Roles And Mechanism Of LncRNA NONMMUT040834 In Cardiomyocyte Necrosis And Cardiac Ischemia/Reperfusion Injury

ObjectiveAcute myocardial infarction is caused by acute coronary artery occlusion and is characterized by high morbidity,mortality and disability rates worldwide.Acute myocardial infarction can cause a large number of myocardial cells to die,resulting in irreversible heart damage,and cell necrosis is one of the main forms of cell death in myocardial infarction.The regulation of cell necrosis has only been revealed in recent years,and its regulatory mechanism has not been fully revealed.The mechanism of programmed necrosis of myocardial cells in acute myocardial infarction has been extensively reported,and inhibition of myocardial cell necrosis in myocardial infarction has become a new myocardial protective strategy.Long non-coding RNA（lncRNA）is a class of non-coding RNA with a length greater than 200 nt.It is widely involved in various pathophysiological processes in cells and plays an important role in cancer,neurological diseases,immune diseases,cardiovascular diseases and other diseases.Its regulatory role in myocardial infarction has also been reported.By constructing myocardial necrosis models,this study screens new lncRNAs that play regulatory roles in myocardial necrosis,and explores the specific functions and molecular mechanisms of lncRNAs in ischemia/reperfusion（I/R）,so as to enrich the theories on how lncRNAs regulate cell necrosis.It provides a new idea for the prevention and treatment of myocardial I/R injury.Experimental methods1.The optimal concentration and time of H₂O₂-induced necrosis of mouse cardiomyocytes HL-1 were screened by propyl iodide（PI）staining.RNA and lncRNA were collected and sequenced,and lncRNA differentially expressed was screened by sequencing data and quantitative Real-time PCR（qRT-PCR）.2.The mouse myocardial infarction model was constructed by ligation of the left anterior descending branch of coronary artery.The expression of lncRNA,necrosis marker genes and related genes at the transcription level after infarction was detected by qRT-PCR,and the expression of related genes at the protein level was detected by Western blot（WB）.3.LncRNA knockdown adenovirus vector was constructed,and the role of lncRNA in cell necrosis was verified by virus infection cell experiment.The detection contents included qRT-PCR to detect the effect of adenovirus knockdown,PI staining to detect the H₂O₂induced myocardial cell necrosis after lncRNA knockdown,and WB to detect the expression of lncRNA necrosis markers after lncRNA knockdown.4.LncRNA overexpression adenovirus vector was constructed,and the effect of adenovirus overexpression was detected by qRT-PCR.The H₂O₂induced myocardial cell necrosis after lncRNA overexpression was detected by PI staining,and the expression of cell necrosis markers after lncRNA overexpression was detected by WB.5.The role of lncRNA in myocardial infarction was verified by constructing a mouse I/R model after injecting adenovirus with lncRNA knockdown into the tail vein of mice.Gene changes of mice injected with lncRNA knockdown adenovirus were detected by qRT-PCR,protein expression of mice injected with lncRNA knockdown adenovirus was detected by WB,myocardial infarction size of mice was observed by Evans Blue/TTC double staining,and cardiac function injury of mice was measured by M-type echocardiography.6.After lncRNA overexpressed adenovirus was injected into the tail vein of mice,gene changes of lncRNA overexpressed adenovirus were detected by qRT-PCR,protein expression of lncRNA overexpressed adenovirus was detected by WB,myocardial infarction size of mice was observed by Evans Blue/TTC double staining.Cardiac function injury was measured by M-mode echocardiography in mice.7.catRAPID website predicted the downstream target protein of lncRNA,and ZMYND8 was initially selected as the downstream target protein of lncRNA through analysis and WB verification.The interaction between lncRNA and the downstream target protein was further identified by RIP experiment.8.qRT-PCR was used to detect the transfection efficiency of transfected si RNA.WB was used to detect the protein expressions of ZMYND8,RIPK3 and p-RIPK3 in cells after transfection of si RNA.PI staining was used to observe the H₂O₂induced cell necrosis after target protein knockdown.Experimental results1.The optimal treatment time and concentration of H₂O₂induced myocardial cell necrosis were 6 h and 600μM.WB verified that RIPK3 and p-RIPK3 were significantly up-regulated in cells induced by 6 h and 600 μM H₂O₂.2.LncRNAs differentially expressed in cardiomyocyte necrosis were screened and identified by qRT-PCR,and the expression of lncRNA NONMMUT044(hereinafter referred to as lnc4 was up-regulated in cardiomyocyte necrosis.3.Lnc4 overexpression adenovirus was constructed,and PI staining showed that H₂O₂-induced cell necrosis significantly increased after lnc4 overexpression.The results of Evans Blue/TTC double staining and echocardiography in mice showed that lnc4 overexpression could aggravate myocardial infarction size caused by I/R and aggravate cardiac function injury.4.Lnc4 knockdown adenovirus was constructed,and PI staining showed that H₂O₂-induced HL-1 cell necrosis significantly decreased after lnc4 knockdown.The results of Evans Blue/TTC double staining and echocardiography in mice showed that lnc4 knockdown adenovirus injected into caudal vein reduced myocardial infarction size and cardiac function injury after I/R in mice.5.catRAPID website predicted the downstream protein of lnc4 and identified the up-regulated expression of ZMYND8 in myocardial cell necrosis through qRT-PCR screening.WB confirmed that the expression of ZMYND8 at the protein level was positively correlated with lnc4,and its expression level was up-regulated with lnc4overexpression.It was also found that ZMYND8 was increased at the protein level in H₂O₂-induced myocyte necrosis.The RIP further confirmed the interaction between ZMYND8 and lnc4.6.After HL-1 transfected ZMYND8-si RNA,the expression level of ZMYND8 in the cells was decreased by WB detection.Further results showed that ZMYND8knockdown could reduce the expression of RIPK3 and p-RIPK3 protein in H₂O₂-treated cells.The results of PI staining showed that ZMYND8 knockdown could reduce the H₂O₂-induced cell necrosis rate and alleviate the degree of myocardial cell necrosis.ConclusionThrough the construction of myocardial cell necrosis model,it was found that lnc4 expression was significantly up-regulated in myocardial cell necrosis.lnc4overexpression can aggravate the H₂O₂-induced cardiomyocyte necrosis and aggravate the functional injury of myocardial infarction center.lnc4 knockdown can inhibit H₂O₂-induced cardiomyocyte necrosis and reduce the degree of functional injury of myocardial infarction center.The expression of ZMYND8 was up-regulated in both H₂O₂-induced cardiomyocyte necrosis and myocardial infarction,and its expression level was positively correlated with lnc4.Moreover,ZMYND8knockdown could alleviate H₂O₂-induced cell necrosis.RIP experiments showed that ZMYND8 and lnc4 could interact,and overexpression of lnc4 could promote the expression of ZMYND8,and knocking down lnc4 could inhibit the expression of ZMYND8.ZMYND8 knockdown inhibited increased cardiomyocyte necrosis caused by lnc4 overexpression.These results suggest that ZMYND8 is a downstream target of lnc4,which is positively regulated by lnc4 and co-regulates the process of cardiomyocyte necrosis.Future studies still need to explore the specific regulatory mechanisms of lnc4 and ZMYND8 in myocardial cell necrosis,so as to provide new ideas and theoretical basis for clinical prevention and treatment of I/R.