| Background: With the development of social economy,cardiovascular events have been increasing year by year.Among them,high lethal,highly disabling acute myocardial infarction(AMI)has become the leading cause of death in humans.In order to fight this myocardial infarction,humans have explored from different directions.For example,in view of the inability of cardiomyocytes to grow,the study of stem cell orientation seeks to form new cardiomyocytes to supplement the cardiomyocytes that die in the infarcted area after myocardial infarction,in order to further restore cardiac function and improve patient prognosis.The other direction is the rescue of myocardium cells in the dying state of the posterior region of the myocardial infarction.It is a popular one to reduce the infarcted area by studying the function of some special non-coding RNA or protein.Long non-coding RNA(Lnc RNA)is a group of transcripts with a length of 200 nt(nucleotides),lacking a specific intact open reading frame and no protein-coding function,in cell cycle,proliferation,Migration and metabolism play an important role.The research on lnc RNA in the past decade has become a research hotspot in various fields.Morrbid(myeloid RNA regulator of Bim-induced death)is a new cell death-related lnc RNA discovered in 2016 and is homologous to human mice.However,no research related to Morrbid has been published in the heart field.In this study,Morrbid may have a certain regulatory effect on cardiomyocyte death.The purpose of this experiment is:(1)The expression of Morrbid in the acute myocardial infarction model of C57 mice was explored by q PCR technique.(2)The expression trend of Morrbid in human and mouse cardiomyocyte hypoxia model was explored by q PCR technique.(3)Regulate the expression of Morrbid at the cellular level and observe its effect on apoptosis.(4)Morrbid expression was regulated in wild-type C57 mice,and its effect on cardiac function after acute myocardial infarction was observed.(5)The effect of Morrbid-KO on cardiac function after acute myocardial infarction in mice knocked out of the Morrbid gene.(6)Search for possible upstream and downstream genes of Morrbid through database screening.(7)The reliability of downstream genes was verified by q PCR and Western blot in the hypoxic model of neonatal rat cardiomyocytes and the acute myocardial infarction model of C57 mice.(8)The reliability of downstream genes was verified by q PCR and Western blot on regulated human and mouse cell samples.(9)The reliability of the downstream gene was verified on the heart of the regulated C57 mouse and the mouse heart knocked out of the Morrbid gene.(10)Verify the reliability of upstream and downstream genes with primary mouse cardiomyocyte apoptosis experiments.Method: The experiment was purchased from the American Type Culture Collection(ATCC)Human Cardiac Myocyte(HCM),purchased from Jackson Lab C57 mice and C57 Neonatal Mouse Cardiomyocyte(NMC),and Morrbid-KO mice purchased from Beijing Biotech Satotu Co.,Ltd.,used q PCR to detect changes in gene expression in samples;Tunel staining was used to observe apoptosis in each group after hypoxia treatment.Morphological changes.The changes of cardiac function in the acute myocardial infarction model of each group were measured by B-ultrasound.The ratio of the heart-slit area to the dangerous area of the heart slice was taken by TTC and Evans blue double staining.At the same time,the same heart tissue was fixed,and after frozen section,the morphological changes of apoptosis of myocardial tissue were observed by Tunel staining.Western blot was used to verify the changes of downstream genes in hypoxia model,myocardial infarction model and post-regulated samples.Result: 1.In the mouse model of acute myocardial infarction,the expression of Morrbid in the myocardial infarction group was significantly higher than that in the Sham group.In the hypoxic model of NMC cells,the expression of Morrbid was lower than that in the control group without hypoxia.(0h)increased;in the hypoxic model of HCM cells,the overall trend of Morrbid was still rising,and the expression of Morrbid in the 48 hours of hypoxia was significantly higher than that of the control group at 0h.2.In the NCM cell apoptosis experiment,the apoptosis rate of Morrbid up-regulation group was significantly lower than that of the control group,and the apoptosis rate of Morrbid down-regulation group was significantly higher than that of the control group,that is,Morrbid played an anti-apoptotic effect in NCM cell apoptosis model.3.In the mouse acute myocardial infarction model,the cardiac function of the Morrbid up-regulation group was significantly better than that of the control group,and the cardiac function of the Morrbid knockout group was also significantly worse than that of the control group;the I/A of the Morrbid up-regulation group was significantly smaller than that of the control group.The I/A of the Morrbid knockout group was also significantly higher than that of the control group;the apoptosis rate of myocardial tissue in the Morrbid up-regulation group was significantly lower than that of the control group,and the myocardial apoptosis rate of the Morrbid knockout group was also significantly higher than that of the control group.4.The Morrbid downstream gene Serpine1 was consistent with the expression of Morrbid in the mouse acute heart model and NMC cell H202 injury model.5.After the intramuscular injection of adenovirus to upregulate Morrbid in mice,the expression of the downstream gene Serpine1 was also up-regulated.After knocking out the Morrbid gene,the expression of the downstream gene Serpine1 was decreased.At the same time,after the Morfbid was regulated by adenovirus in HCM cells,the downstream gene Serpine1 the expressions showed corresponding changes,which were consistent with the experiments in mice.6.In the NCM cell apoptosis assay,after up-regulating Morrbid,down-regulation of Serpine1 with Si RNA partially blocked the anti-apoptotic effect of Morrbid.7.In NCM cells,treatment with PI3 K inhibitor LY294002 can inhibit the increase of Morrbid expression.Conclusion: 1.The long non-coding gene Morrbid plays an anti-apoptotic role in NMC cells.2.The long-chain non-coding gene Morrbid has protective effects on cardiac function in acute myocardial infarction in mice.3.The anti-apoptotic effect of the long-chain non-coding gene Morrbid is partly achieved by regulating its downstream gene Serpine1. |
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