| MicroRNAs for short miRNAs,is a group of highly conservative non-coding RNA in organisms.Studies have found that MicroRNAs can regulate cell proliferation,differentiation,apoptosis and other life phenomena,and is by far the most important cardiovascular physiological function regulation molecule.Autophagy(autophagy)is a ubiquitous metabolic pathway and a highly conservative catabolism process.It removes damaged organelles and protein aggregates through autophagy lysosome pathway and recycles its products to maintain intracellular homeostasis.Previous studies have shown that autophagy can regulate exercise-induced myocardial hypertrophy.Exercise-induced cardiomyocyte hypertrophy is a kind of exercise-induced adaptive response to changes in the expression of genes related to cell growth and proliferation after cardiomyocytes and their interstitial cells are stimulated by mechanical stretch or growth factors.Previous studies have shown that miR-26b-5p plays an important role in the formation of exercise-induced cardiac hypertrophy,but its specific regulation mechanism is not impeccable.Purposes:This study aimed to investigate the expression and regulation of miR-26b-5p in exercise-induced cardiac hypertrophy,and to verify the molecular mechanism of miR-miR-26b-5p regulating exercise-induced cardiac hypertrophy through target genes.Methods:Twenty-four Wistar clean female rats aged 6 weeks were randomly divided into two groups: quiet control group(CON,n=12)and exercise-induced myocardial hypertrophy model group(EX,n=12).The rats in the quiet group were raised in ordinary cages without any exercise.The rats in the model group made swimming training for 10 weeks according to the exercise plan of Fernandes,and the animal model of exercise-induced myocardial hypertrophy was established.Echocardiographic indexes,whole heart index(whole heart weight / body weight,HW/BW)and left ventricular index(left ventricular weight / body weight,LW/BW)were used to reflect cardiac hypertrophy.HE staining was used to observe the morphology of cardiomyocytes,and combined with the detection of the expression of myocardial pathological markers in rats,the establishment of exercise-induced cardiac hypertrophy animal model was comprehensively evaluated.The expression of miR-26b-5p in rat myocardium was detected by biochip technology and RT-qPCR.At the same time,transmission electron microscope,expression of LC3 B and Beclin1 were used to reflect the changes of myocardial autophagy in rats with exercise-induced cardiac hypertrophy.Bioinformatics was used to predict the target gene of miR-26b-5p.After overexpression and interference expression of miR-26b-5p in vitro,the expression of predictive target gene ULK1 was detected by Western Blot and RT-qPCR,and the change of LC3 B was detected by immunofluorescence test to reflect the change of autophagy.Finally,the targeting relationship was verified by double luciferase reporter gene.This proves the expression and regulation of miR-26b-5p in exercise-induced myocardial hypertrophy.Result: 1.Establishment and evaluation of an animal model of exercise-induced cardiac hypertrophy: After 10 weeks of swimming in rats,the heart index of the model group was significantly higher than that of the control group rats(p<0.05),and the left ventricular index was significant increased(p<0.01).The results of HE staining showed that the myocardial volume of cardiomyocytes in the model group was enlarged,the structure was normal,the nucleus was oval,the size distribution was uniform,the collagen fibers proliferated to a certain extent,and the diameter of cardiomyocytes increased significantly(p < 0.05).The results of echocardiography in rats showed that the cardiac pumping function of rats in the model group was significantly higher than that in the control group.The results of pathological examination of cardiac hypertrophy showed that compared with the control group,the expression of ANP and ?-actin mRNA in the myocardium of the model group was not significantly changed compared with the control group.The ?-MHC/?-MHC ratio did not change significantly.Therefore,in this modeling process,hypertrophic changes occurred in the heart of 10-week swimming rats,and left ventricular hypertrophy was more obvious,but did not lead to pathological changes in the rat heart,indicating that exercise-induced cardiac hypertrophy in Wistar rats The model was successfully established.2.Changes of miRNA in animal models of exercise-induced cardiac hypertrophy: We found that miR-26b-5p expression was significantly decreased(p<0.01).Compared with the control group,myocardial miR-26b-5p expression was significantly decreased in the model group(p<0.01)3.Changes in myocardial autophagy in an animal model of exercise-induced cardiac hypertrophy: LC3 II mRNA and protein expression levels in the myocardial tissue of rats in the model group after 10 weeks of swimming compared to rats in the control group,and The LC3II/LC3 I ratio was significantly increased(p<0.01,p<0.01,p<0.01),and the expression of Beclin1 mRNA and protein in the myocardial tissue of model rats was also significantly increased(p<0.01,p<0.01).Transmission electron microscopic examination of autophagosomes revealed that compared with control rats,the number of autophagosomes in the rat model group increased significantly,and the volume increased.The number of autophagosomes in the model group was also in 30 visual fields.Far more than the control group.This shows that: Through 10 weeks of swimming exercise,the level of autophagy in rat myocardial hypertrophy was increased.4.Bioinformatics Prediction Results: Through the bioinformatics prediction of miRNA targets,it was found that in the gene sequence of rat ULK1,the position sequence(tacttgaa)of coding region 1019-1026 and 1120-1127 is miR-miR-26b-5p Binding site.5.MiR-26b-5p regulates exercise-induced myocardial hypertrophy by targeting ULK1: 1 the expression levels of ULK1 protein and mRNA in myocardial tissue of rats were detected.It was found that the expression levels of ULK1 protein and mRNA in model group were significantly higher than those in control group(p < 0.01).2 compared with the control group,the levels of ULK1 mRNA and protein in cardiomyocytes overexpressing miR-26b-5p were significantly decreased(p < 0.01),and immunofluorescence results showed that the expression of LC3 B was down-regulated and myocardial autophagy was decreased,while the levels of ULK1 mRNA and protein in cardiomyocytes interfering with miR-26b-5p expression were significantly increased(p < 0.01).Immunofluorescence results showed that LC3 B expression was up-regulated and autophagy was enhanced.6.The results of double luciferase reporter gene assay showed that the relative luciferase activity after co-transfection of ULK1-WT 3'UTR and miR-26b-5p mimics was significantly lower than that of mimics-NC group,on the contrary,the relative luciferase activity of ULK1-WT 3'UTR and miR-26b-5p inhibitor co-transfection group was significantly higher than that of inhibitor-NC group,but there was no difference between the two groups and NC group after co-transfection with ULK1-MT1+MT2 3'UTR,respectively.To sum up,ULK1 is the target gene of miR-26b-5p,and miR-26b-5p can regulate exercise-induced cardiac hypertrophy through autophagy-related gene ULK1.Analysis conclusion: 1.A 10-week swimming exercise successfully established a model of exercise-induced cardiac hypertrophy in Wistar rats.2.In the rat model of exercise-induced cardiac hypertrophy,the level of myocardial autophagy increased significantly.3.Cardiomyogenic hypertrophy is the result of miRNA targeting on autophagy-related genes.The mechanism may be that exercise can down-regulate the expression level of cardiac miR-26b-5p,and miR-26b-5p targets ULK1(The binding site of ULK1 at position 1019-1026 and 1120-1127 base sequence(tacttgaa)upregulates the expression level,which promotes the level of myocardial autophagy and induces exercise cardiac hypertrophy. |
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