Mol Biol Rep DOI 10.1007/s11033-013-2891-x
MicroRNA-22 targeting CBP protects against myocardial ischemia–reperfusion injury through anti-apoptosis in rats Jian Yang • Lihua Chen • Jun Yang • Jiawang Ding • Song Li • Hui Wu • Jing Zhang Zhixing Fan • Wusong Dong • Xinxin Li
•
Received: 12 July 2013 / Accepted: 2 December 2013 Ó Springer Science+Business Media Dordrecht 2013
Abstract MicroRNAs are extensively involved in the pathogenesis of major cardiovascular diseases by suppressing target gene expression. Recent studies have reported that microRNA-22 (miR-22) may be implicated in ischemia–reperfusion (I/R) induced myocardial injury. However, the specific function of miR-22 in myocardial I/R injury is far from clear nowadays. The present study was designed to determine the role of miR-22 in myocardial I/R injury and investigate the underlying cardio-protective mechanism. The rat myocardial I/R injury model was induced by occluding the left anterior descending coronary artery for 30 min followed by 12 h reperfusion. As predicted, adenovirus-mediated miR-22 overexpression markedly reduced the release of creatine kinase and lactate dehydrogenase, infarct size and cardiomyocytes apoptosis. Moreover, CREB binding protein (CBP) as a potential miR-22 target by bioinformatics was significantly inhibited after miR-22 transfection. We also found that p53 acetylation activity, pro-apoptotic related genes Bax and p21 levels were all decreased associated with the down-regulation of CBP. In conclusion, our data demonstrate that miR-22 could inhibit apoptosis of cardiomyocytes through one of its targets, CBP. Thus, miR-22 may constitute a new
Jian Yang and Lihua Chen contributed equally to this work. J. Yang J. Yang (&) J. Ding S. Li H. Wu J. Zhang Z. Fan W. Dong X. Li Department of Cardiology, The First College of Clinical Medical Sciences, China Three Gorges University, Yichang 443000, Hubei, China e-mail: [email protected] L. Chen Department of Optometry and Ophthalmology, Yichang Central People’s Hospital, Yichang 443000, Hubei, China
therapeutic target for the prevention of myocardial I/R injury. Keywords MicroRNAs Reperfusion injury CREB binding protein Apoptosis Cardiomyocytes
Introduction It is well known that reperfusion is the most important therapeutic strategy for ischemic heart disease [1, 2]. However, reperfusion itself could cause reperfusion-associated myocardial dysfunction and damage [3, 4]. To date, it has been widely accepted that apoptosis plays a key role in myocardial ischemia–reperfusion (I/R) injury [5, 6]. It is therefore necessary to find an effective target against apoptosis for attenuating I/R injury. MicroRNAs (miRNAs) are a class of endogenous, small (=22 nt in size), noncoding single-stranded RNAs that negatively regulate gene expression through binding on the 30 untranslated region (UTR) of target mRNAs [7, 8]. They participate in almost all cellular processes, such as proliferation, differentiation and apoptosis [7–9]. In addition, miRNAs are also recognized as critical regulators of cardiovascular diseases, including myocardial infarction [10], heart failure [11], cardiac hypertrophy and arrhythmia [12, 13]. Thus, miRNAs might be attractive therapeutic targets for the treatment of myocardial I/R injury. Recently, Ren et al. [14] using miRNA arrays have found that the expression of one specific microRNA, miR-22, is decreased in rat hearts on I/R in vivo and ex vivo. These data suggest that miR-22 may be involved in the initiation and progression of myocardial I/R injury. CREB binding protein (CBP), as a general transcriptional co-activator, is essential in cell growth, differentiation and
123
Mol Biol Rep
apoptosis [15]. Because of its histone acetyltransferase activity, CBP could acetylate various sequence-specific transcription factors such as CREB, NF-kB, AP-1 and p53, to regulate their downstream genes expression [16–18]. In fact, CBP is identified as a potential miR-22 target by the bioinformatics of TargetScan. More interestingly, preliminary unpublished work from our laboratory has demonstrated that the level of CBP is strikingly up-regulated in rat hearts after I/R. However, it is entirely unknown whether CBP mediates myocardial I/R injury by miR-22 regulation. In the present study, we used adenoviral overexpression of miR-22 to investigate its effect on I/R injury, and identify the underlying mechanism further. Our data demonstrates for the first time that miR-22 could efficiently attenuate I/R injury by regulating CBP expression and inhibiting apoptosis. Taken together, these findings implicate that miR-22 may be a new therapeutic target for ischemic heart disease.
or saline was respectively injected into six separate sites of the left ventricular anterior wall via a 26-gauge needle. The chest was closed after injection and the rat was allowed to recover. Myocardial I/R treatment was performed four days later. All the rats were re-anesthetized and ventilated artificially with room air. The thorax was reopened through the original intercostal space, and the left anterior descending coronary artery (LAD) was identified. The LAD was ligated using a 6–0 silk suture. Additionally, a medical latex tube (socket, inner diameter, 1.5 mm) was placed between the ligature and the LAD. Myocardial ischemia was induced by tightening the ligature around the latex tube. Ischemia was confirmed by myocardial blanching and electrocardiography evidence of injury. After 30 min ischemia, the latex tube was removed to restore the coronary circulation. At 12 h post-reperfusion, the hearts and blood samples were obtained for further analysis. Shamoperated controls underwent the same procedures, with the exception that the LAD was not ligated.
Materials and methods
Detection of creatine kinase (CK) and lactate dehydrogenase (LDH)
Animal care Adult male Sprague-Dawley rats (250–300 g) received a standard diet and free water. The procedures for experiments and animal care were approved by the Animal Care and Use Committee of China Three Gorges University (CTGU), and conformed to the Guide for the Care and Use of Laboratory Animals by the National Institutes of Health (NIH Publication No. 80-23). miR-22 Expression vector construct Rno-miR-22 precursor DNA (MI0000851) was synthesized by Genechem (Shanghai, China). The adenovirus expressing miR-22 (Ad-miR-22), or control adenovirus expressing Scramble (Ad-Scramble) was generated using the AdMax system (Microbix Biosystems, Canada) according to the manufacturer’s protocols. These resulting adenoviruses were further packaged and amplified in HEK293 cells, and purified using CsCl banding. The final titers were determined in plaque assays. In vivo gene transfer and rat myocardial I/R model The animals were subject to adenovirus-mediated gene transfer, and subsequent myocardial I/R surgical procedure [19]. In brief, rats were anesthetized with pentobarbital (30 mg/kg intraperitoneal). A left parasternal incision was made through the fourth and fifth ribs, and the pericardium was gently opened to expose the heart. A 100 ll solution of Ad-miR-22 (1 9 109 PFU), Ad-Scramble (1 9 109 PFU)
123
Blood samples were collected at the end of reperfusion and centrifuged at 3,000 g for 10 min to isolate serum. Two specific marker enzymes, including the creatine kinase (CK) and lactate dehydrogenase (LDH), were assayed using commercial kits (Beijing Kemeidongya Biotechnology Ltd., China). Results were presented in international units per litre. Assessment of myocardial infarct size Myocardial infarct size was evaluated by Evans Blue/triphenyltetrazolium chloride (TTC) staining as previously described. At the end of 12 h reperfusion, the LAD was religated, and 1 ml of 2 % Evans Blue dye (Sigma-Aldrich, St Louis, MO) was administered intravenously to distinguish the non-ischemic area from the area at risk. The hearts were rapidly excised and sliced parallel to the atrioventricular groove into 2-mm-thick sections. The slices were then incubated in 1 % TTC (Sigma Chemicals, St. Louis, MO, USA) at 37 °C for 20 min to identify the infarct zone. The area at risk (AAR), infarct area (IA) and left ventricular area (LV) were measured digitally using Image-Pro Plus 5.0 software (Media Cybernetic, USA). The percentage of IA/ AAR and AAR/LV was calculated respectively. Determination of myocardial apoptosis Cardiomyocytes apoptosis was assessed by terminal deoxynucleotide transferase dUTP nick end labeling (TUNEL) staining. The obtained heart tissue was washed, dehydrated, and imbedded in paraffin. Then the TUNEL
Mol Biol Rep
assay was performed using an apoptosis detection kit (Roche, Basle, Switzerland) in accordance with instructions. The numbers of TUNEL-positive cells and the total cells in heart sections were counted in five microscopic fields. The ratio of TUNEL-positive cells to total cardiomyocytes was apoptosis index (AI). Total RNA extraction and quantitative RT-PCR Total RNA from the cardiac muscle samples was extracted using Trizol Reagent (Invitrogen, Carlsbad, CA) according to the manufacturer’s protocol. miR-22 levels were measured using the mirVanaTM qRT-PCR miRNA detection kit (Ambion Inc. TX). Amplification and detection of specific products were performed with the ABI Prism 7,500 system (PE Applied Biosystems). U6 was used as an internal control. miR-22 expression was normalized to U6 small nucleolar RNA. 2-DDCt method [20] was used to calculate relative expression levels. Western blot analysis To determine the protein levels of CBP, p53, acetylated p53, Bax, p21 and b-actin in myocardial tissue, proteins extraction and Western blot analysis were performed as described previously. Briefly, equal amounts of protein extracts were subjected to SDS-PAGE, and transferred onto nitrocellulose membranes. Non-specific binding was blocked with 5 % non-fat dry milk. The membrane was rinsed, and immunoblotted with each primary antibody at a recommended dilution. Subsequently, the membrane was washed and incubated with horseradish peroxidase-conjugated secondary antibody. Bands were visualized using an enhanced chemiluminescence (ECL) detection kit (Pierce, Rockford, IL, USA). Statistical analysis All values were presented as mean ± SEM. Differences between groups were analyzed for significance by one-way analysis of variance (ANOVA) and Student–Newman– Keuls (SNK) q test using SPSS software (version 14.0). A value of P \ 0.05 was considered to be statistically significant.
Results Myocardial I/R induced down-regulation of miR-22 expression As shown in Fig. 1, after 12 h of reperfusion, myocardialspecific miR-22 expression was significantly down-
Fig. 1 Regulation of miR-22 after myocardial I/R. miR-22 was down-regulated in I/R group, while adenovirus-mediated miR-22 gene transfer in vivo up-regulated its expression. Values were expressed as mean ± SEM, n = 8 per group, *P \ 0.05
regulated 1.7-fold as compared to sham group without injury. Transfection of miR-22 into the myocardium after four day, could markedly increase miR-22 expression by 81.2 % (Ad-miR-22 vs. Ad-Scramble, P \ 0.05). However, Ad-Scramble had no apparent effect on the expression level of miR-22 compared with I/R group (P [ 0.05). Effects of miR-22 on serum CK and LDH The activities of serum CK and LDH were initially low in the sham group. Myocardial ischemia for 30 min by LAD ligation followed by 12 h of reperfusion significantly increased the leakage of CK and LDH compared with sham group (Fig. 2). However, the elevation in CK and LDH levels was obviously suppressed by Ad-miR-22 transfection (Ad-miR-22 vs. I/R, P \ 0.05). Ad-Scramble did not affect I/R-induced increase leakage of CK and LDH from myocardium (Ad-Scramble vs. I/R, P [ 0.05). miR-22 Reduced infarct size The extent of myocardial infarction was evaluated after reperfusion. No significant difference was found in AAR/ LV among the four groups (data not shown). Infarct size was measured to be 48.3 ± 3.5 % of the area at risk (IA/ AAR) in the I/R group. miR-22 over expression significantly reduced myocardial infarct size compared with that in the I/R group (P \ 0.05, Fig. 3). Interestingly, pretreatment with Ad-Scramble did not affect infarct size (51.4 ± 6.7 % vs. I/R group, P [ 0.05). Our results suggest that transient up-regulation of miR-22 may play a cardio-protective role during myocardial I/R injury.
123
Mol Biol Rep Fig. 2 miR-22 reduced creatine kinase (CK) and lactate dehydrogenase (LDH) release. Ad-miR-22 reduced CK and LDH level after I/R injury in rat serum. a The activity of CK in serum. b The activity of LDH in serum. Data were expressed as mean ± SEM, n = 8, *P \ 0.05
Fig. 3 miR-22 decreased myocardial infarct size in rats subjected to I/R injury. a Representative illustrations of infarct size as stained by Evans Blue and TTC. Dark blue area, nonischemic tissue; red-stained area, area at risk; white area, infracted tissue. b The infarct size normalized to the area at risk in different groups. AAR: area at risk; IA: infarct area. Results were presented as mean ± SEM, n = 6 in each group, *P \ 0.05
miR-22 Attenuated cardiomyocytes apoptosis induced by I/R Terminal deoxynucleotide transferase dUTP nick end labeling assay was performed to evaluate the cardiomyocytes apoptosis in vivo. As shown in Fig. 4, the apoptotic index in sham group was only 3.1 ± 0.9 %. Compared with sham group, TUNEL-positive cardiomyocytes were more frequently observed in the I/R group and Ad-Scramble group (P both \ 0.05). However, this effect was largely alleviated by Ad-miR-22 transfection (23.4 ± 1.8 % in Ad-miR-22 group vs. 32.6 ± 4.1 % in I/R group, P \ 0.05).
123
Fig. 4 Effect of miR-22 on apoptosis in I/R induced myocardial injury. a Representative microphotographs of TUNEL staining from four groups. Arrows indicate TUNEL-positive cardiomyocytes nuclei stained as dark brown. Scale bar represents 25 lm. b Mean apoptotic index was counted in each group. Data were expressed as mean ± SEM, n = 8, *P \ 0.05
miR-22 Regulated the relative protein expression CBP, as the target gene of miR-22 (predicted by the TargetScan software, Fig. 5a), was significantly up-regulated
Mol Biol Rep
Fig. 5 Immunoblots and graphs showing the relative protein levels and fold changes from different groups. a Putative binding sites of miR-22 in the CBP 30 UTR (white sequences) predicted by TargetScan. b Original representative western blots of the proteins obtained from myocardial tissue. c miR-22 suppressed p53 acetylation induced
by I/R injury. d The relative CBP, p21 and Bax protein levels in the four groups. Bars show mean ± SEM, n = 8. NS means not significant at the 0.05 level. *P \ 0.05, HP \ 0.05 vs. I/R or AdScramble group
4.2-fold as compared to sham group without I/R injury (Fig. 5b, d). Delivery of miR-22 into the myocardium could markedly decrease CBP protein expression by 45.2 % (vs. I/R group, P \ 0.05). Ad-Scramble had no inhibitory effect on the expression of CBP (vs. I/R group, P [ 0.05). It was found that I/R induced significant p53 activation and acetylation in the injured myocardium. AdmiR-22 treatment resulted in a 44.1 and 38.7 % reduction of p53 acetylation in Ad-Scramble transfected and saline treated myocardium, respectively (P both \ 0.05). However, the total p53 protein level was not suppressed by AdmiR-22 (P [ 0.05; Fig. 5b, c). In addition, p53 downstream pro-apoptotic proteins Bax and p21, also exhibited the similar change like acetylated p53 after Ad-miR-22 transfection (Fig. 5b, d).
reduction, and down-regulated activity of p53 acetylation could be clearly observed in vivo. These major findings unveil that the cardio-protection of miR-22 against cardiomyocytes apoptosis is, at least in part, functionally attributed to its suppression of CBP. A number of miRNAs, such as miR-1 [21], miR-146a [22]and miR-494 [23] were verified to be involved in myocardial I/R injury by altering the expression of key genes associated with cell apoptosis. miR-22 was considered to be a tumor-suppressing microRNA in various cancers [24]. Moreover, Jovicic et al. [25] also found that miR-22 had a pronounced neuro-protective effect by targeting the related genes and inhibiting neuron apoptosis. Recently, miR-22 has been reported as a critical regulator of cardiomyocyte hypertrophy and cardiac remodeling [26]. However, the role of miR-22 in myocardial I/R injury remains unclear at this point. In this study, we found miR22 was significantly down-regulated in ischemic myocardium subjected to 30 min LAD occlusion followed by 12 h reperfusion in vivo. The adenoviral vector-mediated transfection system could efficiently deliver miR-22 into rat hearts. Overexpression of miR-22 reduced the release of myocardial enzyme, cardiomyocytes apoptosis and infarct size of rats after myocardial I/R. Thus, we presume that the molecular mechanisms involved in the cardio-protective effect of miR-22 are very complicated.
Discussion It is well established that myocardial I/R injury is a complex pathophysiological process in which apoptosis plays a crucial role [6]. In the current study, we found that AdmiR-22 exerted a potent protective effect against I/R induced apoptosis in cardiomyocytes. Meanwhile, increased levels of miR-22, with a concomitant CBP
123
Mol Biol Rep
Indeed, miRNAs elicit their function through post-transcriptional silencing of RNA interference pathway [7, 8]. Individual miRNA may have multiple targets [27]. miR-22 as a cardiac-enriched microRNA, has several different targets in cardiomyocytes by using software applications such as miRanda and TargetScan. Among these targets, the CBP gene attracts researchers’ attention due to its multifunctional coordination ability. In this paper, our experiments confirm that CBP was inversely correlated with miR22 expression in rat heart tissues. In fact, CBP gene is widely expressed and plays pivotal role in cardiovascular system. We have previously reported that CBP mediates the development and progression of neointimal formation after vessel injury [28]. CBP and its close homologue p300 are known to interact with a variety of diverse transcriptional factors [16–18], which lead to distinct biological outcomes. Particulally, the function of CBP in gene transcription is also dependent on its acetylation status. The tumor suppressor protein p53 is one of the important transcriptional factors, which regulates the apoptosisrelated gene expression involved in I/R injury [29]. The activation of p53 could contribute to cardiomyocytes apoptosis via inducing cell cycle arrest and pro-apoptotic proteins [30]. Hence, suppressing p53 activity can reduce I/R induced myocardial injury. In our study, we observed that Ad-miR-22 not only prevented cell apoptosis, but also decreased I/R induced p53 acetylation. Furthermore, p53 downstream target genes, including p21 for cell-cycle arrest and Bax for apoptosis were both markedly inhibited by miR-22 transfection. By repressing CBP, miR-22 attenuates p53 acetylation, leading to the reduction of proapoptotic genes. In this regard, our data suggest that p53dependent apoptosis in myocardial I/R injury, at least in part, through activation of the miR-22/CBP/p53 pathway. Although miR-22 has been shown to have apparent antiapoptotic function, the cardio-protective effect of it might not be similar for all cardiovascular diseases [26, 31]. miR22 has several other targets in cardiomyocytes, which engage multiple signaling pathways. Further studies should investigate other targets of miR-22 to fully elucidate the role of miR-22 in myocardial I/R injury. In summary, our study reveals that dys-regulation of miR-22 expression contributes to ischemic heart disease. Overexpression of miR-22 provides cardiac protection against I/R induced cardiomyocytes apoptosis by directly targeting CBP. These findings support the perspective that enhancing miR-22 expression might be a desirable therapeutic approach for the treatment of myocardial I/R injury. Acknowledgments This project was supported by the National Natural Science Foundation of China (Grant Nos. 81200088, 81201458) and the Natural Science Foundation of Yichang city, China (Grant No. A12301-01).
123
References 1. Yellon DM, Hausenloy DJ (2007) Myocardial reperfusion injury. N Engl J Med 357(11):1121–1135 2. Cokkinos DV, Pantos C (2007) Myocardial protection in man– from research concept to clinical practice. Heart Fail Rev 12(3–4):345–362 3. Yang J, Yang J, Ding JW, Chen LH, Wang YL, Li S, Wu H (2008) Sequential expression of TLR4 and its effects on the myocardium of rats with myocardial ischemia–reperfusion injury. Inflammation 31(5):304–312 4. Eltzschig HK, Eckle T (2011) Ischemia and reperfusion–from mechanism to translation. Nat Med 17(11):1391–1401 5. Matsumura K, Jeremy RW, Schaper J, Becker LC (1998) Progression of myocardial necrosis during reperfusion of ischemic myocardium. Circulation 97(8):795–804 6. Elsa¨sser A, Suzuki K, Schaper J (2000) Unresolved issues regarding the role of apoptosis in the pathogenesis of ischemic injury and heart failure. J Mol Cell Cardiol 32(5):711–724 7. Grishok A, Pasquinelli AE, Conte D, Li N, Parrish S, Ha I, Baillie DL, Fire A, Ruvkun G, Mello CC (2001) Genes and mechanisms related to RNA interference regulate expression of the small temporal RNAs that control C. elegans developmental timing. Cell 106(1):23–34 8. Ambros V (2004) The functions of animal microRNAs. Nature 431(7006):350–355 9. Bartel DP (2004) MicroRNAs: genomics, biogenesis, mechanism, and function. Cell 116(2):281–297 10. Dong S, Cheng Y, Yang J, Li J, Liu X, Wang X, Wang D, Krall TJ, Delphin ES, Zhang C (2009) MicroRNA expression signature and the role of microRNA-21 in the early phase of acute myocardial infarction. J Biol Chem 284(43):29514–29525 11. Goren Y, Kushnir M, Zafrir B, Tabak S, Lewis BS, Amir O (2012) Serum levels of microRNAs in patients with heart failure. Eur J Heart Fail 14(2):147–154 12. Wang J, Yang X (2012) The function of miRNA in cardiac hypertrophy. Cell Mol Life Sci 69(21):3561–3570 13. Lu Y, Zhang Y, Wang N, Pan Z, Gao X, Zhang F, Zhang Y, Shan H, Luo X, Bai Y, Sun L, Song W, Xu C, Wang Z, Yang B (2010) MicroRNA-328 contributes to adverse electrical remodeling in atrial fibrillation. Circulation 122(23):2378–2387 14. Ren XP, Wu J, Wang X, Sartor MA, Qian J, Jones K, Nicolaou P, Pritchard TJ, Fan GC (2009) MicroRNA-320 is involved in the regulation of cardiac ischemia/reperfusion injury by targeting heat-shock protein 20. Circulation 119(17):2357–2366 15. Goodman RH, Smolik S (2000) CBP/p300 in cell growth, transformation, and development. Genes Dev 14(13):1553–1577 16. Ait-Si-Ali S, Ramirez S, Barre FX, Dkhissi F, Magnaghi-Jaulin L, Girault JA, Robin P, Knibiehler M, Pritchard LL, Ducommun B, Trouche D, Harel-Bellan A (1998) Histone acetyltransferase activity of CBP is controlled by cycle-dependent kinases and oncoprotein E1A. Nature 396(6707):184–186 17. McManus KJ, Hendzel MJ (2001) CBP, a transcriptional coactivator and acetyltransferase. Biochem Cell Biol 79(3):253– 266 18. Avantaggiati ML, Ogryzko V, Gardner K, Giordano A, Levine AS, Kelly K (1997) Recruitment of p300/CBP in p53-dependent signal pathways. Cell 89(7):1175–1184 19. Chao J, Yin H, Yao YY, Shen B, Smith RS Jr, Chao L (2006) Novel role of kallistatin in protection against myocardial ischemia–reperfusion injury by preventing apoptosis and inflammation. Hum Gene Ther 17(12):1201–1213 20. Qin Y, Yu Y, Dong H, Bian X, Guo X, Dong S (2012) MicroRNA 21 inhibits left ventricular remodeling in the early phase of
Mol Biol Rep
21.
22.
23.
24.
25.
26.
rat model with ischemia–reperfusion injury by suppressing cell apoptosis. Int J Med Sci 9(6):413–423 Tang Y, Zheng J, Sun Y, Wu Z, Liu Z, Huang G (2009) MicroRNA-1 regulates cardiomyocyte apoptosis by targeting Bcl-2. Int Heart J 50(3):377–387 Wang X, Ha T, Liu L, Zou J, Zhang X, Kalbfleisch J, Gao X, Williams D, Li C (2013) Increased expression of microRNA146a decreases myocardial ischaemia/reperfusion injury. Cardiovasc Res 97(3):432–442 Wang X, Zhang X, Ren XP, Chen J, Liu H, Yang J, Medvedovic M, Hu Z, Fan GC (2010) MicroRNA-494 targeting both proapoptotic and antiapoptotic proteins protects against ischemia/ reperfusion-induced cardiac injury. Circulation 122(13):1308– 1318 Ling B, Wang GX, Long G, Qiu JH, Hu ZL (2012) Tumor suppressor miR-22 suppresses lung cancer cell progression through post-transcriptional regulation of ErbB3. J Cancer Res Clin Oncol 138(8):1355–1361 Jovicic A, Zaldivar Jolissaint JF, Moser R, Silva Santos Mde F, Luthi-Carter R (2013) MicroRNA-22 (miR-22) overexpression is neuroprotective via general anti-apoptotic effects and may also target specific Huntington’s disease-related mechanisms. PLoS ONE 8(1):e54222. doi:10.1371/journal.pone.0054222 Gurha P, Abreu-Goodger C, Wang T, Ramirez MO, Drumond AL, van Dongen S, Chen Y, Bartonicek N, Enright AJ, Lee B,
27.
28.
29.
30.
31.
Kelm RJ Jr, Reddy AK, Taffet GE, Bradley A, Wehrens XH, Entman ML, Rodriguez A (2012) Targeted deletion of microRNA-22 promotes stress-induced cardiac dilation and contractile dysfunction. Circulation 125(22):2751–2761 Huang Y, Zou Q, Wang SP, Tang SM, Zhang GZ, Shen XJ (2011) The discovery approaches and detection methods of microRNAs. Mol Biol Rep 38(6):4125–4135 Yang J, Jiang H, Chen SS, Chen J, Li WQ, Xu SK, Wang JC (2010) Lentivirus-mediated RNAi targeting CREB binding protein attenuates neointimal formation and promotes re-endothelialization in balloon injured rat carotid artery. Cell Physiol Biochem 26(3):441–448 Lin CL, Tseng HC, Chen RF, Chen WP, Su MJ, Fang KM, Wu ML (2011) Intracellular zinc release-activated ERK-dependent GSK-3b-p53 and Noxa-Mcl-1 signaling are both involved in cardiac ischemic-reperfusion injury. Cell Death Differ 18(10): 1651–1663 Zhang C, Feng Y, Qu S, Wei X, Zhu H, Luo Q, Liu M, Chen G, Xiao X (2011) Resveratrol attenuates doxorubicin-induced cardiomyocyte apoptosis in mice through SIRT1-mediated deacetylation of p53. Cardiovasc Res 90(3):538–545 Xu XD, Song XW, Li Q, Wang GK, Jing Q, Qin YW (2012) Attenuation of microRNA-22 derepressed PTEN to effectively protect rat cardiomyocytes from hypertrophy. J Cell Physiol 227(4):1391–1398
123
MicroRNA-22 targeting CBP protects against myocardial ischemia–reperfusion injury through anti-apoptosis in rats Jian Yang • Lihua Chen • Jun Yang • Jiawang Ding • Song Li • Hui Wu • Jing Zhang Zhixing Fan • Wusong Dong • Xinxin Li
•
Received: 12 July 2013 / Accepted: 2 December 2013 Ó Springer Science+Business Media Dordrecht 2013
Abstract MicroRNAs are extensively involved in the pathogenesis of major cardiovascular diseases by suppressing target gene expression. Recent studies have reported that microRNA-22 (miR-22) may be implicated in ischemia–reperfusion (I/R) induced myocardial injury. However, the specific function of miR-22 in myocardial I/R injury is far from clear nowadays. The present study was designed to determine the role of miR-22 in myocardial I/R injury and investigate the underlying cardio-protective mechanism. The rat myocardial I/R injury model was induced by occluding the left anterior descending coronary artery for 30 min followed by 12 h reperfusion. As predicted, adenovirus-mediated miR-22 overexpression markedly reduced the release of creatine kinase and lactate dehydrogenase, infarct size and cardiomyocytes apoptosis. Moreover, CREB binding protein (CBP) as a potential miR-22 target by bioinformatics was significantly inhibited after miR-22 transfection. We also found that p53 acetylation activity, pro-apoptotic related genes Bax and p21 levels were all decreased associated with the down-regulation of CBP. In conclusion, our data demonstrate that miR-22 could inhibit apoptosis of cardiomyocytes through one of its targets, CBP. Thus, miR-22 may constitute a new
Jian Yang and Lihua Chen contributed equally to this work. J. Yang J. Yang (&) J. Ding S. Li H. Wu J. Zhang Z. Fan W. Dong X. Li Department of Cardiology, The First College of Clinical Medical Sciences, China Three Gorges University, Yichang 443000, Hubei, China e-mail: [email protected] L. Chen Department of Optometry and Ophthalmology, Yichang Central People’s Hospital, Yichang 443000, Hubei, China
therapeutic target for the prevention of myocardial I/R injury. Keywords MicroRNAs Reperfusion injury CREB binding protein Apoptosis Cardiomyocytes
Introduction It is well known that reperfusion is the most important therapeutic strategy for ischemic heart disease [1, 2]. However, reperfusion itself could cause reperfusion-associated myocardial dysfunction and damage [3, 4]. To date, it has been widely accepted that apoptosis plays a key role in myocardial ischemia–reperfusion (I/R) injury [5, 6]. It is therefore necessary to find an effective target against apoptosis for attenuating I/R injury. MicroRNAs (miRNAs) are a class of endogenous, small (=22 nt in size), noncoding single-stranded RNAs that negatively regulate gene expression through binding on the 30 untranslated region (UTR) of target mRNAs [7, 8]. They participate in almost all cellular processes, such as proliferation, differentiation and apoptosis [7–9]. In addition, miRNAs are also recognized as critical regulators of cardiovascular diseases, including myocardial infarction [10], heart failure [11], cardiac hypertrophy and arrhythmia [12, 13]. Thus, miRNAs might be attractive therapeutic targets for the treatment of myocardial I/R injury. Recently, Ren et al. [14] using miRNA arrays have found that the expression of one specific microRNA, miR-22, is decreased in rat hearts on I/R in vivo and ex vivo. These data suggest that miR-22 may be involved in the initiation and progression of myocardial I/R injury. CREB binding protein (CBP), as a general transcriptional co-activator, is essential in cell growth, differentiation and
123
Mol Biol Rep
apoptosis [15]. Because of its histone acetyltransferase activity, CBP could acetylate various sequence-specific transcription factors such as CREB, NF-kB, AP-1 and p53, to regulate their downstream genes expression [16–18]. In fact, CBP is identified as a potential miR-22 target by the bioinformatics of TargetScan. More interestingly, preliminary unpublished work from our laboratory has demonstrated that the level of CBP is strikingly up-regulated in rat hearts after I/R. However, it is entirely unknown whether CBP mediates myocardial I/R injury by miR-22 regulation. In the present study, we used adenoviral overexpression of miR-22 to investigate its effect on I/R injury, and identify the underlying mechanism further. Our data demonstrates for the first time that miR-22 could efficiently attenuate I/R injury by regulating CBP expression and inhibiting apoptosis. Taken together, these findings implicate that miR-22 may be a new therapeutic target for ischemic heart disease.
or saline was respectively injected into six separate sites of the left ventricular anterior wall via a 26-gauge needle. The chest was closed after injection and the rat was allowed to recover. Myocardial I/R treatment was performed four days later. All the rats were re-anesthetized and ventilated artificially with room air. The thorax was reopened through the original intercostal space, and the left anterior descending coronary artery (LAD) was identified. The LAD was ligated using a 6–0 silk suture. Additionally, a medical latex tube (socket, inner diameter, 1.5 mm) was placed between the ligature and the LAD. Myocardial ischemia was induced by tightening the ligature around the latex tube. Ischemia was confirmed by myocardial blanching and electrocardiography evidence of injury. After 30 min ischemia, the latex tube was removed to restore the coronary circulation. At 12 h post-reperfusion, the hearts and blood samples were obtained for further analysis. Shamoperated controls underwent the same procedures, with the exception that the LAD was not ligated.
Materials and methods
Detection of creatine kinase (CK) and lactate dehydrogenase (LDH)
Animal care Adult male Sprague-Dawley rats (250–300 g) received a standard diet and free water. The procedures for experiments and animal care were approved by the Animal Care and Use Committee of China Three Gorges University (CTGU), and conformed to the Guide for the Care and Use of Laboratory Animals by the National Institutes of Health (NIH Publication No. 80-23). miR-22 Expression vector construct Rno-miR-22 precursor DNA (MI0000851) was synthesized by Genechem (Shanghai, China). The adenovirus expressing miR-22 (Ad-miR-22), or control adenovirus expressing Scramble (Ad-Scramble) was generated using the AdMax system (Microbix Biosystems, Canada) according to the manufacturer’s protocols. These resulting adenoviruses were further packaged and amplified in HEK293 cells, and purified using CsCl banding. The final titers were determined in plaque assays. In vivo gene transfer and rat myocardial I/R model The animals were subject to adenovirus-mediated gene transfer, and subsequent myocardial I/R surgical procedure [19]. In brief, rats were anesthetized with pentobarbital (30 mg/kg intraperitoneal). A left parasternal incision was made through the fourth and fifth ribs, and the pericardium was gently opened to expose the heart. A 100 ll solution of Ad-miR-22 (1 9 109 PFU), Ad-Scramble (1 9 109 PFU)
123
Blood samples were collected at the end of reperfusion and centrifuged at 3,000 g for 10 min to isolate serum. Two specific marker enzymes, including the creatine kinase (CK) and lactate dehydrogenase (LDH), were assayed using commercial kits (Beijing Kemeidongya Biotechnology Ltd., China). Results were presented in international units per litre. Assessment of myocardial infarct size Myocardial infarct size was evaluated by Evans Blue/triphenyltetrazolium chloride (TTC) staining as previously described. At the end of 12 h reperfusion, the LAD was religated, and 1 ml of 2 % Evans Blue dye (Sigma-Aldrich, St Louis, MO) was administered intravenously to distinguish the non-ischemic area from the area at risk. The hearts were rapidly excised and sliced parallel to the atrioventricular groove into 2-mm-thick sections. The slices were then incubated in 1 % TTC (Sigma Chemicals, St. Louis, MO, USA) at 37 °C for 20 min to identify the infarct zone. The area at risk (AAR), infarct area (IA) and left ventricular area (LV) were measured digitally using Image-Pro Plus 5.0 software (Media Cybernetic, USA). The percentage of IA/ AAR and AAR/LV was calculated respectively. Determination of myocardial apoptosis Cardiomyocytes apoptosis was assessed by terminal deoxynucleotide transferase dUTP nick end labeling (TUNEL) staining. The obtained heart tissue was washed, dehydrated, and imbedded in paraffin. Then the TUNEL
Mol Biol Rep
assay was performed using an apoptosis detection kit (Roche, Basle, Switzerland) in accordance with instructions. The numbers of TUNEL-positive cells and the total cells in heart sections were counted in five microscopic fields. The ratio of TUNEL-positive cells to total cardiomyocytes was apoptosis index (AI). Total RNA extraction and quantitative RT-PCR Total RNA from the cardiac muscle samples was extracted using Trizol Reagent (Invitrogen, Carlsbad, CA) according to the manufacturer’s protocol. miR-22 levels were measured using the mirVanaTM qRT-PCR miRNA detection kit (Ambion Inc. TX). Amplification and detection of specific products were performed with the ABI Prism 7,500 system (PE Applied Biosystems). U6 was used as an internal control. miR-22 expression was normalized to U6 small nucleolar RNA. 2-DDCt method [20] was used to calculate relative expression levels. Western blot analysis To determine the protein levels of CBP, p53, acetylated p53, Bax, p21 and b-actin in myocardial tissue, proteins extraction and Western blot analysis were performed as described previously. Briefly, equal amounts of protein extracts were subjected to SDS-PAGE, and transferred onto nitrocellulose membranes. Non-specific binding was blocked with 5 % non-fat dry milk. The membrane was rinsed, and immunoblotted with each primary antibody at a recommended dilution. Subsequently, the membrane was washed and incubated with horseradish peroxidase-conjugated secondary antibody. Bands were visualized using an enhanced chemiluminescence (ECL) detection kit (Pierce, Rockford, IL, USA). Statistical analysis All values were presented as mean ± SEM. Differences between groups were analyzed for significance by one-way analysis of variance (ANOVA) and Student–Newman– Keuls (SNK) q test using SPSS software (version 14.0). A value of P \ 0.05 was considered to be statistically significant.
Results Myocardial I/R induced down-regulation of miR-22 expression As shown in Fig. 1, after 12 h of reperfusion, myocardialspecific miR-22 expression was significantly down-
Fig. 1 Regulation of miR-22 after myocardial I/R. miR-22 was down-regulated in I/R group, while adenovirus-mediated miR-22 gene transfer in vivo up-regulated its expression. Values were expressed as mean ± SEM, n = 8 per group, *P \ 0.05
regulated 1.7-fold as compared to sham group without injury. Transfection of miR-22 into the myocardium after four day, could markedly increase miR-22 expression by 81.2 % (Ad-miR-22 vs. Ad-Scramble, P \ 0.05). However, Ad-Scramble had no apparent effect on the expression level of miR-22 compared with I/R group (P [ 0.05). Effects of miR-22 on serum CK and LDH The activities of serum CK and LDH were initially low in the sham group. Myocardial ischemia for 30 min by LAD ligation followed by 12 h of reperfusion significantly increased the leakage of CK and LDH compared with sham group (Fig. 2). However, the elevation in CK and LDH levels was obviously suppressed by Ad-miR-22 transfection (Ad-miR-22 vs. I/R, P \ 0.05). Ad-Scramble did not affect I/R-induced increase leakage of CK and LDH from myocardium (Ad-Scramble vs. I/R, P [ 0.05). miR-22 Reduced infarct size The extent of myocardial infarction was evaluated after reperfusion. No significant difference was found in AAR/ LV among the four groups (data not shown). Infarct size was measured to be 48.3 ± 3.5 % of the area at risk (IA/ AAR) in the I/R group. miR-22 over expression significantly reduced myocardial infarct size compared with that in the I/R group (P \ 0.05, Fig. 3). Interestingly, pretreatment with Ad-Scramble did not affect infarct size (51.4 ± 6.7 % vs. I/R group, P [ 0.05). Our results suggest that transient up-regulation of miR-22 may play a cardio-protective role during myocardial I/R injury.
123
Mol Biol Rep Fig. 2 miR-22 reduced creatine kinase (CK) and lactate dehydrogenase (LDH) release. Ad-miR-22 reduced CK and LDH level after I/R injury in rat serum. a The activity of CK in serum. b The activity of LDH in serum. Data were expressed as mean ± SEM, n = 8, *P \ 0.05
Fig. 3 miR-22 decreased myocardial infarct size in rats subjected to I/R injury. a Representative illustrations of infarct size as stained by Evans Blue and TTC. Dark blue area, nonischemic tissue; red-stained area, area at risk; white area, infracted tissue. b The infarct size normalized to the area at risk in different groups. AAR: area at risk; IA: infarct area. Results were presented as mean ± SEM, n = 6 in each group, *P \ 0.05
miR-22 Attenuated cardiomyocytes apoptosis induced by I/R Terminal deoxynucleotide transferase dUTP nick end labeling assay was performed to evaluate the cardiomyocytes apoptosis in vivo. As shown in Fig. 4, the apoptotic index in sham group was only 3.1 ± 0.9 %. Compared with sham group, TUNEL-positive cardiomyocytes were more frequently observed in the I/R group and Ad-Scramble group (P both \ 0.05). However, this effect was largely alleviated by Ad-miR-22 transfection (23.4 ± 1.8 % in Ad-miR-22 group vs. 32.6 ± 4.1 % in I/R group, P \ 0.05).
123
Fig. 4 Effect of miR-22 on apoptosis in I/R induced myocardial injury. a Representative microphotographs of TUNEL staining from four groups. Arrows indicate TUNEL-positive cardiomyocytes nuclei stained as dark brown. Scale bar represents 25 lm. b Mean apoptotic index was counted in each group. Data were expressed as mean ± SEM, n = 8, *P \ 0.05
miR-22 Regulated the relative protein expression CBP, as the target gene of miR-22 (predicted by the TargetScan software, Fig. 5a), was significantly up-regulated
Mol Biol Rep
Fig. 5 Immunoblots and graphs showing the relative protein levels and fold changes from different groups. a Putative binding sites of miR-22 in the CBP 30 UTR (white sequences) predicted by TargetScan. b Original representative western blots of the proteins obtained from myocardial tissue. c miR-22 suppressed p53 acetylation induced
by I/R injury. d The relative CBP, p21 and Bax protein levels in the four groups. Bars show mean ± SEM, n = 8. NS means not significant at the 0.05 level. *P \ 0.05, HP \ 0.05 vs. I/R or AdScramble group
4.2-fold as compared to sham group without I/R injury (Fig. 5b, d). Delivery of miR-22 into the myocardium could markedly decrease CBP protein expression by 45.2 % (vs. I/R group, P \ 0.05). Ad-Scramble had no inhibitory effect on the expression of CBP (vs. I/R group, P [ 0.05). It was found that I/R induced significant p53 activation and acetylation in the injured myocardium. AdmiR-22 treatment resulted in a 44.1 and 38.7 % reduction of p53 acetylation in Ad-Scramble transfected and saline treated myocardium, respectively (P both \ 0.05). However, the total p53 protein level was not suppressed by AdmiR-22 (P [ 0.05; Fig. 5b, c). In addition, p53 downstream pro-apoptotic proteins Bax and p21, also exhibited the similar change like acetylated p53 after Ad-miR-22 transfection (Fig. 5b, d).
reduction, and down-regulated activity of p53 acetylation could be clearly observed in vivo. These major findings unveil that the cardio-protection of miR-22 against cardiomyocytes apoptosis is, at least in part, functionally attributed to its suppression of CBP. A number of miRNAs, such as miR-1 [21], miR-146a [22]and miR-494 [23] were verified to be involved in myocardial I/R injury by altering the expression of key genes associated with cell apoptosis. miR-22 was considered to be a tumor-suppressing microRNA in various cancers [24]. Moreover, Jovicic et al. [25] also found that miR-22 had a pronounced neuro-protective effect by targeting the related genes and inhibiting neuron apoptosis. Recently, miR-22 has been reported as a critical regulator of cardiomyocyte hypertrophy and cardiac remodeling [26]. However, the role of miR-22 in myocardial I/R injury remains unclear at this point. In this study, we found miR22 was significantly down-regulated in ischemic myocardium subjected to 30 min LAD occlusion followed by 12 h reperfusion in vivo. The adenoviral vector-mediated transfection system could efficiently deliver miR-22 into rat hearts. Overexpression of miR-22 reduced the release of myocardial enzyme, cardiomyocytes apoptosis and infarct size of rats after myocardial I/R. Thus, we presume that the molecular mechanisms involved in the cardio-protective effect of miR-22 are very complicated.
Discussion It is well established that myocardial I/R injury is a complex pathophysiological process in which apoptosis plays a crucial role [6]. In the current study, we found that AdmiR-22 exerted a potent protective effect against I/R induced apoptosis in cardiomyocytes. Meanwhile, increased levels of miR-22, with a concomitant CBP
123
Mol Biol Rep
Indeed, miRNAs elicit their function through post-transcriptional silencing of RNA interference pathway [7, 8]. Individual miRNA may have multiple targets [27]. miR-22 as a cardiac-enriched microRNA, has several different targets in cardiomyocytes by using software applications such as miRanda and TargetScan. Among these targets, the CBP gene attracts researchers’ attention due to its multifunctional coordination ability. In this paper, our experiments confirm that CBP was inversely correlated with miR22 expression in rat heart tissues. In fact, CBP gene is widely expressed and plays pivotal role in cardiovascular system. We have previously reported that CBP mediates the development and progression of neointimal formation after vessel injury [28]. CBP and its close homologue p300 are known to interact with a variety of diverse transcriptional factors [16–18], which lead to distinct biological outcomes. Particulally, the function of CBP in gene transcription is also dependent on its acetylation status. The tumor suppressor protein p53 is one of the important transcriptional factors, which regulates the apoptosisrelated gene expression involved in I/R injury [29]. The activation of p53 could contribute to cardiomyocytes apoptosis via inducing cell cycle arrest and pro-apoptotic proteins [30]. Hence, suppressing p53 activity can reduce I/R induced myocardial injury. In our study, we observed that Ad-miR-22 not only prevented cell apoptosis, but also decreased I/R induced p53 acetylation. Furthermore, p53 downstream target genes, including p21 for cell-cycle arrest and Bax for apoptosis were both markedly inhibited by miR-22 transfection. By repressing CBP, miR-22 attenuates p53 acetylation, leading to the reduction of proapoptotic genes. In this regard, our data suggest that p53dependent apoptosis in myocardial I/R injury, at least in part, through activation of the miR-22/CBP/p53 pathway. Although miR-22 has been shown to have apparent antiapoptotic function, the cardio-protective effect of it might not be similar for all cardiovascular diseases [26, 31]. miR22 has several other targets in cardiomyocytes, which engage multiple signaling pathways. Further studies should investigate other targets of miR-22 to fully elucidate the role of miR-22 in myocardial I/R injury. In summary, our study reveals that dys-regulation of miR-22 expression contributes to ischemic heart disease. Overexpression of miR-22 provides cardiac protection against I/R induced cardiomyocytes apoptosis by directly targeting CBP. These findings support the perspective that enhancing miR-22 expression might be a desirable therapeutic approach for the treatment of myocardial I/R injury. Acknowledgments This project was supported by the National Natural Science Foundation of China (Grant Nos. 81200088, 81201458) and the Natural Science Foundation of Yichang city, China (Grant No. A12301-01).
123
References 1. Yellon DM, Hausenloy DJ (2007) Myocardial reperfusion injury. N Engl J Med 357(11):1121–1135 2. Cokkinos DV, Pantos C (2007) Myocardial protection in man– from research concept to clinical practice. Heart Fail Rev 12(3–4):345–362 3. Yang J, Yang J, Ding JW, Chen LH, Wang YL, Li S, Wu H (2008) Sequential expression of TLR4 and its effects on the myocardium of rats with myocardial ischemia–reperfusion injury. Inflammation 31(5):304–312 4. Eltzschig HK, Eckle T (2011) Ischemia and reperfusion–from mechanism to translation. Nat Med 17(11):1391–1401 5. Matsumura K, Jeremy RW, Schaper J, Becker LC (1998) Progression of myocardial necrosis during reperfusion of ischemic myocardium. Circulation 97(8):795–804 6. Elsa¨sser A, Suzuki K, Schaper J (2000) Unresolved issues regarding the role of apoptosis in the pathogenesis of ischemic injury and heart failure. J Mol Cell Cardiol 32(5):711–724 7. Grishok A, Pasquinelli AE, Conte D, Li N, Parrish S, Ha I, Baillie DL, Fire A, Ruvkun G, Mello CC (2001) Genes and mechanisms related to RNA interference regulate expression of the small temporal RNAs that control C. elegans developmental timing. Cell 106(1):23–34 8. Ambros V (2004) The functions of animal microRNAs. Nature 431(7006):350–355 9. Bartel DP (2004) MicroRNAs: genomics, biogenesis, mechanism, and function. Cell 116(2):281–297 10. Dong S, Cheng Y, Yang J, Li J, Liu X, Wang X, Wang D, Krall TJ, Delphin ES, Zhang C (2009) MicroRNA expression signature and the role of microRNA-21 in the early phase of acute myocardial infarction. J Biol Chem 284(43):29514–29525 11. Goren Y, Kushnir M, Zafrir B, Tabak S, Lewis BS, Amir O (2012) Serum levels of microRNAs in patients with heart failure. Eur J Heart Fail 14(2):147–154 12. Wang J, Yang X (2012) The function of miRNA in cardiac hypertrophy. Cell Mol Life Sci 69(21):3561–3570 13. Lu Y, Zhang Y, Wang N, Pan Z, Gao X, Zhang F, Zhang Y, Shan H, Luo X, Bai Y, Sun L, Song W, Xu C, Wang Z, Yang B (2010) MicroRNA-328 contributes to adverse electrical remodeling in atrial fibrillation. Circulation 122(23):2378–2387 14. Ren XP, Wu J, Wang X, Sartor MA, Qian J, Jones K, Nicolaou P, Pritchard TJ, Fan GC (2009) MicroRNA-320 is involved in the regulation of cardiac ischemia/reperfusion injury by targeting heat-shock protein 20. Circulation 119(17):2357–2366 15. Goodman RH, Smolik S (2000) CBP/p300 in cell growth, transformation, and development. Genes Dev 14(13):1553–1577 16. Ait-Si-Ali S, Ramirez S, Barre FX, Dkhissi F, Magnaghi-Jaulin L, Girault JA, Robin P, Knibiehler M, Pritchard LL, Ducommun B, Trouche D, Harel-Bellan A (1998) Histone acetyltransferase activity of CBP is controlled by cycle-dependent kinases and oncoprotein E1A. Nature 396(6707):184–186 17. McManus KJ, Hendzel MJ (2001) CBP, a transcriptional coactivator and acetyltransferase. Biochem Cell Biol 79(3):253– 266 18. Avantaggiati ML, Ogryzko V, Gardner K, Giordano A, Levine AS, Kelly K (1997) Recruitment of p300/CBP in p53-dependent signal pathways. Cell 89(7):1175–1184 19. Chao J, Yin H, Yao YY, Shen B, Smith RS Jr, Chao L (2006) Novel role of kallistatin in protection against myocardial ischemia–reperfusion injury by preventing apoptosis and inflammation. Hum Gene Ther 17(12):1201–1213 20. Qin Y, Yu Y, Dong H, Bian X, Guo X, Dong S (2012) MicroRNA 21 inhibits left ventricular remodeling in the early phase of
Mol Biol Rep
21.
22.
23.
24.
25.
26.
rat model with ischemia–reperfusion injury by suppressing cell apoptosis. Int J Med Sci 9(6):413–423 Tang Y, Zheng J, Sun Y, Wu Z, Liu Z, Huang G (2009) MicroRNA-1 regulates cardiomyocyte apoptosis by targeting Bcl-2. Int Heart J 50(3):377–387 Wang X, Ha T, Liu L, Zou J, Zhang X, Kalbfleisch J, Gao X, Williams D, Li C (2013) Increased expression of microRNA146a decreases myocardial ischaemia/reperfusion injury. Cardiovasc Res 97(3):432–442 Wang X, Zhang X, Ren XP, Chen J, Liu H, Yang J, Medvedovic M, Hu Z, Fan GC (2010) MicroRNA-494 targeting both proapoptotic and antiapoptotic proteins protects against ischemia/ reperfusion-induced cardiac injury. Circulation 122(13):1308– 1318 Ling B, Wang GX, Long G, Qiu JH, Hu ZL (2012) Tumor suppressor miR-22 suppresses lung cancer cell progression through post-transcriptional regulation of ErbB3. J Cancer Res Clin Oncol 138(8):1355–1361 Jovicic A, Zaldivar Jolissaint JF, Moser R, Silva Santos Mde F, Luthi-Carter R (2013) MicroRNA-22 (miR-22) overexpression is neuroprotective via general anti-apoptotic effects and may also target specific Huntington’s disease-related mechanisms. PLoS ONE 8(1):e54222. doi:10.1371/journal.pone.0054222 Gurha P, Abreu-Goodger C, Wang T, Ramirez MO, Drumond AL, van Dongen S, Chen Y, Bartonicek N, Enright AJ, Lee B,
27.
28.
29.
30.
31.
Kelm RJ Jr, Reddy AK, Taffet GE, Bradley A, Wehrens XH, Entman ML, Rodriguez A (2012) Targeted deletion of microRNA-22 promotes stress-induced cardiac dilation and contractile dysfunction. Circulation 125(22):2751–2761 Huang Y, Zou Q, Wang SP, Tang SM, Zhang GZ, Shen XJ (2011) The discovery approaches and detection methods of microRNAs. Mol Biol Rep 38(6):4125–4135 Yang J, Jiang H, Chen SS, Chen J, Li WQ, Xu SK, Wang JC (2010) Lentivirus-mediated RNAi targeting CREB binding protein attenuates neointimal formation and promotes re-endothelialization in balloon injured rat carotid artery. Cell Physiol Biochem 26(3):441–448 Lin CL, Tseng HC, Chen RF, Chen WP, Su MJ, Fang KM, Wu ML (2011) Intracellular zinc release-activated ERK-dependent GSK-3b-p53 and Noxa-Mcl-1 signaling are both involved in cardiac ischemic-reperfusion injury. Cell Death Differ 18(10): 1651–1663 Zhang C, Feng Y, Qu S, Wei X, Zhu H, Luo Q, Liu M, Chen G, Xiao X (2011) Resveratrol attenuates doxorubicin-induced cardiomyocyte apoptosis in mice through SIRT1-mediated deacetylation of p53. Cardiovasc Res 90(3):538–545 Xu XD, Song XW, Li Q, Wang GK, Jing Q, Qin YW (2012) Attenuation of microRNA-22 derepressed PTEN to effectively protect rat cardiomyocytes from hypertrophy. J Cell Physiol 227(4):1391–1398
123
