MiR-133a And MiR-760 Participate In The Protective Effects Of H₂S Against Myocardial Ischemia-reperfusion Injury

Acute myocardial infarction（AMI）is the main cause of death and disability worldwide.Thrombolysis and percutaneous coronary intervention（PCI）are the main strategies of reperfusion treatment at present,which are often accompanied by myocardial ischemia-reperfusion injury（MIRI）.MIRI is myocardial damage or dysfunction after blood flow is restored after the blood supply to the heart patient is interrupted,which can cause apoptosis,necrosis and even cardiac arrest.It has been proved that apoptosis and endoplasmic reticulum stress play an indispensable role in the occurrence and development of MIRI.At present,many experimental and clinical studies have confirmed that the heart injury caused by ischemia-reperfusion（I/R）is related to a variety of micro RNAs（miRNAs）,and have verified and reported some mechanisms of action.However,these findings are not enough for clinical application,and detail mechanisms of MIRI still need to be further studied.MiRNAs are small,endogenous,non-coding RNAs subgroup containing18 to 23 nucleotides.In general,miRNAs silence their m RNA targets by binding to complementary recognition sequences of m RNA and inhibiting their expression.In the past few decades,miRNAs have been shown to play a regulatory role in different biological processes,such as myocardial remodeling,tumorigenesis,cell differentiation and apoptosis.In particular,its regulatory role in myocardial ischemia-reperfusion injury has attracted more and more attention.At present,it has been found that hydrogen sulfide（H₂S）has an endogenous protective effect on myocardial ischemia-reperfusion injury,but the specific mechanism is not fully clear.H₂S can also affect the growth and apoptosis of tumor cells by regulating the expression of miRNAs.Therefore,we speculate that some miRNAs may be involved in the protective effect of H₂S on myocardial ischemia-reperfusion injury.In this study,rat and H9C2 cardiomyocytes were used as research objects.RT-PCR,Western blot and flow cytometry were used to study the expression of mir-133a and mir-760 and their effects on myocardial ischemia-reperfusion injury.Part one miR-133a participated in the protective effect of hydrogen cardiomyocytes induced by ischemia-reperfusionObjective:To investigate whether miR-133a is involved in the protective effect of hydrogen sulfide on endoplasmic reticulum stress and apoptosis of H9C2 cardiomyocytes induced by ischemia-reperfusion.Methods:The model of hypoxia/reoxygenation injury of H9C2cardiomyocytes was established.The neonatal cardiomyocytes were prepared to treated with hydrogen sulfide（H₂S）or transfected with miR-133a activator or miR-133a inhibitor,either separately,or in combination.Non-treated cardiomyocytes served as control.The ER stress biomarker GRP78,CHOP and e IF2αexpression level were measured by Western blot.Cell apoptosis was assessed by flow cytometry after staining with the Annexin V-FITC.Proliferation was monitored by Brd U labeling,while cell migration and invasion were determined by Transwell assays,respectively.Results:1. H₂S can reverse the apoptosis and endoplasmic reticulum stress induced by ischemia/reperfusion,especially at 200μmol.2.Ischemia/reperfusion can regulate the expression of miR-133a in cardiomyocytes.While ischemia/reperfusion can increase apoptosis,the expression level of miR-133a is significantly reduced.In contrast,the expression level of miR-133a increased in a dose-dependent manner in the ischemia/reperfusion and H₂S combined treatment groups.3.Overexpression of miR-133a and H₂S treatment inhibits H/R-induced endoplasmic reticulum stress and apoptosis in cardiomyocyte.4.MiR-133a plays an important role in cell migration and alleviates the inhibition of H/R on H9C2 cell proliferation.Summary:1. At the level of isolated cells,H₂S has a clear protective effect on cardiomyocytes.2.miR-133a is involved in the inhibition of cardiomyocyte apoptosis by H₂S.High expression of H₂S and miR-133a reversed H/R-induced ER stress and cardiomyocyte apoptosis.3.miR-133a has protective effect on H/R-induced endoplasmic reticulum stress and cardiomyocyte apoptosis,and can enhance the ability of cell movement.Part two MiR-133a is involved in the protective effect of hydrogen sulfide ischemia-reperfusion in vivoObjective:To investigate whether miR-133a is involved in the protective effect of hydrogen sulfide on endoplasmic reticulum stress and apoptosis induced by ischemia-reperfusion in rat cardiomyocytes.Methods:Establishment of rat myocardial I/R in vivo model.SD rats were given NaHS intraperitoneal injection to rats before suffered from I/R injury.The miR-133a mimic was transferred into the myocardium before I/R injury.The leakage of CK,CK-MB and LDH in serum of rats was detected by automatic chemical method,and the left ventricular pressure was measured,miR-133a expression,GRP78,chop and e IF2αexpression were detected by TUNEL method,RT-PCR method and Western blot method respectively.Results:1. NaHS pretreatment and miR-133a mimic pretreatment can reduce CK,CK-MB,LDH leakage,and improve left ventricular systolic pressure.2. After NaHS pretreatment,miR-133a expression was significantly up-regulated.3.NaHS pretreatment and miR-133a mimic pretreatment inhibit I/R-induced ER stress and cardiomyocyte apoptosis.Summary:1.H₂S and miR-133a mimic can significantly reduce the leakage of CK,CK-MB and LDH,and protect the heart function.2.Both H₂S and miR-133a mimic can reduce I/R-induced apoptosis and endoplasmic reticulum stress.Part three Micro RNA-760-targeted DUSP1 participates in the protectiveObjective:To investigate whether microrna-760 is involved in the protective effect of hydrogen sulfide on myocardial ischemia-reperfusion injury and its possible mechanism.Methods:Establishment of rat myocardial I/R in vivo model.In vivo experiments,twenty rats were randomly divided into two groups（n=10）:Sham group and Ischemia/Reperfusion（I/R）group.In vitro experiments,H9C2 cells were exposed to hypoxia condition for 6h,and then reoxygenated under normal condition to establish hypoxia/reoxygenation（H/R）model.TTC staining was applied to measure myocardial infarct size,and CCK8 was used to investigate cell viability.Target Scan and Dual luciferase reporter assay were employed to confirm the targeting relationship between miR-760 and DUSP1.RT-PCR and western blotting were used to detect the expression of relative proteins.Results:1.This study revealed high expression of miR-760 was observed in MIRI rats and H/R-H9C2 cells,miR-760 may aggravate MIRI.2.MiR-760 low-expression enhanced the inhibitory effect of NaHS on apoptosis of H/R-H9C2 cells as well as the expression of cleaved caspase 3and cleaved PARP.3. Target Scan and dual luciferase reporter assay further confirmed the targeting relationship between dual-specificity protein phosphatase（DUSP1）and miR-760.4. Mir-760 participated in the protection of MIRI by H₂S by reducing the level of DUSP1.5.DUSP1 enhanced the protective effect of NaHS on MIRI.Summary:1.High expression of miR-760 in rats may be associated with MIRI.2.Low expression of miR-760 enhanced the inhibitory effect of H₂S on the apoptosis of H/R-H9C2 cells.3.DUSP1 is a target gene of miR-760.4.miR-760 participates in the protective effect of H₂S on MIRI by decreasing the level of DUSP1.5. miR-760 enhances the protective effect of H₂S aganist MIRI.