Identiyfing And Bioinformatics Analysis Of The Specific MicroRNAs Involved In Morphine Preconditioning Protecting Cardiomyocytes From Hypoxia-reoxygenation Injury In Rats With Heart Failure

Background Heart failure is the serious phase of a variety of cardiovascular diseasessuch as coronary atherosclerotic cardiopathy, rheumatic heart disease and so on.Cardiac surgery is a pivotal therapy for these disease. While, perioperativeischemia-reperfusion injury is more sensitive to these patients, and it is very necessaryto find some valid protective measures against the perioperative IRI. In our previousstudy, we found that hypoxic preconditioning impaired its protective effect of cardiomyocytes from hypoxia-reoxygenation injury in failed heart while morphinepreconditioning conserved it. MiRNA participated in the process of hypoxia-reoxygenation injury, ischemic preconditioning, heat failure as well as the biologicalfunction of morphine. While it’s unknown whether miRNA involved in thecardioprotection of morphine preconditioning from hypoxia-reoxygenation injury in ratwith heart failure.Methods Ventricular myocytes were isolated from normal and failed adult rat hearts.The cardiomyocytes models of morphine preconditioning and hypoxia-reoxygenation(H/R) injury were used, respectively, in two series of experiments. In experiment one,the cardiomyocytes were divided into three groups:(1) normal control group (nCON),the cardiomyocytes isolated from normal heart were cultured in normal condition;(2)failed control group (fCON), the cardiomyocytes isolated from failed heart werecultured in normal condition;(3) failed MPC group (fMPC), the cardiomyocytesisolated from failed heart were administered with morphine for10min followed bymorphine free buffer for30min. After that, the cells were collected and the total RNAwas extracted for miRNA microarray to identified the differentially expressed miRNAinduced by MPC. Targetscan software was used to predict the target genes ofdifferentially expressed miRNAs. GO and KEGG pathway analysis were performed tofind the enrichment gene functions and pathways; then the core miRNAs and targetgenes were identified by miRNA-gene-network. In experiment two, the isolatednormal and failed cardiomyocytes were divided into four groups:(1) normal controlgroup (nCON), the normal cardiomyocytes were treated as experiment one;(2) failedcontrol group (fCON), the failed cardiomyocytes were treated as experiment one;(3)failed HR group (fHR), the failed cardiomyocytes were treated in hypoxic conditionfor90min followed by reoxygenation for120min.(4) failed MPC+HR group(fMPC+HR): after pretreated with MPC as before the failed cardiomyocytes were also treated in hypoxic condition for90min followed by reoxygenation for120min. Afterthe all treatment the cells were collected and the total RNA and protein were extractedfor qRT-PCR and western blot analysis to validated the results of miRNA microarrayas well as the putative target genes.Results1. The reults of miRNA microarrary The results of miRNA microarray showed thatsome miRNAs were differetially expressed between each two groups. Comared withnCON,5miRNAs were up-regulated while7miRNAs down-regulated in fCON.Compared to fCON,8miRNAs were up-regulated while10miRNAs down-regulatedin fMPC (p<0.01, signal value>500). Among them, expression level of miR-133b-5p,miR-6216, miR-664-1-5p and let7e-5p were significantly different (fold change>2or<0.5), miR-133b-5p was the most downregulated miRNA in fCON group whencompared to nCON, while fMPC induced it upregulated; miR-6216, let7e-5p wereupregulated in fCON group when compared to nCON, but fMPC induced itdownregulated. For miR-664-1-5p it was no significant difference in fCON comparedto nCON group while downregulated in fMPC compared to fCON group.2. The results of bioinformatic analysis for the target genes of the differentiallyexpressed miRNAs GO and Pathway analysis revealed that the target genes regulatedby differentially expressed miRNAs induced by MPC in failed cardiomyocytes weremainly focused on apoptosis, calcium ion transport and MAPK signaling pathway. BymiRNA-gene-network, we concluded the core miRNAs and target genes, and we foundthat miR-133b-5p may play an important role in the process of MPC protecting thefailed cardiomyocytes from HR injury through inhibiting apoptosis, and Fas might beits potential target genes.3. Validation of miRNA microarrary data by qRT-PCR analysis The expressionpatterns of miR-133b-5p, miR-6216, let7e-5p were detected in the groups of nCON, fCON and fMPC by qRT-PCR, which were agreed with those of the microarray data.4. The expression level of target gene Fas by HR injury as well as MPC+HR treatmentin failed cardiomyocytes By qRT-PCR, the level of Fas mRNA was increased infCON compared with nCON group, decreased in fHR compared with fCON, and stilldecreased in fMPC+HR compared with fHR group. By western blot, the level of Fasprotein was increased in fCON compared with nCON group, unceasingly increased infHR compared with fCON, while decreased in fMPC+HR compared with fHR group.The expression pattern of Fas protein showed a negative correlationship between Fasand miR-133b-5p in each group after HR injury.Conclusions The results of miRNA microarray and qRT-PCR revealed that MPCinduced the changes of miRNA profile in failed cardiomyocytes, among themmiR-133b-5p, miR-6216, let7e-5p were the most differentially expressed miRNAs.The functions of target genes of the differentially expressed miRNAs mainly involvedin apoptosis, calcium homeostasis and MAPK signaling pathway. And miR-133b-5p aswell as Fas might be the core miRNA and core target gene respectively involving in theprocess of MPC on failed cardiomyocytes from HR injury.