| Coronary microembolization(CME)is the most predominant complication in percutaneous coronary intervention(PCI)therapy.The risk of CME in perioperative PCI has been revealed as up to 45% in high-risk patients.The transient "no blood flow" or "slow blood flow" caused by CME is an independent predictor of long-term poor prognosis in patients with acute coronary syndrome.CME may result in myocardial malfunction,including myocardial infarction,arrhythmias,heart attack,and reduction of coronary flow reserve fraction.However,effective preventative and therapeutic approaches have yet to be identified in clinic application.Therefore,it is an urgent task to study the intrinsic pathogenesis and possible treatment of CME.Pyroptosis is an inflammatory-mediated cell death,which is stimulated via downstream activated caspase-1 and ultimately results in cellular rupture and the release of proinflammatory cytokines.Pyroptotic cell death is tightly regulated and has been linked to the incidence of a range of cardiovascular diseases(CVDs).Our previous study found that NLRP3 inflammasome is activated in myocardial tissue undergoing CME,and inhibition of NLRP3 inflammasome alleviates CMEinduced myocardial damage.Therefore,suppressing cardiomyocyte pyroptosis might be a candidate target for CME.However,the molecular mechanisms governing cardiomyocyte pyroptosis in the context of CME are not clearly understood.Long non-coding RNAs(lncRNAs)are a series of RNAs comprising more than200 nucleotides and are not able to encode proteins.Initially,lncRNAs were considered to be “garbage” generated during gene expression,but emerging evidence indicates that lncRNAs contributes to many processes of the cell,including genomic imprinting,evaluation of cell fate,alternative splice of RNA,and chromatin modification.Currently,many lncRNAs have been reported significantly contributing to the pathogenesis of CVDs.In our previous study,the lncRNA taurine-upregulated gene 1(TUG1)was abnormally expressed in CME heart tissue,was found by lncRNA expression microarray screening.Current studies revealed the dynamic expression of lncRNA TUG1 in the progression and diseases of the heart.It is,however,not known whether lncRNA TUG1 may also regulate CME-induced myocardial damage and if so,what mechanisms are involved.In the current study,we determined the role of lncRNA TUG1 in CME-induced myocardial injury and investigated the mechanisms underlying this process.This study might give deeper insight into regulatory mechanisms underlying CME and provide a candidate target for prevention and treatment of CME.PATR Ⅰ Dynamic expression of lncRNA TUG1 in myocardial tissue after coronary microembolization and its relationship with cardiac dysfunction and pyroptosis Objective: To observe the dynamic changes of lncRNA TUG1 in myocardial tissue after CME and its relationship with cardiac dysfunction and myocardial pyroptosis.Methods: Fifty SD rats were randomly divided into Sham group(n=10)and CME group(n=40).The CME group rats were randomly divided into 3h,6h,9h,12 h observation time point groups after CME,with 10 rats in each group.The ascending aorta was clamped and polyester microspheres were injected into the left ventricle to establish a CME model,while the Sham group was injected with an equal volume of normal saline.HE staining was used to observe myocardial pathological changes,and transmission electron microscopy(TEM)was used to observe the ultrastructure of myocardial tissue.Echocardiography was used to detect cardiac function in rats.qRT-PCR was used to detect the relative expression level of lncRNA TUG1,NLRP3 mRNA,IL-1β mRNA,and IL-18 mRNA.The relative expression level of NLRP3,IL-1β,and IL-18 protein was detected by Western blot.Results:1.A rat CME model was established using microspheres,which were visible in the myocardial tissue following injection.The inflammatory cell infiltration,disordered or ruptured myocardial fibers,and interstitial edema were visible surrounding microspheres in myocardium following CME.The analyses of TEM revealed that myocardial sarcomeres were partially ruptured and disordered following CME,with unaligned Z-rays,mitochondrial vacuolation and swelling,and damaged mitochondrial cristae consistent with pyroptotic cell death.2.The CME group revealed a significant decline in LVEF and LVFS,and the functions of the heart got the worst value at 9 h post-CME(all P<0.05).3.Compared with the Sham group,the expression level of lncRNA TUG1 was significantly decreased,while the expression level of NLRP3 mRNA,IL-1βmRNA,and IL-18 mRNA was significantly upregulated in each time point(3h,6h,9h,and 12h)group after CME(all P<0.05).The expression of these mRNAs reached the lowest or peak value at 9 h post-CME.4.Compared with the Sham group,the expression level of NLRP3,IL-1β,and IL-18 protein was significantly up-regulated in each time point(3h,6h,9h,and 12h)group after CME(all P<0.05).The expression of these proteins reached the peak value at 9 h post-CME.5.The expression of lncRNA TUG1 was considerably associated with LVEF,but negatively correlated with the expression of NLRP3,IL-18,and IL-1β mRNA(all P<0.05).Conclusion: The CME rat model was successfully established.The expression level of lncRNA TUG1 in myocardial tissue after CME was significantly downregulated,which may be closely related to CME-caused cardiac dysfunction and myocardial pyroptosis.PART Ⅱ The role of lncRNA TUG1 in coronary microembolization-induced myocardial damage and pyroptosis in rats Objective: To investigate the role of lncRNA TUG1 in CME-induced myocardial damage and NLRP3 inflammasome mediated pyroptosis.Methods: Sixty SD rats were randomly divided into Sham group,CME group,CME+Ad-NC group,CME+Ad-TUG1 group,CME+Ad-sh NC group,CME+Adsh TUG1 group,with 10 rats in each group.The ascending aorta was clamped and polyester microspheres were injected into the left ventricle to establish a CME model,while the Sham group was injected with an equal volume of normal saline.Fluorescence microscope was used to observe the efficiency of r AVV9 transfection.Echocardiography was used to detect and calculate LVEF and LVFS.HBFP staining was used to observe and calculate the micro-infarcted area.Serum cTnI concentration was evaluated via ELISA.qRT-PCR was used to detect the relative expression level of lncRNA TUG1.The relative expression level of pyroptosis-related proteins(NLRP3,ASC,Caspase-1 p20,GSDMD,IL-1β,and IL-18)were detected by Western blot.Results:1.Under the fluorescence microscope,it can be observed that the myocardial tissue successfully transfected with adeno-associated virus emits green fluorescence.Compared with the CME+Ad-NC group,the expression level of lncRNA TUG1 in myocardial tissue of the CME+Ad-TUG1 group was significantly up-regulated(P<0.05);compared with the CME+Ad-sh NC group,the expression level of lncRNA TUG1 in the myocardial tissue of the CME+Adsh TUG1 group Significantly down(P<0.05).2.Compared with the Sham group,LVEF and LVFS in the CME group were significantly decreased(all P<0.05).Overexpression of lncRNA TUG1 significantly increased LVEF and LVFS after CME(all P<0.05),while knockdown of lncRNA TUG1 further reduced LVEF and LVFS after CME(all P<0.05).3.Compared with the Sham group,the micro-infarcted area of myocardium in the CME group was significantly increased(P<0.05).Overexpression of lncRNA TUG1 significantly reduced the micro-infarcted area after CME(P<0.05),while knockdown of lncRNA TUG1 further increased the micro-infarcted area(P<0.05).4.Compared with the Sham group,the serum cTnI concentration in the CME group was significantly increased(P<0.05).Overexpression of lncRNA TUG1 significantly reduced the serum cTnI concentration after CME(P<0.05),while knockdown of lncRNA TUG1 further increased the serum cTnI concentration(P<0.05).5.Compared with the Sham group,the expression levels of myocardial pyroptosis-related proteins in the CME group were significantly increased(P<0.05).Overexpression of lncRNA TUG1 significantly reduced expression levels of myocardial pyroptosis-related proteins after CME(P<0.05),while knockdown of lncRNA TUG1 further increased expression levels of myocardial pyroptosis-related proteins(P<0.05).Conclusion: Overexpression of lncRNA TUG1 alleviates CME-caused myocardial damage and NLRP3 inflammasome mediated pyroptosis,whereas knockdown of lncRNA TUG1 exerts the opposite effects.PART Ⅲ LncRNA TUG1 regulates coronary microembolization-induced myocardial damage and pyroptosis via sponging miR-186-5p Objective: To investigate the mechanism of lncRNA TUG1 regulates CMEinduced myocardial injury and pyroptosis.Methods: The ascending aorta was clamped and polyester microspheres were injected into the left ventricle to establish a rat CME model.LPS stimulated H9C2 cells to establish an in vitro cardiomyocyte inflammatory injury model.LncRNA TUG1 overexpression and shRNA plasmids or AAVs,miR-186-5p agomiR and antagomiR were used for TUG1 and miR-186-5p overexpression and knockdown in rats or H9C2 cells.Fluorescence in situ hybridization(FISH)was used to observe the intracellular localization of lncRNA TUG1.Dual luciferase reporter gene detection,anti-AGO2 RIP,and RNA pull-down were conducted to validate the associations between lncRNA TUG1 and miR-186-5p.Echocardiography was used to detect and calculate LVEF and LVFS.HBFP staining was used to observe and calculate the micro-infarcted area.Serum and cell culture supernatant samples were evaluated via ELISA.The cell viability was evaluated via CCK-8 assay.qRT-PCR was used to detect the relative expression level of lncRNA TUG1 and miR-186-5p.The relative expression level of pyroptosis-related proteins(NLRP3,ASC,Caspase-1 p20,GSDMD,IL-1β,and IL-18)were detected by Western blot.Results:1.RNA FISH results revealed that TUG1 is a lncRNA that is abundant in the cytoplasm.The findings of the dual luciferase reporter gene detection,anti-AGO2 RIP,and RNA pull-down revealed that miR-186-5p was a directly target of lncRNA TUG1.2.Compared with the Sham group,the miR-186-5p level was significantly upregulated in each time point(3h,6h,9h,and 12h)group after CME(all P<0.05),and the expression level of miR-186-5p reached the peak value at 9 h post-CME.The expression of miR-186-5p was negatively correlated with the expression of lncRNA TUG1(P<0.05).3.MiR-186-5p was highly expressed via miR-186-5p agomiR but was silenced by miR-186-5p antagomiR in heart tissue(all P<0.05).Knockdown of miR-186-5p elevated the lncRNA TUG1 level in the myocardium undergoing CME,while miR-186-5p overexpression suppressed lncRNA TUG1(all P<0.05).Overexpression of lncRNA TUG1 potentially suppressed miR-186-5p expression in the myocardium following CME,while knockdown of lncRNA TUG1 elevated miR-186-5p level(all P<0.05).4.MiR-186-5p depression significantly improved LVEF and LVFS following CME(all P<0.05).Knockdown of miR-186-5p reduced micro-infarcted size significantly(P<0.05).Knockdown of miR-186-5p decreased serum cTnI concentration following CME significantly(P<0.05).MiR-186-5p inhibition decreased the expression of myocardial pyroptosis-related proteins significantly(all P<0.05).5.The results of CCK-8 revealed that overexpression of miR-186-5p decreased the elevated H9C2 cell viability caused by lncRNA TUG1 overexpression(P<0.05).Meanwhile,the overexpression of miR-186-5p increased lncRNA TUG1 overexpression reduced LDH,IL-1β,and IL-18 release(all P<0.05).Moreover,the expression level of pyroptosis associated proteins was dramatically down-regulated by overexpression of lncRNA TUG1,which were reversed by overexpression of miR-186-5p(all P<0.05).Conclusion:1.Knockdown of miR-186-5p ameliorates CME-induced myocardial injury and NLRP3 inflammasome mediated pyroptosis.2.Overexpression of lncRNA TUG1 alleviates CME-induced myocardial damage via sponging miR-186-5p.PART Ⅳ MiR-186-5p regulates coronary microembolization-induced myocardial damage and pyroptosis by directly targeting XIAP Objective: This study aims to explore the specific molecular mechanism of miR-186-5p regulating myocardial injury and pyroptosis after CME.Methods: The ascending aorta was clamped and polyester microspheres were injected into the left ventricle to establish a rat CME model.LPS stimulated H9C2 cells to establish an in vitro cardiomyocyte inflammatory injury model.XIAP overexpression and shRNA plasmids or AAVs,miR-186-5p agomiR and antagomiR were used for XIAP and miR-186-5p overexpression and knockdown in rats or H9C2 cells.Dual luciferase reporter gene detection and RNA pull-down were conducted to validate the associations between XIAP mRNA and miR-186-5p.Echocardiography was used to detect and calculate LVEF and LVFS.HBFP staining was used to observe and calculate the micro-infarcted area.Serum and cell culture supernatant samples were evaluated via ELISA.The cell viability was evaluated via CCK-8 assay.qRT-PCR was used to detect the relative expression level of XIAP mRNA and miR-186-5p.The relative expression level of pyroptosis-related proteins(NLRP3,ASC,Caspase-1 p20,GSDMD,IL-1β,and IL-18)were detected by Western blot.Results:1.The findings of the dual luciferase reporter gene assay and RNA pull-down revealed that XIAP mRNA was a directly target of miR-186-5p.2.Compared with the Sham group,the XIAP mRNA and protein level were significantly downregulated in each time point(3h,6h,9h,and 12h)group after CME(all P<0.05),and the expression level of XIAP mRNA and protein reached the lowest value at 9 h post-CME.The expression of XIAP mRNA was positively correlated with miR-186-5p,while negatively correlated with lncRNA TUG1(all P<0.05).3.XIAP was highly expressed via Ad-XIAP but was silenced by Ad-sh XIAP in heart tissue(all P<0.05).Knockdown of miR-186-5p elevated the XIAP mRNA level in the myocardium undergoing CME,while miR-186-5p overexpression suppressed XIAP mRNA(all P<0.05).Overexpression of XIAP mRNA potentially suppressed miR-186-5p expression in the myocardium following CME,while knockdown of XIAP mRNA elevated miR-186-5p level(all P<0.05).4.XIAP overexpression significantly improved LVEF and LVFS following CME(all P<0.05).XIAP overexpression reduced micro-infarcted size significantly(P<0.05).XIAP overexpression decreased serum cTnI concentration following CME significantly(P<0.05).XIAP overexpression decreased the expression of myocardial pyroptosis-related proteins significantly(all P<0.05).5.The results of CCK-8 revealed that knockdown of XIAP decreased the elevated H9C2 cell viability caused by miR-186-5p depression(P<0.05).Meanwhile,the knockdown of XIAP increased miR-186-5p depression reduced LDH,IL-1β,and IL-18 release(all P<0.05).Moreover,the expression level of pyroptosis associated proteins was dramatically down-regulated by inhibition of miR-186-5p,which were reversed by knockdown of XIAP(all P<0.05).Conclusion:1.Overexpression of XIAP ameliorates CME-induced myocardial injury and NLRP3 inflammasome mediated pyroptosis.2.knockdown of miR-186-5p alleviates CME-induced myocardial damage via directly targeting XIAP. |
PDF Full Text Request