MicroRNA-92a Inhibition Attenuates Hypoxia/Reoxygenation-Induced Myocardiocyte Apoptosis by Targeting Smad7

Background MicroRNAs (miRNAs) regulate a lot of physiological and pathological processes, including myocardial ischemia/reperfusion. Recent studies reported that knockdown of miR-92a could attenuate ischemia/reperfusion-induced myocardial injury. In the present study, we examined the potential anti-apoptotic effects of miR-92a in a rat myocardiocyte cell line, and the possible role of Smad7 in such actions. Methodology and Results In a preliminary bioinformatic analysis, we identified SMAD family member 7 (Smad7) as a potential target for miR-92a. A luciferase reporter assay indeed demonstrated that miR-92a could inhibit Smad7 expression. Myocardial ischemia/reperfusion was simulated in rat H9c2 cells with 24-h hypoxia followed by 12-h reoxygenation. Prior to hypoxia/reoxygenation, cells were transfected by miR-92a inhibitor. In some experiments, cells were co-transfected with siRNA-Smad7. The miR-92a inhibitor dramatically reduced the release of lactate dehydrogenase and malonaldehyde, and attenuated cardiomyocyte apoptosis. The miR-92a inhibitor increased SMAD7 protein level and decreased nuclear NF-κB p65 protein. Effects of the miR-92a inhibitor were attenuated by co-transfection with siRNA-Smad7. Conclusion Inhibiting miR-92a can attenuate myocardiocyte apoptosis induced by hypoxia/reoxygenation by targeting Smad7.


Introduction
Myocardial ischemia/reperfusion (I/R) injury contributes to the damage after ischemic events in patients with coronary heart disease (CHD) [1,2]. I/R injury is also implicated in cardiac procedures that require cardio-pulmonary bypass, and in CHD patients receiving percutaneous coronary intervention or coronary artery bypass surgery. I/R injury is mediated by a variety of factors, including oxidative stress, intracellular Ca 2+ overload, rapid restoration of physiological pH upon reperfusion, the mitochondrial permeability transition pore (MPTP), and exaggerated inflammation [3].
MicroRNAs (miRNAs) are a class of endogenous, small noncoding single-stranded RNAs, typically 18-24 nucleotides in length, that negatively regulate gene expression through binding to the 39-untranslated region (UTR) of target mRNAs [4]. MiRNAs play critical roles in a variety of heart diseases, including cardiac hypertrophy [5], heart failure [6], arrhythmia [7], myocardial infarction [8] and I/R injury [9]. Growing evidence also supports a pivotal role for miR-92a in multiple processes, including tumorigenesis and metastasis [10], cell proliferation and apoptosis [11]. In the study, we found that transfection with miR-92a inhibitor could attenuate myocardial injury and apoptosis induced by hypoxia/reoxygenation (H/R) in cultured rat H9c2 myocardiocytes cells. A preliminary bioinformatics analysis identified Smad7 as a target for miR-92a. Accordingly, we also examined the possible involvement of Smad7 in the protective action of miR-92a.

Cell Culture
The H9c2 cells (ventricular myocardiocyte, rat in origin; Cell Bank of the Chinese Academy of Sciences, Shanghai, China) were seeded at a density of 2610 4 cells/cm 2 in 6-well plates and cultured in Dulbecco's modified Eagle's medium (DMEM, Sigma, St. Louis, MO, USA) containing 10% (v/v) fetal bovine serum (FBS, HyClone, Logan, UT, USA) in a humidified atmosphere of 95% air and 5% CO 2 at 37uC.

Transient Transfection with Oligonucleotides
Transfection was carried out using Lipofectamine 2000 (Invitrogen, Carlsbad, CA, USA). The ratio of oligonucleotide vs. the Lipofectamine 2000 transfection reagent was 1:5. MiR-92a mimic, inhibitor and matched negative control (NC) were synthesized by GenePharma, Shanghai, China. For RNA interference, cells were transiently transfected with a siRNA specific for Smad7 or NC (GenePharma). All transfections were carried out after 12-h serum starvation, and lasted for 48-h prior to the H/R experiments.

H/R in H9c2 Cardiomyocytes
Hypoxia was induced by exposing the cells to 1% O 2 , 94% N 2 , and 5% CO 2 for 24 h using a modular incubator (Model 3131, Forma Scientific, Marietta, OH, USA). Reoxygenation (95% air, 5% CO 2 , 37uC) lasted for 12 h. Cells under normoxia throughout the experiments were included as a control. All experiments were repeated three times.

Quantitative Real-time Polymerase Chain reaction (qRT-PCR)
Total RNA was extracted using Trizol reagent (Invitrogen). Bulge-loop miRNA qRT-PCR primer sets (one RT primer and a pair of qRT-PCR primers for each set) specific for miR-17, miR-18a, miR-19a, miR-20a, miR-19b and miR-92a were designed by RiboBio (Guangzhou, China). MiRNAs were reverse transcribed using the stem-loop RT primer. The primers for Smad7 were also designed by RiboBio. qRT-PCR was carried out to examine the expression of specific miRNAs or mRNA on a Rotor-Gene 3,000 real-time DNA detection system (Corbett Research, Sydney, Australia) using SYBR Green (Qiagen, Shanghai, China). All samples were analyzed in triplicate. Gene expression was determined by comparing the data against the standard curve, and normalized against U6.

Determination of Cell Injury and Apoptosis
Structural integrity of cultured H9c2 cardiomyocytes was evaluated by measuring the concentration of lactate dehydrogenase (LDH) and malonaldehyde (MDA) in the culture media by ELISA using an automatic biochemical analyzer (Model 7150, Hitachi, Tokyo, Japan). Apoptosis was detected by annexinV-FITC/propidium iodide (AV/PI) dual staining (Bender MedSystems, Burlingame, CA, USA).

DNA Constructs and Reporter Gene Assays
To examine whether miR-92a regulates the expression of Smad7, we used a dual luciferase psiCheck-2 reporter plasmid (Promega, Madison, WI, USA) to generate a reporter plasmid harboring the Smad7 39-UTR. For luciferase reporter experiments, the 39UTR of the Smad7 gene was amplified by PCR from rat genomic DNA and cloned into psiCHECK-2 (Promega) between the Not1 and Sgf1 sites. The construct with a mutated targeting fragment (TATACCG) in the 39-UTR of Smad7 lacking the putative miR-92a binding sequence was used as a mutated control. 293T cells were co-transfected with psiCheck2 containing the Smad7 39-UTR and the miR-92a mimic using Lipofectamine 2000 (Invitrogen). Co-transfection with non-targeting negative control RNA was performed as a control. The cells were harvested 24 h after transfection for luciferase activity using a dual luciferase reporter assay kit (Promega) on a luminometer (Lumat LB9507).

Statistics and Data Analysis
All data are expressed as the mean6SEM. Comparisons between groups were made by one-way analysis of variance or two-tailed student's t-test. Differences were considered statistically significant at P,0.05. SPSS software version 19.0 (SPSS, Chicago, IL, USA) was used for data analysis. All experiments were performed at least three times.

MiR-17-92 Expression Profiles in H/R H9c2 Cardiomyocytes
In our previous study [12], we found that miR-17, miR-19a, miR-20a, miR-19b and miR-92a, but not miR-18a, were highly expressed in the heart of C57BL/6 mice. In the current study, the expression of the miR-17-92 cluster was up-regulated in H/R H9c2 cardiomyocytes: the expression of miR-92a was significantly up-regulated by 2.78-fold over the control (P,0.01 vs. control) (Figure 1). Based on the most remarkable change in response to hypoxia/reoxygenation as reflected by qRT-PCR, miR-92a was selected for subsequent experiments.

Inhibition of miR-92a Protects against H/R-induced Injury and Apoptosis
H/R treatment increased LDH in the culture media (16.3660.74 vs. 8.1660.47 ng/mL in normoxic condition, P, 0.01) ( Figure 3A). The miR-92a inhibitor significantly decreased LDH release in response to H/R (10.9361.35 ng/mL, P,0.01 vs. the H/R group). Co-transfection with siRNA-Smad7 attenuated the effects of the miR-92a inhibitor.
H/R treatment increased MDA release (38.8363.70 vs. 20.3362.05 ng/mL in normoxic condition, P,0.01) ( Figure 3B). The H/R-induced MDA release was significantly decreased by the miR-92a inhibitor (26.9361.59 ng/mL, P,0.01 vs. the H/R group). The observed effects of the miR-92a inhibitor were also attenuated by co-transfection with siRNA-Smad7.    Figure 4C). Transfection with miR-92a inhibitor significantly decreased the percentage of necrosis induced by H/R (6.1660.35%, P,0.05 vs. the H/R group). The effects of miR-92a inhibitor were also attenuated by co-transfection with siRNA-Smad7.

Smad7 is a Target of miR-92a
Bioinformatic analysis using MiRanda, miRDB, miRwalk and TargetScan suggested Smad7 as a target of miR-92a. Specifically, Figure 7. Inhibition of miR-92a promotes SMAD7 expression and activates the Smad7/NF-kB signaling pathway. A. qRT-PCR analysis of endogenous Smad7 mRNA levels in the cardiomyocytes transfected with miR-92a inhibitor, or co-transfected with miR-92a inhibitor and siRNA-Smad7. B. Western blotting assays for the SMAD7 protein level in the cardiomyocytes transfected with miR-92a inhibitor, or co-transfected with miR-92a inhibitor and siRNA-Smad7. C. Western blotting assays for the cytosolic NF-kB p65 protein levels in the cardiomyocytes transfected with miR-92a inhibitor, or co-transfected with miR-92a inhibitor and siRNA-Smad7. D. Western blotting assays for the nuclear NF-kB p65 protein levels in the cardiomyocytes transfected with miR-92a inhibitor, or co-transfected with miR-92a inhibitor and siRNA-Smad7. (*P,0.05 and **P,0.01 vs. the H/R group). H/R, hypoxia/reoxygenation. doi:10.1371/journal.pone.0100298.g007 the 39-UTR of the Smad7 mRNA contains one binding site for miR-92a ( Figure 5A).
In comparison with the mutated control, the miR-92a mimic reduced the activity of the luciferase reporter fused with the Smad7 39-UTR by 41% ( Figure 5B). Inmunocytofluorescent staining ( Figure 6) revealed very low level of SMAD7 in cells exposed to the H/R treatment. The protein level of SMAD7 was increased by the miR-92a inhibitor.
The miR-92a inhibitor did not affect the level of Smad7 mRNA ( Figure 7A). Co-transfection with siRNA-Smad7 significantly decreased the level of Smad7 mRNA (P,0.01). Western blotting ( Figure 7B) revealed increased level of SMAD7 by the miR-92a inhibitor (in comparison to H/R alone, P,0.05). The effects of miR-92a inhibitor were attenuated by co-transfection with siRNA-Smad7.
Cytosolic NF-kB p65 was not affected by transfection with miR-92a inhibitor, or co-transfection with miR-92a inhibitor and siRNA-Smad7 ( Figure 7C). Nuclear NF-kB p65 was significantly decreased by the miR-92a inhibitor (in comparison with H/R alone, P,0.05). The effects of the miR-92a inhibitor on nuclear NF-kB p65 were attenuated by co-transfection with siRNA-Smad7 ( Figure 7D).

Discussion
Apoptosis plays a crucial role in myocardial I/R injury [13,14]. A number of miRNAs, including miR-1 [15], miR-15b [16], miR-21 [17] and miR-145 [18], have been implicated in myocardial I/ R injury due to their effects on key genes associated with apoptosis. MiR-92a has been implicated in myocardial I/R injury in variety of experimental models. Bonauer et al. demonstrated that the expression level of miR-92a was up-regulated 24 h after coronary artery ligation in mice [19]. They also showed that injection of antagomir-92a after permanent coronary artery ligation in mice improved left ventricular function, reduced myocardial infarction size and apoptosis, and increased the number of new blood vessels, especially in the border areas of the infarction. Hinkel and colleagues demonstrated that inhibiting miR-92a protects against myocardial I/R injury in a porcine model [20].
Studies of miRNAs in various models of myocardial I/R injury [21][22][23][24] indicated varying changes of miRNA expression across different species, indicating the complexity of miRNA responses, as well as the complexity of miRNA functions. For example, Hinkel et al demonstrated that inhibition of miR-92a significantly reduced I/R-induced cell apoptosis and necrosis in HL-1 cells [20]. Conversely, Bonauer et al. reported antagomir-92a did not affect cell apoptosis induced by I/R in cultured neonatal ventricular cardiomyocytes from Wistar rats [19]. Zhang et al. demonstrated that both overexpression and down-regulation of miR-92a could have pro-angiogenic effects in human umbilical endothelial cells (HUVEC) [25].
In the current study, cultured H9c2 cardiomyocytes were subjected to 24-h hypoxia followed by 12-h reoxygenation. qRT-PCR analysis revealed increased expression of all miRNAs in the miR-17-92 cluster upon H/R treatment. Increased expression of miR-92a was the most prominent at 2.78-fold.
The present study showed that the inhibition of miR-92a could significantly reduce H/R-induced myocardiocyte injury and apoptosis. Based on bioinformatic analyses, Smad7 was identified as a target of miR-92a. Such a prediction was confirmed by a dual luciferase reporter assay.
Through imperfect sequence-specific binding to the 39-UTR of target mRNAs, miRNAs down-regulate gene expression by degrading target mRNAs [26,27] and/or inhibiting translation [28]. The present study demonstrated that inhibition of miR-92a significantly increased protein levels of SMAD7, but did not affect Smad7 mRNA levels, indicating that miR-92a inhibits the protein translation at the post-transcriptional level, but does not promote Smad7 mRNA degradation.
SMAD7 is an important transcriptional factor that regulates the expression of apoptosis-related genes involved in myocardial I/R injury [29,30]. SMAD7 protects against apoptosis through inhibiting the NF-kB signaling pathway [31,32]. Put together, our findings suggest that apoptosis in myocardial I/R injury, is mediated, at least partly, through the miR-92a/Smad7/NF-kB p65 pathway.
Despite of our findings, whether Smad7 is the most important target of miR-92a in cardiomyocytes (and thus the therapeutic potentials) remains unknown. Other potential candidates included (but not limited to) Pten and MKK4 [33,34] Also, the current study did not provide direct evidence for the interaction between Smad7 and NF-kB p65. Another limitation of the current study is the use of myocardiocytes from a single species (rats), without the presence of endothelial and inflammatory cells. Based on the Hinkel et al. study that demonstrated reduced I/R-induced cell apoptosis and necrosis in HL-1 cells upon miR-92a inhibition in a murine myocyte-like cell line [20], we boldly speculate that our findings may be generalized to other species although such a generalization clearly needs to be verified.
Taken together, the current study indicated that inhibition of miR-92a can attenuate cardiomyocyte apoptosis induced by H/R via the up-regulation of SMAD7 and down-regulation of nuclear NF-kB p65.