| Background and Objection:Acute myocardial infarction(AMI)with subsequent left ventricular dysfunction and heart failure is a major cause of morbidity and mortality in the Western world[1].Clinical interventions such as angioplasty or thrombolytic agents have been effective in disrupting the occlusion and re-establishing the coronary flow.However,these treatments to reduce the injuries during the ischaemic period or reperfusion are not available.A large number of studies have confirmed that mitochondrial enzyme aldehyde dehydrogenase 2(aldh2)is a crucial enzyme involved in protecting the heart from ischemic by mediating both the detoxification of reactive aldehydes such as acetaldehyde and 4-hydroxy-2-nonenal(4-HNE)and the bioactivation of nitroglycerin(GTN)[2,3].Over-expression of ALDH2 wild-type enzymes can confer multiple beneficial effects to the heart tissue and cardiac functions.Aldh2 plays a role in protecting the heart from aldehyde toxicity and against myocardial damages caused by acute ethanol toxicity[4,5].Moreover,aldh2 knockouted transgenic mice lacked any detectable aldh2 enzyme activity and thus accumulated a high level of acetaldehyde after ethanol gavages[6,7].So it was significantly more sensitive to alcohol and acetaldehyde-induced toxicity and damage than wild type mice[6,7].Previous experimental and clinical investigations have uncovered that ethanol consumption and acetaldehyde inhalation are higher risk to ALDH2-inactive humans than those with functional aldh2.Aldh2-deficient humans who habitually consume alcohol have a high cancer risk[8-10].However,the actual regulatory mechanism of aldh2 wild-type gene is not clear.MicroRNAs(miRNAs)inhibit translation or induce mRNA degradation in general by binding to the complementary sequences in 3’-untranslated regions(3’-UTR)of target mRNAs[11].Whether miRNAs are involved in this aldh2 gene regulation is unclear.Many of the protein-coding genes regulated by miRNAs are presently not defined.And bioinformatic approaches may help to recognize them.A search for miRNAs regulatory targets by bioinformatic tools led to the identification of aldh2 as a possible target of mmu-miR-28(miR-28).But mir-28 regulation aldh2 expression has not yet been reported in mus musculus.This study was to investigate the role of miR-28 in the regulation of aldh2 and to explore the mechanism of miR-28 promotes cardiac ischemia in mus musculus.Materials and methodsCell lines and reagentsMouse cardiac myocytes was established from the heart of mice 1-4 days after birth,human embryonic kidney cells-derived cell lines 293T(American Type Culture Collection(ATCC)number:CRL-11268),were grown in Dulbecco’s modified Eagle’s medium with 10%fetal bovine serum(Gibco)and penicillin/streptomycin(Sigma).All cells were cultured at 370C in a humidified chamber supplemented with 5%C02.Cells were grown under hypoxic(1%O2)or normoxic(21%O2)conditions at 37℃/5%CO2.Mmu-miR-28,anti-mmu-miR-28,and the negative control were purchased from Ambion(Applied Biosystems,Foster City,CA,USA).Anti-aldh2 antibody was purchased from Santa Cruz Biotechnology(Santa Cruz,CA,USA).Preparation of buyanghuanwu(BYHW)decoction Shenghuangqi,dangguiwei,chishao,dilong,chuanxiong,honghua,taoren were prepared in accordance with the proportion 10:8:6:5:5:5:5.MiRNA target predictionThe analysis of potential mmu-miR-28 targets was performed by the algorithms TargetScan 5.1(http://targetscan.org/),PicTar(http://pictar.mdc-berlin.de/),and miRanda(http://microRNA.org).To identify the genes,we predicted by the 3 different common algorithms.Results of the forecast targets were intersected by miRGen(http://www.diana.pcbi.upenn.edu/miRGen/v3/miRGen.html)website tools[12].Vector constructionMus musculus mitochondrial aldehyde dehydrogenase 2(Aldh2)3’UTR target site was amplified by PCR using the primers 5’-TCTAGAGCTTTTTCTTAATTTCATT-3’(forward)and 5’-TCTAGATTTTAAAATGATGCTACTGC-3’(reverse).DNA products were then cloned into pUC18(TaKaRa).After verification by DNA sequencing,the Aldh23’TUTR was digested by Xba I and Xba I,then cloned into the pGL3 luciferase vector(Promega)which was with the same restriction enzyme.This construct,named pGL3-Aldh2,was used to transfect for 293T cell lines.To make mmu-miR-28 binding site delete,we used the two-step PCR approach where the seed sequences were deleted in the primers used for PCR reactions.Transfection with mmu-miR-28The day before transfection,293T cells were seeded in antibiotic-free medium.Transfection was carried out using 100 nM of mmu-miR-28,anti-mmu-miR-28 and negative control 1 precursor miRNAs using Lipofectamine 2000 in accordance with the manufacturer’s procedure(Invitrogen).The level of mmu-miR-28 expression in transfected 293T cell line was assessed by real-time RT-PCR(Taqman MicroRNA Assays)24 h after transfection as described above.Cell apoptosis detectioncells were seeded in 6-well plates at a concentration of 2×105 cells/ml.After culturing for 48 h,cells were collected by trypsinization and washed with ice-cold PBS twice.Then they were resuspended in 100 μ1 of 1 ×Annexin-binding buffer,5 μ1 of Annexin V/FITC and 20 μ1 of PI solution(20μg/ml).After incubation,the cells were left at room temperature in the dark for 15 min.Fluorescence was measured with a flow cytometer(Becton,Dickinson and Co.,San Jose,CA)and the data were analyzed with Cell Quest software.All samples were assayed in triplicate,and the apoptotic fraction was calculated as follows:apoptotic cell number/total cell number×100.Luciferase activity assay293T cells were transfected with 0.1 μg of pGL3-Aldh2 along with 0.01 μg of pRL-TK vector(Promega)containing Renilla luciferase and 30 pmol of mmu-miR-28 or negative control 1 or anti-mmu-miR-28 oligonucleotides,and split into 12-well plates in duplicates.After 24 h,the cells were lysed.And luciferase activity was measured by the assay kit from Promega.pGL3 was cotransfected and used for normalization.Each transfection was repeated twice in triplicate.Real-time RT-PCR analysis of miRNA and mRNATotal RNA was isolated by Trizol reagent(Invitrogen).The detection of expression levels of mature mmu-miR-28 through the stem-loop method was performed by the TaqMan MicroRNA Assays(Applied Biosystems,Foster City,CA,USA),as described[13].Relative expression levels of mmu-miR-28 were calculated by the ΔCT method and normalized with internal control U6 RNA(Applied Biosystems).To detect relative levels of Aldh2 mRNA,Taqman real-time qRT-PCR(Invitrogen)was performed by Cyber Green method.For normalization,we used β-actin.PCR primers were Aldh2 sense,5’-CCCCTCACCCAACGAAAAATTAC-3’,and Aldh2 antisense,primer 5’-GATCCTGAATCTGGGGCATGG-3’.All qRT-PCRs were performed in triplicate,and the data are presented as means ± standard errors of the means(SEM).Western blotMouse cardiac myocytes and 293T cells were transfected with 100 pmol of mmu-miR-28 or negative control 1 in 6-well plates.After transfection,cells were cultured at 37℃C.And intermediate samples at 24 and 48 h were collected and analyzed by Western blot to assess Aldh2 expression.The cells were incubated overnight with polyclonal antibody against anti-Aldh2 alpha(BD Biosciences)and anti-β-actin(Santa Cruz Biotechnology,Santa Cruz,CA,USA)at 4℃.And they were incubated with secondary antibodies conjugated to horseradish peroxidase(HRP).The signals were detected by enhanced chemiluminescence(ECL;Pierce,Rockford,IL,USA).Digital images of auto-radiographs were acquired by Fluor-S MultiImager,and band signals were quantified using a Quantity-one build-in specific densitometric algorithm(Quantity-one;Bio-Rad).Statistical analysisStatistical analysis was performed using the statistical package for social sciences(SPSS)17.0 software.Values were expressed as the mean ± SEM.Differences/correlations between groups were calculated by Student’s t test.P<0.05 was considered to be statistically significant.ResultsmiR-28 promotes cardiac ischemiaTo investigate whether miR-28 promote myocardial ischemia in musculus,the effect of acceleration of miR-28 on myocardial damage was examined in vitro.We found that the miR-28 expression of cells increased,while aldh2 expression reduced in a time-dependent manner under normoxic conditions(Fig.1A-1C).To further examine the effect of miR-28 inhibition on cell apoptosis,the Annexin V-FITC stainingassay was performed.The miR-28-transfected cells showed a significant increase in the cellular apoptosis.Compared with the negative control 1 precursor molecules,miR-28 silencing by anti-miR-28 attenuated about 41%myocardial apoptosis under hypoxic conditions(p=0.017,t-test).And miR-28 over-expression caused a 55%increasing in apoptosis(Fig.1D).In comparison with negative control 1 precursor miRNAs,transfection of miR-28 into cardiomyocytes caused a 54%decrease of ALDH2 protein levels(Fig.1E).This finding suggests that ALDH2 is downregulated by miR-28 in cardiac cells.Aldh2 is a target of miR-28The role of miR-28 in promoting myocardial ischemia was unclear.To identify the potential role of miR-28 in myocardial cells,we identified putative target genes by a bioinformatic analysis:microRNA,TargetScan,and PicTar algorithms.At least 2 of the algorithms showed that Aldh2 was a potential mmu-miR-28 target gene(Fig.2A,2B).Mouse myocardial cells were used to determine whether miR-28 down-regulated aldh2 expression.In comparison with negative control 1 precursor miRNAs,transfection of miR-28 into myocardial cells caused a 53%decrease in aldh2 protein levels.On the contrary,anti-miR-28-transfected cells exhibited a 1.8-fold increase in aldh2 protein levels,compared with negative control 1 miRNA inhibitors.(Fig.2C)This finding suggests that ALDH2 is down-regulated by miR-28 in mouse myocardial cells.MiR-28 interacts with 3’UTR of ALDH2 mRNATo determine whether the 3’untranslated region of ALDH2 mRNA was a functional target of miR-28,we cloned a 106-bp aldh2 3’-UTR,which includes a potential target site for miR-28,into the downstream of the luciferase reporter gene to generate the pGL3-aldh2 vector.This vector was cotransfected into 293T cells along with miR-28 or negative control 1.A Renilla luciferase vector(pRL-TK)was used to normalize the differences in the transfection efficiency.As is shown in Figure 3,miR-28 suppressed the luciferase activity in 293T cells by 60%compared with the negative control(P<0.001,t-test).So,immunoassay and luciferase results provided strong indications that aldh2 is a target of miR-28 in mouse myocardial cells.BYHW decoction pretect cardiac cellsBYHW decoction caused a significantly increase in aldh2 mRNA and protein levels in myocardial cells.Compared with the negative control,BYHW decoction decreased about 41%myocardial apoptosis under hypoxic conditions.DiscussionCardiac ischemia is a major cause of cardiac diseases-related death.Hence,identifying the role of miRNAs in promoting cardiac ischemia has direct clinical implications.Although it is now known that miRNAs might have a key role in diverse cellular processes including differentiation,proliferation and apoptosis[14],the underlying mechanism in cardiac ischemia is largely unknown.We show here that the over-expression of mus-miR-28 promotes myocardial ischemic damage in vitro,and identify ALDH2 as a direct functional target for miR-28 through interacting with their 3’UTR.More importantly,aldh2 is expressed in mitochondrial but barely detectable in the blood.It is known to us,miRNAs expression can very easily and quickly detected in tissues and serum[15,16].Therefore,we hypothesis that miRNAs seems to be as a probable index in the identification of cardiac ischemia after acute myocardial infarction.Similar to our results,Croce CM[17]found the potential for miRNA biology to clarify both the molecular pathogenesis of cancer and the inherent complexities in translating its biology to clinics.We are recognizing the potential for miRNA biology to clarify both the molecular pathogenesis of cancer and the inherent complexities.An increasing evidence has indeed proved the importance of miRNAs in cancer,suggesting their possible use as diagnostic biomarkers and miRNA-based anticancer therapies[18].The mitochondrial Aldh2 is one of 19 members of the ALDH gene family,which plays a crucial role in the oxidation and detoxification of reactive aldehydes involved in protecting the heart from ischemic injury[4,19].Aldh2 is a key metabolic enzyme in catalyzing the oxidation of acetaldehyde to acetic acid in ethanol metabolism and detoxification of other reactive aldehydes such as 4-hydroxy-2-nonenal(4-HNE).In addition to its dehydrogenase activity,ALDH2 has an esterase activity that catalyses the conversion of nitroglycerin(glyceryl trinitrate,GTN)to 1,2 glyceryl dinitrate(1,2-GDN),and thus mediates bioactivation of GTN.Activation of ALDH2 could significantly reduce ischemia/reperfusion damage by attenuating elevation in cardiac aldehydes,creatine kinase isoenzyme MB leakage and protein carbonyl formation.In addition,ALDH2 also alleviated ischemia/reperfusion-induced myocardial contractile function impairment by improved maximal velocity of pressure development and decline,left ventricular developed pressure,and heart rate[20].On the contrary,a functional ALDH2 decrease causes constitutive myocardial apoptosis and injury[21].Therefore,ALDH2 down-regulation by miR-28 may contribute to ischemia/reperfusion(I/R)injury.To gain further insight into whether miR-28 promotes cardiac ischemia,we transferred miR-28 precursor or anti-miR-28 inhibitor into musculus myocardial cells.The data in this study shows that the miR-28 expression increased,while aldh2 expression reduced in a time-dependent manner under normoxic conditions.The miR-28-transfected myocardial cells showed a significant increase in the cellular apoptosis.Compared with the negative control 1 precursor molecules,miR-28 over-expression caused a 55%increasing in apoptosis under hypoxic condition.On the contrary,the effect of cardiac ischemia protection was almost abolished by anti-miR-28.The myocardial protective effect of ALDH2 against ischaemia/reperfusion injury possibly through detoxification of toxic aldehyde and the differential regulation of autophagy through AMPK-and Akt-mTOR signalling during ischaemia and reperfusion were previously reported in knockout mice[22].In this article,we describe miR-28 as a direct regulator of ALDH2 expression in myocardial cells,and show a new mechanism responsible for ALDH2 regulation.Our data suggest that the myocardial damaging effect of miR-28 is involved in miR-28-ALDH2 signal during ischaemia.BYHW decoction is representative of traditional Chinese medicine Yiqihuoxue therapy.We found that BYHW decoction could decrease ALDH2 expression,prevent myocardial ischemia.However,the mechanisms of BYHW decoction for the cardial protection remains to be further explored.In the future,we will determine whether BYHW decoction contribute to the inhibition of ALDH2 via the miR-28 pathway,and consequently prevent cardiac ischemia.Conclusion:In summary,our results suggest that miR-28 promotes myocardial ischemia through the inhibition of ALDH2 expression in mus.BYHW decoction may be effective to protect cardiac ischemia.Our results also indicate that miRNAs is as a probable index in the identification of myocardial ischemia after acute myocardial infarction.Innovations of this study:1.We confirmed that ALDH2 is a target of miR-28 in mus.2.We found that miR-28 promotes myocardial ischemia through the inhibition of ALDH2 expression.3.BYHW decoction may be effective to protect cardiac ischemia. |
