Inhibition Of LncRNA Gpr19 Attenuates Myocardial Ischemia/Reperfusion Injury Via The MiR-324-5p/Mtfr1 Axis

Reperfusion therapy after myocardial infarction(MI)can effectively restore the blood supply and nutritional support of ischemic myocardium and save the dying myocardium.However,myocardial ischemia-reperfusion(I/R)injury has become a new threat to reperfusion therapy for MI.In most cases,I/R is beneficial for functional recovery of ischemic organs and repairs damaged structures.But in some cases I/R can increase dysfunction and structural damage in the tissues and organs.The heart is more vulnerable by irreversible I/R injury,which leads to increase myocardial infarction and seizure fatal arrhythmia.These conditions are called myocardial ischemic/reperfusion injury(MIRI).There are many potential mechanisms for MIRI,including intracellular calcium overload,matrix metalloproteinases(MMP),inflammation and neutrophil infiltration,mito-chondrial dysfunction/i ncreased permeability,oxidative stress and cardio-myocyte apoptosis.A large number of studies have confirmed that oxidative stress and apoptosis play an important role in the pathophysiological process of I/R injury.Multitudinous dysregulated Long-chain non-coding RNAs(lnc RNAs)exposed to I/R damage have been identified.In these disordered lnc RNAs,lnc RNA Gpr19 was selected as a potential gene based on its high fold expression difference.We aimed to explore the function role and molecular mechanism of lnc RNA Gpr19 in I/R injury of MI.In this study,the expression levels of mir-324 in serum of patients with acute myocardial infarction and healthy controls were first compared.Then,C57BL/6 mice were performed I/R injury as in vivo models.The neonatal rat ventricular cardiomyocytes(NRCMs)exposed to oxygen glucose deprivation/ recovery(OGD/R)systerm were used as in vitro model.TUNEL assay,RT-PCT,western blot and oxidative stress analysis were conducted in this study to determine apoptosis and oxidativestress levels.Our results indicated that expression level of miR-324 significantly increases in serum of patients with acute myocardial infarction and suppression of lnc RNA Gpr19 attenuates oxidative stress and apoptosis in NRCMs exposed to OGD/R.We also indicated that inhibition of lnc RNA Gpr19 improves cardiac function and reduces apoptosis and myocardial fibrosis scar formation in vivo.We further demonstrated that inhibition of lnc RNA Gpr19 decreases oxidative stress and apoptosis in OGD/R induced NRCMs via regulating miR-324-5p and mitochondrial fission regulator1(Mtfr1).We elucidated the function role and potential molecular mechanism of lnc RNA Gpr19 in I/R injury of MI,provided theoretical basis for the important role of lnc RNA Gpr9 in I/R injury,and provided a new perspective for the next clinical treatment of I/R injury of MI.Our study includes the following parts:Part One Expression of miR-324 in serum of patients with acute myocardial infarctionObjectives:1.To collecte the serum in patients with acute myocardial infarction and healthy controls.2.To observed and compare the expression level of miR-324 in serum.Methods: By collecting the serum of patients with acute myocardial infarction and healthy controls,RNA was extracted for RT-PCR detection.The expression levels of mir-324 in the serum of two groups were observed and compared.Results: The expression level of miR-324 in serum of patients with acute myocardial infarction was significantly higher than healthy controls.Conclusion: The expression level of miR-324 significantly increases in serum of patients with acute myocardial infarction.4Part Two Expression of LncRNA Gpr19 in myocardium after ischem-ia-reperfusion injuryObjectives:1.To build up mice model of I/R injury after acute myocardial infarction and simulate I/R injury by NRCMs exposed to OGD/R.2.To explore the expression of lnc RNA Gpr19 in I/R injury mice of AMI and NRCMs exposed to OGD/R.Methods:The I/R model of 8-10 week-old male C57BL/6 mice was selected and divided into sham operation group and I/R group.RT-PCR was used to detect the expression of lnc RNA Gpr19 in I/R injured mice after AMI.Oxygen glucose deprivation/recovery(OGD/R)system was used to induce injury of neonatal rat ventricular cardiomyocytes(NRCMs)to simulate in vitro I/R injury model.They were randomly divided into four groups : NC group,OGD2 h group,OGD4 h group and OGD6 h group.The expression of lnc RNA Gpr19 in NRCMs exposed to OGD/R was detected by RT-PCR,western-blot and other molecular biological techniques.Results:1.The mouse model of I/R injury after AMI was successfully established,and the I/R injury was simulated by NRCMs exposed to OGD/R.2.RT-PCR analysis showed that expression of lnc RNA Gpr19 was significantly increased in mice with AMI I/R injury compared with the sham group.In the model of myocardial I/R injury simulated by NRCMs exposed to OGD/R,the expression of lnc RNA Gpr19 was significantly enhanced in the OGD2 h group,OGD4 h group and OGD6 h group.Conclusions:1.Expression of lnc RNA Gpr19 is upregulated in I/R injury mice of AMI2.Expression of lnc RNA Gpr19 is upregulated in NRCMs exposed to OGD/R.Part Three Protective effect of inhibition of LncRNA Gpr19 on ischemia reperfusion injury in myocardial infarctionObjectives:1.To explore the effects of lnc RNA Gpr19 on oxidative stress and apoptosis in NRCMs exposed to OGD/R.2.To investigate the effects on myocardial injury in MIRI mice by intervening lnc RNA Gpr19.Methods:8-10 week-old male C57BL/6 mice were selected and adenoassociated virus(AAV)coni or Gpr19 RNAi were injected into caudal vein 14 days before I/R operation.The mice were randomly divided into four groups:Sham group,I/R group,I/R+Ad-coni group and I/R+Gpr19 RNAi group and given corresponding operational treatment.The effects of lnc RNA Gpr19 on myocardial cell viability,apoptosis,oxidative stress and other functions were evaluated by Western blot,TUNEL staining and oxidative stress test.The degree of myocardial fibrosis was evaluated by masson staining,and the myocardial function of mice was evaluated by echocardiography.Results:1.Western blot,TUNEL staining and oxidative stress test results demonstrated that the apoptosis and oxidative stress in NRCMs induced by OGD/R was suppressed by lnc RNA Gpr19 inhibition.2.Masson staining,echocardiography,and LDH detection results confirmed that the MIRI in mice was significantly reduced by lnc RNA Gpr19 inhibition.Conclusions:1.Inhibition of lnc RNA Gpr19 reduces oxidative stress and apoptosis in NRCMs exposed to OGD/R.2.Inhibition of lnc RNA Gpr19 improves myocardial injury after ischemia-reperfusion injury.Part Four The role of miR-324-5p/Mtfr1 axis in the attenuation of myo-cardial infarction ischemia-reperfusion injury via inhibition of LncRNA Gpr19Objectives:1.To explore the role of miR-324-5p in mediating the inhibition oflnc RNA Gpr19 to reduce oxidative stress and apoptosis in NRCMs exposed to OGD/R.2.To observe the direct regulation of miR-324-5p on Mtfr1 at the transcription and translation levels.3.To explore the role of Mtfr1 in mediating the inhibition of lnc RNA Gpr19 to reduce oxidative stress and apoptosis in NRCMs exposed to OGD/R.Methods:The target miRNA of lnc RNA Gpr19 and the target of miRNA were analyzed by bioinformatics.By overexpression or inhibition of lnc RNA Gpr19,the Gpr19/target miRNA/target protein signaling pathway was verified by using dualluciferase reporter genes.By oxidative stress test,western blot and TUNEL,the regulatory effect of Gpr19/target miRNA/target protein signaling pathway on oxidative stress and apoptosis in NRCMs exposed to OGD/R was detected.Results:1.LncRNA Gpr19 regulates oxidative stress and apoptosis in NRCMs exposed to OGD/R by regulating miR-324-5p.We performed dualluciferase reporter gene assay to further experimentally demonstrate that miR-324-5p was a target of lnc RNA Gpr19.We further confirmed by RT-PCR that there is a negative regulatory relationship between lnc RNA Gpr19 and miR-324-5p.The results of oxidative stress assay showed that the oxidative stress level induced by OGD/R inhibited by lnc RNA Gpr19 inhibition could be partially blocked by miR-324-5p inhibitor.Western blot and TUNEL analysis results showed that inhibition of lnc RNA Gpr19 inhibited the apoptotic level of NRCMs induced by OGD/R was partially reversed by miR-324-5p inhibitor.2.Mtfr1 is a direct target of miR-324-5p.The predicted binding site of miR-324-5p on the 3?-UTR of Mtfr1 m RNA by bioinformatics analysis.The results of dualluciferase assay indicated that miR-324-5p,binds directly to putative Mtfr1 3?-UTR regions as predicted.We further confirmed that miR-324-5p can negatively regulate the expression of Mtfr1 at transcriptional and translation levels.3.LncRNA Gpr19 mediates oxidative stress and apoptosis of NRCMs induced by OGD/R by regulating Mtfr1.Western blot,TUNEL and oxidative stress analysis were performed in this study.Western blot and TUNEL assay results demonstrated that the apoptosis and oxidative stress in NRCMs induced by OGD/R was suppressed by lnc RNA Gpr19 inhibition,whereas was partially blocked after Mtfr1 overexpression.Suppression of lnc RNA Gpr19 reduced oxidative stress elevated by OGD/R was significantly reversed by overexpression of Mtfr1.Conclusions:1.Mi R-324-5p inhibitor can partially reverse the inhibition of lnc RNA Gpr19 in oxidative stress and apoptosis in NRCMs exposed to OGD/R.2.Mi R-324-5p inhibits the expression of Mtfr1 at the transcription and translation levels.3.Overexpression of Mtfr1 can partially block the inhibition of lnc RNA Gpr19 in oxidative stress and apoptosis of NRCMs induced by OGD/R.