| Background:Myocardial infarction caused the ischemic death of cardiomyocytes and inflammatory reaction which lead to high mortality in modern society. Restoration blood flow to the ischemic tissue has been demonstrated to be an effective approach of saving the risk region of heart. However, the reperfusion therapy also causes the death of cardiomyocytes and the generation of reactive oxygen species (ROS) which is called ischemia reperfusion injury (IRI). Recent studies revealed that high-mobility group box-1(HMGB1), an novel inflammatory factor which is passively released from necrotic and apoptosis cells or actively secreted by activated immune cells, played an important role in the pathogenesis of cardiac IRI. MicroRNAs (miRNAs) are a group of small non-coding molecules that negatively regulate gene expression via RNA-induced silencing complexes (RISC), which cause translational repression or mRNA degradation of their target genes. As an individual miRNA has been emerged as a regulator that controls the expression of hundreds of proteins, they have been proved to regulate the expression of key proteins in different signaling pathways involved in physiological processes such as cardiac ischemia reperfusion. However, the relationship between miRNAs and HMGB1is poorly investigated. Recently, we found an potential relationship between miR-451and HMGB1by the Targetscan software. In this study, we attempt to test the hypothesis whether miR-451induced cardioprotection in I/R injury by attenuating HMGB1induced pro-inflammotory effect.We observedMethods:The recombinant plasmids containing pre-miR-451and asmiR-451were constructed, packaged, amplified and purified by HEK293cells. The recombinant viruses were used for efficient transfection. In vitro:Neonatal rat ventricular myocytes were isolated and cultured for96h before transfecting with Ad-GFP/Ad-miR-451/Ad-asmiR-451, then cardiomyocytes were incubated in DMEM F12for3h anoxia followed by2h reoxygenation. The cell survival rate was detected by trypan blue, apoptosis index was assessed by flow cytometric analysis of propidium Iodide (PI) and Annexin V double staining, SOD activity were measured, The expression of HMGB1was detected by Western Blotting, Real-Time PCR and immunofluorescence assay. The expression of cleaved-Caspase3was detected by Western Blotting, and miR-451was detected by Real-Time PCR. The luciferase activity was used for detecting the relationship between HMGB13'-UTR seed region with miR-451. In vivo:Male Sprague-Dawley rats with weight from200to250g were randomly assigned into5groups receiving the following treatments:Group1:Control (n=12):rats were subjected to surgical manipulation without the induction of myocardial ischemia; Group2: Ischemia and reperfusion (I/R)(n=12):rats were injected Phosphate Buffered Saline (PBS)(150?1) at six different sites in the anterior wall of the left ventricle3days before and then subjected to the left anterior descending coronary artery occlusion for30min followed by reperfusion for24h; Group3:I/R+Ad-GFP (n=12):rats were injected Ad-GFP (150?1,1×1011pfu/ml) and subjected to I/R model; Group4:I/R+Ad-miR-541(n=12):rats were injected Ad-miR-451(150?1,1×1011pfu/ml) and subjected to I/R model; Group4:I/R+Ad-asmiR-541(n=12):rats were injected Ad-asmiR-451(150?1,1×1011pfu/ml) and subjected to I/R model. At24h after reperfusion, the infarcted area and the risk area of heart were defined by TTC and Evans'Blue; Transfection efficiency was evaluated by fluorecent microscope; The inflammatory status of heart was evaluated by HE staining; Apoptosis in heart sections were measured by TUNEL staining; The MDA concentration and SOD activity contained in tissue were measured; The concentration of LDH and CK contained in serum were measured; The expression of HMGB1was detected by Western Blotting and Real-Time PCR. The expression of cleaved-Caspase3was detected by Western Blotting, and miR-451was detected by Real-Time PCR.Results:1. Recombiant Ad-miR-451and Ad-asmiR-451were constructed successfully with the titer of3×1011pfu/ml.2. In vitro experiments:Compared with the control group, the cell viability and the SOD activity were significantly decreased, the apoptosis rate of cells was increased in A/R group and A/R+Ad-GFP group, Real-Time PCR and Western Blotting analysis revealed that the mRNA and protein expression level of HMGB1were significantly increased, the expression of cleaved-Caspase3protein was increased, the expression of miR-451was decreased (all P<0.05). However, when compared with A/R group and A/R+Ad-GFP group, Ad-miR-451significantly prevented the loss of cardiomyocyte viability and the SOD activity, Ad-miR-451also prevented the cell apoptosis and suppress the expression level of cleaved-Caspase3; Real-Time PCR and Western Blotting analysis revealed that the protein expression level of HMGB1and cleaved-Caspase3were significantly decreased, the expression of miR-451was increased (all P<0.05). But the mRNA expression level of HMGB1was not changed significantly. When compared with A/R group and A/R+Ad-GFP group, Ad-asmiR-451failed to impact the cell viability, SOD activity, the expression and distribution of HMGB1, the expression level of cleaved-Caspase3and miR-451(all P>0.05).3. In vivo experiments:Compared with the control group, the inflammatory status was more serious, the apoptisis index was higher, the serum concentration of CK and LDH were significantly increased, the MDA concentration was increased and the SOD activity was decreased in heart tissue in I/R group and I/R+Ad-GFP group. Real-Time PCR and Western Blotting analysis revealed that the mRNA and protein expression level of HMGB1were significantly increased, the expression of cleaved-Caspase3protein was increased, the expression of miR-451was decreased in I/R group and I/R+Ad-GFP group (all P<0.05). When compared with I/R group and I/R+Ad-GFP group, Ad-miR-451significantly prevented the inflammatory status of heart tissue. The serum concentration of CK and LDH were significantly decreased, the MDA concentration was decreased and the SOD activity was increased in heart tissue in Ad-miR-451group. Ad-miR-451also prevented the cell apoptosis and suppress the expression level of cleaved-Caspase3; Real-Time PCR and Western Blotting analysis revealed that mRNA expression and the protein expression level of HMGB1were significantly decreased, the expression of miR-451was increased (all P<0.05). When compared with I/R group and I/R+Ad-GFP group, Ad-asmiR-451failed to impact cell apoptosis, the expression and distribution of HMGB1protein(P>0.05). But the serum concentration of CK and LDH, the MDA concentration, the SOD activity in heart tissue were significantly changed, the expression level of cleaved-Caspase3was significantly increased, the expression of miR-451was significantly decreased, the mRNA expression level of HMGB1was significantly increased(P<0.05).Conclusions:1. The recombinant plasmids containing miR-451and asmiR-451were constructed, packaged, amplified by HEK293cells. After purified, the Recombiant Ad-miR-451and Ad-asmiR-451were constructed successfully with the titer of3X1011pfu/ml. 2. we confirmed the binding site and regulated relationship between miR-451and HMGB1. Upregulation of miR-451may protect against the A/R induced oxidant stress injury and apoptosis of cardiac myocytes via its target gene HMGB1.3. miR-451could regulate the expression of HMGB1on both transcriptional and post-transcriptional levels in the rat ischemia/reperfusion models. Upregulation of miR-451may protect against the I/R induced oxidant stress injury and apoptosis of cardiac myocytes via its target gene HMGB1. |
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