| BackgroundAs the changes of lifestyle and the ageing of population, ischemic heart disease has been one of the major reasons for the threat to the life and health of human. Now, in our country, the amount of patients with ischemic heart disease is more than20million, and there are about1million new patients with ischemic heart disease each year. In ischemic heart disease, acute myocardial infarction which is caused by coronary artery occlusion is a very serious disease, and opening of infarction related artery timely to make the return of blood supply as soon as possible is the key to save the dying myocardium. But, it was found that the return of blood supply may product additional myocardial injury, such as myocardial stunning, diminished cardiac function and malignant arrhythmia, which is the myocardial ischemia/reperfusion injury (I/RI). The concept of ischemia/reperfusion injury was put forward in1960by Jennings for the first time. Studies found that myocardial ischemia/reperfusion injury was related to the release of oxygen free radical, overload of intracellular calcium, metabolic disorders of myocardial energy, invasion of neutrophils, injury of vascular endothelial cell and apoptosis and so on. It is a new challenge to study the pathogenetic mechanism of myocardial ischemia/reperfusion injury and to investigate how to reduce or eliminate the ischemia/reperfusion injury.MicroRNA (miRNA) is a class of endogenous non-coding single and small RNA which is composed with21-23bases that negatively regulating the gene expression at post-transcriptional level by degrading target mRNA or translation repression. Studies found that the expression of miRNA was rich in the cardiovascular system, involved in heart development, myocardial remodeling, myocardial hypertrophy, myocardial cell apoptosis, arrhythmia, heart failure and other pathological and physiological processes. In recent years, the function of miRNA in myocardial ischemia/reperfusion injury was paid to more attention. Van Rooij etc. found that injection of miRNA-1, miRNA-21or miRNA-24into mice can reduce myocardial infarction area in myocardial ischemia/reperfusion injury. miRNA-1, as a kind of myocardial specificity miRNA, plays an important role in myocardial ischemia/reperfusion injury. Ren etc. found that the expression of miRNA-320was significantly decreased in myocardial ischemia/reperfusion injury and knockout of miRNA-320increased the expression of HSP20, reducing myocardial cell apoptosis induced by myocardial ischemia/reperfusion.Ren etc. found that the expression of miRNA-7was increased in myocardial ischemia/reperfusion injury with gene chip. In recent years, studies found that miRNA-7was a kind of tumor suppressor which inhibited tumor cell proliferation and promoted tumor cell apoptosis in various tumors, such as lung cancer, tongue squamous cell carcinoma and malignant neuroblastomas. But, the expression change, the role and mechanism of miRNA-7in myocardial ischemia/reperfusion injury are not clear.Recently, more attention was paid to the poly(ADP-ribose) polymerases (PARP), a kind of enzymes related to cellular injury. PARP is a family of protease existing in eukaryotes which plays a part in protein modification and nucleotide polymerization. PARP mainly exists in the nucleus and there are18superfamily, including PARP-1、PARP-2、PARP-3、PARP-4/VPARP、Tankyrase1, Tankyrase2, and so on. PARP is involved in many physiological and pathological processes, such as the repair of DNA damage, gene expression, the stability of chromatin, cellular proliferation, differentiation, apoptosis, necrosis and gene transcription. PARP is activated and participated in DNA repair when DNA is damaged. PARP is cleaved by activated Caspase-3to avoid exceeding consumption of the NAD+and ATP, leading to cell apoptosis when cells are damaged severely. Studies have shown that PARP plays an important role in the development of some diseases, such as myocardial ischemia/reperfusion injury, myocardial infarction, heart failure, cerebrovascular disease, diabetes and cancer and so on.Though the functions of miRNA and PARP in myocardial ischemia/reperfusion injury were studied, the mechanism is not clear. Is the expression of miRNA-7changed in myocardial ischemia/reperfusion injury? Does miRNA-7have an effect on myocardial ischemia/reperfusion injury by regulating PARP? In our studies, we established myocardial cell ischemia/reperfusion injury model and the rat heart ischemia/reperfusion injury model, investigated the role and the mechanism of miRNA-7in myocardial ischemia/reperfusion injury, so that we can provide a new therapeutic target for myocardial ischemia/reperfusion injury.This study includes three parts:Part I In vitro:The change of expression of microRNA-7a/b and the effect of microRNA-7a/b on cell apoptosis in ischemia/reperfusion injuryPart II In vitro:MicroRNA-7a/b reduces myocardial cell apoptosis in ischemia/reperfusion injury by inhibiting PARPPart III In vivo:MicroRNA-7a/b reduces myocardial injury induced by myocardial ischemia/reperfusion in ratsPart I In vitro:The change of expression of microRNA-7a/b and the effect of microRNA-7a/b on cell apoptosis in ischemia/reperfusion injuryObjectiveTo investigate the change of expression of microRNA-7a/b and the effect on myocardial cell apoptosis in myocardial ischemia/reperfusion injury.Methods1. The extraction of primary myocardial cells:obtain the heart of neonatal Wistar rat, shred to lmm3, and then digest with trypsin and collagenase Ⅱ. After differential adhesion, the myocardial cells are put in incubator at37℃.2. Establish the hyoxia-reoxygenation injury model of myocardial cell.The following experimental groups were studied in primary myocardial cells:(1) control group;(2) SI/R group;The following experimental groups were studied in H9C2myocardial cells:(1) control group;(2) control+mir-7a mimic group;(3) control+mir-7b mimic group;(4) control+mir-7a inhibitor group;(5) control+mir-7b inhibitor group;(6) SI/R group;(7) SI/R+mir-7a mimic group;(8) SI/R+mir-7b mimic group;(9) SI/R+mir-7a inhibitor group;(10) SI/R+mir-7b inhibitor group.3. Real-time quantitative PCR for miR-7a and miR-7b:myocardial cells were harvested and miRNAs were extracted by use of mirVanaTM miRNA extraction kit. The miR-7a/b expression level was determined by real-time quantitative PCR using Taqman miRNA reverse transcription kit and TaqMan Universal PCR Master Mix according to the manufactures’ instruction. The primers of miR-7a/b and U6were obtained from Ambion, and U6as an internal reference for miR-7a/b expression.4. Transfection of miR-7a/b mimic and miR-7a/b inhibitor:For miR-7a/b over-expression or inhibition, miR-7a/b mimic or inhibitor was transfected into H9C2myocardial cells with Lipofectamine2000according to the manufactures’ instruction.5. Detection of apoptosis of myocardial cells:Flow cytometry was used.6. Detection of LDH in myocardial cells:LDH detection kit was used.Results1. The expression of miR-7a/b was increased after myocardial ischemia/reperfusion injury:as compared with controls, the expression of miR-7a/b was significantly increased after myocardial ischemia/reperfusion injury. This indicates that miR-7a/b is involved in the myocardial ischemia/reperfusion injury;2. After myocardial ischemia/reperfusion injury, myocardial cell apoptosis rate was increased and the level of LDH was increased:compared with the control group, the cell apoptosis rate was increased and the level of LDH was increased significantly after H9C2myocardial cell was in hypoxia for10hours and reoxygenation for2hours;3. The effects of mir-7a/b on the damage of myocardial cell in myocardial ischemia/reperfusion injury:compared with the SI/R group, the transfection of mir-7a/b mimic significantly reduced the apoptosis rate of myocardial cells and the release of LDH; On the contrary, the transfection of mir-7a/b inhibitor increased the apoptosis rate of myocardial cells and the release of LDH;The results show that mir-7a/b plays an important role in myocardial ischemia/reperfusion injury and overexpression of mir-7a/b can significantly reduce the myocardial cell injury caused by ischemia/reperfusion.Conclusion1. The expression of miR-7a/b was increased significantly in myocardial stimulated ischemia/reperfusion model;2. In myocardial stimulated ischemia/reperfusion, transfection of miR-7a/b mimic reduced the myocardial cell apoptosis rate and the level of LDH, on the contrary, transfection of miR-7a/b inhibitor increased the myocardial cell apoptosis rate and the level of LDH.Part II In vitro:MicroRNA-7a/b reduces myocardial cell apoptosis in ischemia/reperfusion injury by inhibiting PARPObjectiveTo investigate the target gene of mir-7a/b and the regulatory mechanism of mir-7a/b in myocardial ischemia/reperfusion injury.Methods1. Establish the hyoxia-reoxygenation injury model of myocardial cell.The following experimental groups were studied in H9C2myocardial cells.(1) control group;(2) control+mir-7a mimic group;(3) control+mir-7b mimic group;(4) control+mir-7a inhibitor group;(5) control+mir-7b inhibitor group;(6) SI/R group;(7) SI/R+mir-7a mimic group;(8) SI/R+mir-7b mimic group;(9) SI/R+mir-7a inhibitor group;(10) SI/R+mir-7b inhibitor group.2. Transfection of miR-7a/b mimic and miR-7a/b inhibitor:For miR-7a/b over-expression or inhibition, miR-7a/b mimic or inhibitor was transfected into H9C2myocardial cells with Lipofectamine2000according to the manufactures’ instruction. Scramble control miRNA was used as the negative control. miR-7a/b mimic, miR-7a/b inhibitor and scramble control miRNA were purchased from GenePharm.3. Dual-luciferase reporter assay:To confirm that miR-7a/b can directly bind to the3’-untranslated region (3’-UTR) of PARP, the dual-luciferase reporter assay was performed. After transfection of miR-7a/b mimic and GV126-PARP-3’-UTR-WT (wild type)/MU (mutant type) vectors into HEK293cells, firefly and renilla luciferase activities were measured using the dual-luciferase reporter assay system according to the manufactures’ instruction.The following experimental groups were studied in HEK293cells.(1) MiR control+GV126+pRL-TK;(2) Mimic-7a+GV126-3’UTR-WT+pRL-TK;(3) Mimic-7b+GV126-3’ UTR-WT+pRL-TK;(4) Mimic-7a+GV126-3’ UTR-MU+pRL-TK;(5) Mimic-7b+GV126-3’UTR-MU+pRL-TK.4. Investigating the effect of miR-7a/b on the target gene and the role of miR-7a/b in myocardial ischemia/reperfusion injury:After miR-7a/b mimic or miR-7a/b inhibitor was transfected into H9C2cells, the expression of the target gene was measured by western blot analysis. After transfection of mir-7a/b mimic or miR-7a/b inhibitor, we made myocardial ischemia/reperfusion injury model and detected the expression of PARP, Caspase-3, cleaved PARP and Caspase-3using western blot analysis.Results1. miR-7a/b directly targeted PARP in H9C2cells:The result of dual-luciferase reporter assay showed that compared with the control group, transfection with the miR-7a/b mimic strongly inhibited luciferase activity of the GV126-PARP3’-UTR-WT, but not the GV126-PARP3’-UTR-MU. The expression of PARP was down-regulated with miR-7a/b mimic and up-regulated with miR-7a/b inhibitor by use of western blot analysis.These results demonstrate that miR-7a/b can regulate the expression of PARP directly and PARP is the target gene of miR-7a/b in H9C2cells.2. The effect of miR-7a/b on myocardial cell apoptosis in myocardial hypoxia-reoxygenation injury:PARP was reduced and cleaved PARP and Caspase-3were increased during the hypoxia-reoxygenation injury model. Overexpression of miR-7a/b significantly reduced the levels of cleaved PARP and Caspase-3as compared with SI/R group. In contrast, miR-7a/b inhibitor increased the expression of cleaved PARP and Caspase-3as compared with SI/R group.This indicates that miR-7a/b reduces myocardial cell apoptosis in hypoxia-reoxygenation injury.Conclusion1. PARP is the target gene of miR-7a/b in H9c2cells.2. Mir-7a/b inhibits the expression of PARP via interacting with its3’-UTR and reduces the apoptosis of myocardial cells during the myocardial hypoxia-reoxygenation injury model. Part Ⅲ In vivo:MicroRNA-7a/b reduces myocardial injury induced by myocardial ischemia/reperfusion in ratsObjectiveTo investigate the change of microRNA-7a/b and explore the effect of microRNA-7a/b in rat myocardial ischemia/reperfusion injury.Methods1. Establish the rat model of cardiac I/R.The rat heart I/R model was induced with a4-0silk suture ligating left anterior descending artery (LAD) to block blood flow. After30min of ischemia, the knot was relaxed to establish rat heart I/R model.The following experimental groups for the experiment one were studied (n=8):(1) sham group;(2) I/R group.The following experimental groups for the experiment two were studied (n=8):(1) NC group;(2) mir-7a mimic group;(3) miR-7b mimic group;(4) miR-7a inhibitor group;(5) miR-7b inhibitor group.The following experimental groups for the experiment three were studied (n=16):(1) sham group;(2) I/R group;(3) I/R+NC group;(4) I/R+mir-7a mimic group;(5) I/R+miR-7b mimic group;(6) I/R+miR-7a inhibitor group;(7) I/R+miR-7b inhibitor group.2. Transfection of lentivirus:Lentivirus vector with miR-7a/b mimic or miR-7a/b inhibitor was transfected into the rat myocardium by direct intramyocardial injection. Transfection of vacancy lentivirus was used as a negative control (NC).3. Real-time quantitative PCR for miR-7a and miR-7b:The total RNA was extracted with TRIzol. The miR-7a/b expression level was determined by real-time quantitative PCR using Taqman miRNA reverse transcription kit and TaqMan Universal PCR Master Mix according to the manufactures’ instruction. The primers of miR-7a/b and U6were obtained from Ambion, and U6as an internal reference for miR-7a/b expression.4. Real-time quantitative PCR for PARP:The total RNA was extracted with TRIzol. The expression of mRNA was detected by cDNA synthesis kit and PCR kit of TaKaRa according to the manufactures’ instruction.β-actin was a reference for PARP expression.5. Detect the expression of PARP:After injection of lentivirus into the rat myocardium for7days, western blot analysis was performed to evaluate the expression of PARP.6. Determination of myocardial infarct size:Evans blue and triphenyl tetrazolium chloride (TTC) were used to evaluate the infarcted and noninfarcted areas. Infarct size was expressed as the percentage of the area at risk (AAR).7. Detection of apoptosis of myocardial cells:The TUNEL technique was used.8. Detect the level of LDH in blood serum:LDH detection kit was used.Results1. The expression of miR-7a/b was increased after I/R:As compared with controls, the expression of miR-7a/b was significantly increased after I/R. This shows that miR-7a/b is involved in I/R injury;2. Transfection of lentivirus successfully by direct intramyocardial injection: Lentivirus with miR-7a/b mimic or miR-7a/b inhibitor was transfected into the rat myocardium by direct intramyocardial injection to up-regulate or down-regulate the expression of miR-7a/b;3. Mir-7a/b inhibits the protein expression of PARP:After transfection of lentivirus with mir-7a/b mimic or mir-7a/b inhibitor into rat myocardium for7days, the protein expression level of PARP was detected using western blot method. The protein expression of PARP was inhibited by miR-7a/b mimic, but, increased by miR-7a/b inhibitor. These results demonstrate that PARP is the target gene of miR-7a/b;4. Mir-7a/b negatively regulates the target gene in I/R:As compared with I/R+NC, the expression of PARP was down-regulated with miR-7a/b mimic and up-regulated with miR-7a/b inhibitor in I/R. This indicates that PARP is the target gene of miR-7a/b in I/R.5. The effects of mir-7a/b on the damage of myocardial cell in I/R:As compared with I/R+NC, overexpression of miR-7a/b significantly reduced the infarct size, myocardial cell apoptosis rate and the level of LDH; Conversely, inhibition of miR-7a/b increased the infarct size, myocardial cell apoptosis rate and the level of LDH. This demonstrates that miR-7a/b plays an important role in I/R and overexpression of miR-7a/b protects myocardial cells from I/R injury. Conclusion1. In rat I/R model, the expression of miR-7a/b was increased;2. In rat myocardium, PARP is the target gene of miR-7a/b;3. In rat I/R model, mir-7a/b inhibits the expression of PARP and protects myocardial cells. |
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