were used. The expression levels of the mRNA or miRNA were quantified by qRT-PCR using SYBR Green PCR Master Mix and the Eppendorf Mastercycler Realplex Sequence Detection System. Melting curve analysis was performed after the final amplification step to identify the PCR products ␤-actin was used as an internal control gene. 2.6. Data analysis All data were expressed as the mean ± S.D. values of at least four animals. Statistical analysis was conducted using one-way analysis of variance, followed by the Newman–Keuls test. Comparisons between two groups were performed using the t-test; P-values < 0.05 were considered significant. 3. Results 3.1. Cerebral ischemia induced time curve of several miRNA and MMP9 protein levels in rat hippocampus Recent studies have suggested that miRNAs play a key role in the regulation of gene expression following cerebral ischemia, and miRNAs may also be involved in CNS dysfunction (Kosik, 2006). However, it was not known whether miRNAs are involved in the regulation of MMP9 following cerebral ischemia-reperfusion (I/R) injury. To examine the possible interactions between miRNAs and MMP9, we investigated whether 10 min ischemia induced expression of MMP9 in the rat hippocampus at various times (6, 12 and 24 h) after I/R. As shown in Fig. 1A, MMP9 expression was not significantly increased at 6 or 12 h after I/R (P > 0.05); however, MMP9 protein level increased significantly 24 h after I/R (P < 0.05). And then we quantified the expression of miR-122, -21, -224 and 338-3p in the rat hippocampus at various times (6, 12 and 24 h) following I/R using real-time RT-PCR. As shown in Fig. 1C and D, 10 min ischemia increased expression of miR-21 and -224 at 24 h after I/R (P < 0.05; Fig. 1D and E). However, expression of miR-122 and -338-3p did not increase 24 h after I/R (P > 0.05, Fig. 1B and E), and expression of miR-122 actually unexpectedly decreased 6 h

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Fig. 1. Western blot analysis of MMP9 protein expression and real-time RT-PCR analysis of miR-122, -21, -224 and -338-3p expression following ischemia-reperfusion (I/R). Samples were taken from the hippocampus of rats subjected to sham surgery, or 6, 12 and 24 h after 10 min ischemia (A) MMP9, (B) miR-122, (C) miR-21, (D) miR-224 and (E) miR-338-3p Western blot optical density (O.D.) values and the relative miRNA expression values are presented as the fold-increase compared to sham control animals. Data are expressed as the mean ± S.D. (n = 4); *P < 0.05 versus sham control animals.

after I/R (P < 0.05, Fig. 1B and C). These findings suggested that cerebral ischemia induced increment of miR-21 and miR-224, which may be involved in regulation of MMP9 protein level in rat hippocampi.

Fig. 2A), indicating that miR-21 regulates the expression of MMP9 following ischemia. Unexpectedly, the miR-224 antagomir did not affect the expression of MMP9 at 24 h after I/R (P > 0.05, Fig. 2B). This data indicated that silencing of miR-21 but not -224 may result in the downregulation of MMP9 protein levels following ischemia.

3.2. Antagomir of miR-21 but not miR-224 result in downregulation of MMP9 protein level following ischemia-reperfusion

3.3. The MEK/ERK inhibitor U0126 contributed to a decrease in miR-21 and MMP9 protein level in response to cerebral ischemia

Next, we utilized antagomirs to silence miR-21 and -224 (Chang et al., 2012), to investigate whether the expression of MMP9 is regulated by miR-21 and -224 after cerebral ischemia. The miR-21 antagomir leads to a downregulation of MMP9 after I/R (P < 0.05,

Previous studies have shown that MMP9 may be upregulated in vitro via the ERK signaling pathway (Yen et al., 2011). To determine the relationship between miRNAs, ERK signaling and the regulation of MMP9 expression, the specific MEK/ERK inhibitor

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U0126 (Saboor et al., 2011) was administered prior to ischemia As shown in Fig. 3B, U0126 inhibited the ischemia-induced upregulation of miR-21 24 h after I/R (P < 0.05) but had no effect on the expression of miR-122, -224 and-338-3p at 24 h after I/R (P > 0.05, Fig. 3A, C and D), indicating that the ERK signaling pathway regulates expression of miR-21, but not-122, -224 or -338-3p at 24 h reperfusion postischemia. The effect of U0126 on MMP9 expression was also examined, and we observed that U0126 inhibited the ischemia-induced upregulation of MMP9 (P < 0.05, Fig. 3E). These results suggest that inhibition of MEK/ERK is closely associated with the downregulation of miR-21 and MMP9 protein level in the rat hippocampus following ischemia. 3.4. NMDAr inhibitor ketamine can reduce levels of miR-21 and MMP9 protein expression during ischemia-reperfusion The ERK signaling cascade can be activated via NMDA receptorcoupled calcium signaling (Zhao et al., 2008). The selective NMDAr inhibitor ketamine (Keta) was used to determine the role of calcium signaling in ERK-mediated miRNA expression (Wessler et al., 2011). As shown in Fig. 4A, B, C and D, administration of Keta lead to a significant decrease in the expression of miR-21 (P < 0.05), but not miR-122, -224 or -338-3p at 24 h after I/R (P > 0.05). Interestingly, expression of miR-224 peaked after the administration of ketamine 24 h after I/R (Fig. 4D, P < 0.05), suggesting that an unknown mechanism involving calcium signaling may regulate miR-224 after cerebral ischemia. Keta significantly reduced the expression of MMP9 in response to ischemia (Fig. 4E, P < 0.05). These results suggest that blockage of NMDA receptor-coupled calcium signaling is involve in downregulation of miR-21 and MMP9 protein levels in response to cerebral ischemia. 4. Discussion

Fig. 2. Effect of miR-21 and -224 antagomirs on MMP9 protein level following ischemia-reperfusion (I/R). The antagomirs were administered into the rat cerebral ventricle 24 h before vessel occlusion. Samples were taken from the rat hippocampus 24 h after sham surgery or 24 h after 10 min ischemia in control (Con) animals or animals treated with (A) antagomir-21 or (B) antagomir-224. The samples were subjected to Western blotting for MMP9. The band optical densities (O.D.) are presented as the fold-increase compared to sham control animals. Data are expressed as the mean ± S.D. (n = 4); # P < 0.05 versus I/R 24 h group.

Recently, MMP9 has attracted much attention because of its function in brain ischemia/reperfusion injury MMP9 can degrade main components of the basement membrane surrounding the capillaries leading to disruption of the blood brain barrier (BBB) and it also participates in the angiogenesis after the ischemic brain injury (Ishigaki et al., 2008; He et al., 2009). Previous study indicated that ERK signaling cascade could trigger MMP-9 expression through binding of transcription factor to ets-1 site in MMP-9 promoter. Meanwhile, ERK cascade activated by Ras-GRF following cerebral ischemia was associated with calcium influx through the NMDA coupled channel, which is different from the Grb2/Sos/Ras pathway reported previously (Cao et al., 2011). In the present study, we proved that ERK activation after ischemia was associated with the upregulation of MMP-9 by the application of NMDAr inhibitor ketamine and MEK/ERK inhibitor U0126. Furthermore, a novel mechanism was identified whereby miR-21 plays a role in ERK-regulated MMP-9 expression in calcium-dependent manner following cerebral ischemia. Several studies suggested that the expression of miR-21 was closely linked to MAPK/ERK cascade in acute myeloid leukemia (AML) (Thum et al., 2008). Here, we found that miR-21 was regulated by ERK signaling pathway via calcium-dependent mechanism following cerebral ischemia. Firstly, we detected the expressions of miRNAs and observed that miR-21 and -224 were upregulated at 24 h postischemia, indicating that miR-21 and -224 may be involved in the regulation of cerebral ischemia/reperfusion (Ziu et al., 2011). Then, the MEK inhibitor U0126 was applied to block ERK activity directly, and as expected miR-21 but not-122, -224 or -338-3p reduced significantly, suggesting that miR-21 but not -122, -224 or -338-3p could be regulated by ERK. Finally, pretreatment with NMDAr inhibitor ketamine effectively attenuated

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Fig. 3. Effect of the ERK signaling inhibitor U0126 on miR-122, -21, -224 and -338-3p expression and MMP9 protein level following ischemia-reperfusion (I/R). U0126 was administered into the rat right cerebral ventricle 30 min before vessel occlusion. Samples were taken from the hippocampus 24 h after sham surgery, or 24 h after 10 min ischemia in animals treated with U0126 or vehicle (Veh). (A) miR-122, (B) miR-21, (C) miR-224, (D) miR-338-3p, (E) MMP9 protein levels Western blot optical density (O.D.) values and the relative miRNA expression values are presented as the fold-increase compared to sham control animals. Data are expressed as the mean ± S.D. (n = 4); # P < 0.05 versus I/R 24 h group.

the expression of miR-21 but not-122, -224 or -338-3p. These results suggested that miR-21 but not-122, -224 or -338-3p could be upregulated by ERK cascade in the calcium-dependent manner following cerebral ischemia.

In order to further evaluate the involvement of miR-21 in MMP9 expression, the antagomirs of miR-21 and -224 were administered to downregulate the levels of miRNAs specifically. It was shown that the antagomir of miR-21 but not -224 could reduce the protein

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Fig. 4. Effects of the NMDAr inhibitor ketamine on miR-122, -21, -224 and -338-3p expression and MMP9 protein level following ischemia-reperfusion (I/R). Rats were treated with an intraperitoneal injection of ketamine (Keta) 30 min before vessel occlusion. Samples were taken from the rat hippocampus 24 h after sham surgery, or 24 h after 10 min ischemia in animals treated with Keta or vehicle (Veh). (A) miR-122, (B) miR-21, (C) miR-224, (D) miR-338-3p, (E) MMP9 protein levels Western blot optical density (O.D.) values and the relative miRNA expression values are presented as the fold-increase compared to sham control animals. Data are expressed as the mean ± S.D. (n = 4); # P < 0.05 versus I/R 24 h group.

level of MMP9, suggesting that miR-21 plays a key role in ischemiainduced upregulation of MMP9. Previous studies have showed that inhibition of miR-21 expression repressed cell migration and invasion through downregulation of MMP-9 expression (Meng et al., 2007; Moriyama et al., 2009; Giovannetti et al., 2010; Zhu et al., 2012), which might be associated with downregulaton of RECK as a target of miR-21 (Reis et al., 2012). Our findings provided evidence of a possible functional linkage between miR-21 and MMP9 in cerebral ischemia.

Taken together, the present study demonstrates that miR 21, but not -122, -224 or -338-3p, plays a significant role in ERK cascade-mediated increment of MMP9 protein level following ischemia. Calcium signaling stimuli may trigger the ERK signaling pathway and lead to upregulation of MMP9 after cerebral ischemia, via a mechanism regulated by miR-21. As miR-21 plays a key role in the development of MMP9-mediated cerebral ischemia, it is conceivable that anti-miR-21 could emerge as new class of molecular targets, and these agents could potentially function as an effective

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therapeutic intervention to prevent the formation of lesions after cerebral ischemia. Conflict of interest statement The authors declare that there are no conflicts of interest. Acknowledgments This work was supported by grants from the Program for the Science and Technology of Nanjing (200901081), the National Natural Science Foundation of China (No. 81170714) and the Priority Academic Program Development of Jiangsu Higher Education Institutions. References Cao, Q., Qian, M., Wang, X.F., Wang, B., Wu, H.W., Zhu, X.J., Wang, Y.W., Guo, J., 2011. Negative feedback regulation of Raf/MEK/ERK cascade after sublethal cerebral ischemia in the rat hippocampus. Neurochemical Research 36, 153–162. Chang, C.I., Lee, T.Y., Kim, S., Sun, X., Hong, S.W., Yoo, J.W., Dua, P., Kang, H.S., Kim, S., Li, C.J., Lee, D.K., 2012. Enhanced intracellular delivery and multi-target gene silencing triggered by tripodal RNA structures. Journal of Gene Medicine 14, 138–146. Giovannetti, E., Funel, N., Peters, G.J., Del, Chiaro, M., Erozenci, L.A., Vasile, E., Leon, L.G., Pollina, L.E., Groen, A., Falcone, A., Danesi, R., Campani, D., Verheul, H.M., Boggi, U., 2010. MicroRNA-21 in pancreatic cancer: correlation with clinical outcome and pharmacologic aspects underlying its role in the modulation of gemcitabine activity. Cancer Research 70, 4528–4538. He, Z.J., Huang, Z.T., Chen, X.T., Zou, Z.J., 2009. Effects of matrix metalloproteinase 9 inhibition on the blood brain barrier and inflammation in rats following cardiopulmonary resuscitation. Journal of Chinese Medicine (Engl) 122, 2346–2351. Huang, X.H., Chen, J.S., Wang, Q., Chen, X.L., Wen, L., Chen, L.Z., Bi, J., Zhang, L.J., Su, Q., Zeng, W.T., 2011. miR-338-3p suppresses invasion of liver cancer cell by targeting smoothened. Journal Pathology 225, 463–472. Jiang, Y., Xu, W., Lu, J., He, F., Yang, X., 2001. Invasiveness of hepatocellular carcinoma cell lines: contribution of hepatocyte growth factor, c-met, and transcription factor Ets-1. Biochemical and Biophysical Research Communications 286, 1123–1130. Kim, J., Krichevsky, A., Grad, Y., Hayes, G.D., Kosik, K.S., Church, G.M., Ruvkun, G., 2004. Identification of many microRNAs that copurify with polyribosomes in mammalian neurons. Proceedings of the National Academy of Sciences United States of America 101, 360–365. Kim, S., Shin, J.K., Yoon, H.S., Kim, J.H., 2011. Blockade of ERK phosphorylation in the nucleus accumbens inhibits the expression of cocaine-induced behavioral sensitization in rats. Korean Journal of Physiology Pharmacology 15, 389–395. Kosik, K.S., 2006. The neuronal microRNA system. Nature Review Neuroscience 7, 911–920. Kutay, H., Bai, S., Datta, J., Motiwala, T., Pogribny, I., Frankel, W., Jacob, S.T., Ghoshal, K., 2006. Down-regulation of miR-122 in the rodent and human hepatocellular carcinomas. Journal of Cell Biochemistry 99, 671–678. Ishigaki, D., Ogasawara, K., Suga, Y., Saito, H., Chida, K., Kobayashi, M., Yoshida, K., Otawara, Y., Ogawa, A., 2008. Concentration of matrix metalloproteinase9 in the jugular bulb during carotid endarterectomy correlates with severity of intraoperative cerebral ischemia. Cerebrovascular Disease 25, 587–592. Meng, F., Henson, R., Wehbe-Janek, H., Ghoshal, K., Jacob, S.T., Patel, T., 2007. MicroRNA-21 regulates expression of the PTEN tumor suppressor gene in human hepatocellular cancer. Gastroenterology 133, 647–658.

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