| Background:With the continuous improvement of residents’ living standards,people’s pursuit of health is increasing.However,the incidence rate of cardiovascular disease(CVD)is still rising,which has become one of the main causes of national death in China,causing serious public health and socio-economic losses.Therefore,the study of cardiovascular disease has always been a hot spot in medical research,and the more serious cardiovascular disease is the sudden death caused by myocardial infarction.The related identification is also the focus and hot issue of forensic research.According to statistics,in recent years,the incidence rate and mortality of myocardial infarction in China are generally on the rise.Therefore,the study found that the related markers of myocardial infarction are a problem that needs to be solved by the medical research community,and can also provide basis and convenience for forensic identification.Myocardial infarction is the local myocardial ischemic necrosis caused by the interruption of myocardial oxygen supply or imbalance of demand due to the formation of atherosclerotic thrombus in coronary artery.Myocardial cell apoptosis caused by ischemia and hypoxia during myocardial infarction is an important cause of myocardial cell loss.Long noncoding RNA(lncRNA)is an RNA molecule with a length of more than 200 nt,without the function of coding protein,with a 5’cap,stem ring structure,and 3’poly(A)tail,but without an open reading frame.LncRNA is widely distributed and can play a regulatory role at the level of transcription,post-transcription and translation.Myocardial infarction related transcript(Miat)is a non-coding RNA that was first identified as lncRNA in 2006,located on chromosome 22.It is named because of its multiple single nucleotide polymorphisms,which are closely related to the susceptibility to myocardial infarction,and has the potential to become a candidate biomarker of diagnostic and therapeutic significance.MicroRNAs(miRNAs)are a class of single-stranded non-coding RNA molecules encoded by endogenous genes with a length of about 22 nt,which can play a role by assembling with the 3’-UTR region of downstream mRNA,thus inducing mRNA degradation/inhibiting translation.MiR-182 is an evolutionarily conserved microRNA molecule located on human chromosome 7.Studies have shown that miR-182 plays a regulatory role in breast cancer,liver cancer,kidney injury,pulmonary hypertension and other pathological processes,but its regulatory role in the process of myocardial infarction is still unclear.Nogo protein family is a member of the reticular protein family(Rtn),which is encoded by Rtn4 gene and is located in the transmembrane protein of the endoplasmic reticulum of eukaryotic cells,including three subtypes of Nogo-A,Nogo-B and Nogo-C,of which Nogo-A is the largest protein among the members of the reticular protein family.The function of Nogo-A plays an important role in the process of central nervous system diseases,and is also closely related to the damage caused by cerebral ischemia and hypoxia.Its role and regulatory mechanism in the process of cardiomyocyte apoptosis induced by hypoxia have not been clarified.Objective:1.To explore the expression changes of lncRNA Miat,miR-182 and Nogo-A in hypoxic cardiomyocytes.2.Explore the regulatory relationship between lncRNA Miat and miR-182 and the related mechanism with cell apoptosis.3.Explore the regulatory relationship between miR-182 and Nogo-A and the related mechanism with cell apoptosis.4.Explore the regulatory relationship among lncRNA Miat,miR-182 and Nogo-A.5.To explore the regulatory mechanism of lncRNA Miat/miR-182/nogo-A axis in the process of cardiomyocyte apoptosis due to hypoxia.Methods:1.Use H2O2treatment to simulate hypoxic injury of myocardial cells.First,H9c2 cells are treated with different H2O2 concentration gradients,and the CCK-8 experiment detects the cell viability to determine the best simulated conditions for subsequent tests.2.The expression of Miat,miR-182 and Nogo-A mRNA in H9c2 cells after H2O2 treatment was detected by qRT-PCR;Western blotting was used to detect the expression of pro-apoptotic protein Bax,Caspase-3,anti-apoptotic protein Bcl-2 and Nogo-A after H2O2 treatment;Hoechst apoptosis staining was used to detect the apoptosis rate.3.The biological prediction software RNAhybrid predicts that there may be a targeted binding relationship between Miat and miR-182,and the biological information website Targescan predicts the binding relationship between miR-182 and Nogo-A.4.Transfected si-Miat,NC,miR-182 mimic and then treated with H2O2,qRT-PCR was used to test the transfection efficiency and the expression of miR-182 Nogo-A mRNA;Western blotting was used to detect the expression of apoptosis-related proteins Bax,Caspase-3,Bcl-2 and Nogo-A;Hoechst apoptosis staining was used to detect the change of apoptosis rate in each group.5.si-Miat and miR-182 were co-transfected,miR-182 mimic and up-regulated Nogo-A were co-transfected,and the expression of apoptosis-related proteins Bax,Caspase-3,Bcl-2 and Nogo-A were detected by Western blotting after H2O2 treatment;Hoechst apoptosis staining was used to detect the change of apoptosis rate in each group.Result:1.Use H2O2 treatment to simulate hypoxic injury of myocardial cells.First,treat H9c2 cells with different H2O2 concentration gradients and finally select 800 μ The best simulation condition is that the concentration of mol/L is treated for 18 hours.The expression of Miat in H9c2 cells after H2O2 treatment was increased by qRT-PCR,the expression of pro-apoptotic protein Bax and Caspase-3 was increased by Western blotting,and the expression of anti-apoptotic protein Bcl-2 was decreased,and the apoptosis rate was increased by Hoechst apoptosis staining.2.After verifying the down-regulation efficiency,si-Miat was down-regulated.Compared with H2O2+NC group,the expression of pro-apoptotic protein Bax and Caspase-3 in si-Miat+H2O2 group was decreased,and the expression of anti-apoptotic Bcl-2 was increased.Apoptosis staining showed that the down-regulation of Miat inhibited the apoptosis of H2O2 treated cells.Compared with H2O2+NC group,RT-qPCR detection showed that the expression of Nogo-A mRNA and Nogo-A decreased after down-regulation of Miat.3.Bioprediction software RNAhybrid predicts the existence of targeted binding sites between Miat and miR-182;Down-regulate Mi at and increase the expression of miR-182;After the cells were transfected with miR-182 mimics,qRT-PCR showed that the expression of Nogo-A mRNA was decreased,and the expression of Nogo-A protein in H2O2+miR-182 mimics group was lower than that in H2O2+NC group.4.After co-transfection of si-Miat and miR-182 inhibitor,it was found that compared with the si-Miat+inhibitor NC group,the expression of pro-apoptotic protein Bax and caspase-3 in the co-transfection group of si-Miat and miR-182 inhibitor increased,the expression of anti-apoptotic Bcl-2 decreased,and the apoptosis rate increased by apoptosis staining,indicating that the apoptosis rate in the co-transfection group of si-Miat and miR-182 inhibitor NC group was higher than that in the si-Miat+inhibitor NC group,That is,down-regulation of miR-182 offset the inhibition of apoptosis by down-regulation of Miat.5.Targescan,a bioinformatics website,predicted that there was a hypothetical binding site between miR-182 and Nogo-A.After co-transfection of miR-182-mimics and up-regulated Nogo-A,it was found that compared with miR-182 mimics+Nogo-A NC group,the expression of pro-apoptotic protein Bax and Caspase-3 in miR-182-mimics and up-regulated Nogo-A co-transfection group increased,the expression of anti-apoptotic Bcl-2 decreased,and the apoptosis rate increased by apoptosis staining,indicating that the apoptosis of miR-182-mimics and up-regulated Nogo-A co-transfection group was higher than that of miR-182-mimics+Nogo-A NC group,That is,the inhibition of apoptosis by part of miR-182 mimics was counteracted after the regulation of Nogo-A.6.After co-transfection of si-Miat and miR-182 inhibitor,it was found that the expression of Nogo-A protein in the co-transfection group of si-Miat and miR-182 inhibitor was higher than that in the si-Miat+inhibitor NC group,which was consistent with the trend of apoptosis,indicating that the apoptosis of the co-transfection group of si-Miat and miR-182 inhibitor was higher than that of the si-Miat+inhibitor NC group,that is,the inhibition of the expression of Nogo-A by the down-regulation of miR-182 was offset after down-regulation of miR-182.Conclusions:1.The expression of lncRNA Miat increased in the process of myocardial cell apoptosis induced by hypoxia.2.Inhibition of lncRNA Miat can reduce cardiomyocyte apoptosis induced by hypoxia.3.lncRNA Miat negatively regulates miR-182;MiR-182 regulates Nogo-A negatively.4.lncRNA Miat can promote cardiomyocyte apoptosis induced by hypoxia through miR-182/nogo-A axis. |
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