The Function And Mechanism Of MiR-148a In Cardiac Hypertrophy

Background:Cardiac failure is a syndrome caused by a variety of diseases and physiological factors,leading to an important cause and pathological changes of chronic heart failure is pathological myocardial hypertrophy,with the continuous study of its pathogenesis,the regulation of molecular biology gradually More attention.In recent years,studies have found that microRNA is one of the important mechanisms involved in regulating the pathological process of heart failure.MicroRNAs(miRNAs)are single-stranded non-coding small RNAs with a length of 18-25 nucleotides and have post-transcriptional regulatory properties.miRNAs can form an RNA-induced silencing complex(RISC)that specifically binds to the 3' non-coding region of a target gene mRNA in a completely or incompletely complementary pairing manner,negatively modulating the target gene protein at the post-transcriptional level Expression,thereby regulating a variety of physiological processes of organisms,can participate in and regulate angiogenesis,cardiac hypertrophy,myocardial cell and interstitial fibrosis,inflammation,apoptosis and necrosis in cardiovascular diseases.Cardiac hypertrophy is a pathological process characterized by increased cell volume,increased protein secretion,and increased sarcomere tissue.The underlying triggers are hemodynamic disorders,ischemic injuries,and neuroendocrine imbalances,and other pathological conditions.For fetal gene reexpression,excitatory contraction coupling changes,and changes in energy metabolism balance,studies have shown that many miRNAs function positively or negatively to regulate cardiac hypertrophy.We established a rat abdominal aorta constriction model and cardiomyocyte hypertrophy model to detect the expression level of miR-148 a and compared with the control group;we further constructed miR-148 a overexpressing adenovirus and miR-148 a inhibitor to In rat cardiomyocytes,the changes in cell-associated heart failure marker molecules were observed to clarify the role of miRNA-148 a in heart failure and further explore the possible target genes for heart failure induced by miRNA-148 a.Materials and Methods:1.Construction of rat abdominal aorta constriction modelOrder 180g~200g SD rats,fasting water for 24 h and then anaesthetize with 10% chloral hydrate.Open the abdomen in a sterile environment.Use No.4 silk thread and a needle tube with an outer diameter of 0.6mm to fasten and remove the needle tube.The control group does not ligation.After 4 weeks of feeding,the hearts were sacrificed,HE staining was used to observe the myocardial morphological changes,parallel quantitative PCR was used to detect hypertrophy-related marker molecules,and it was determined that the model was successfully constructed.2.Construction of Primary Cardiomyocyte Hypertrophy ModelRemove SD rat pups born for 1-2 days,disinfect 70% alcohol,remove the ventricles under aseptic conditions,digest with collagenase,and centrifuge three times to separate cardiomyocytes from fibroblasts.After two days of routine culture of cardiomyocytes,Brdu was added and cultured for 24 hours in a serum-free environment.Phenylephrine and angiotensin II were added to induce cardiomyocyte hypertrophy.3.Detection of miR-148 a expression in the hypertrophic modelThe RNA of myocardial tissue and myocardial RNA of hypertrophic cardiomyocytes were routinely extracted by Trizol method.Quantitatively,microRNA was reverse-transcribed using specific primers of the cervical loop structure and was detected by quantitative PCR.SYRB GREEN MIX kit was used for qRT-PCR.4.Construction of overexpressed adenovirus and miR-148 a inhibitorThe corresponding microRNA precursor sequence was cloned and inserted into the pADTrack-CMV shuttle plasmid.After pmeI linearized,it was transformed into BJ5183 competent bacteria.It was applied to Carnagar-resistant LB plates overnight.Sganer sequencing screened the correct recombinant plasmid and used lip 2000.Plasmids were transfected into 293 A cells and virus was packaged and expanded to determine virus titers.The mature sequence of miR-148 a was searched at www.mirbase.org,the complementary sequence of the sequence was designed,and the base was subjected to a 2' methoxy modification.The synthesis and modification sequence was performed by Shanghai Jema Corporation.5.Dual luciferase reporter geneBioinformatics methods were used to predict its target genes.The 3?UTR region of MAP3K4 mRNA was inserted into the luciferase plasmid,and the plasmid and miRNA148 a overexpression plasmid were co-transfected into 293 T cells.After 24 hours,the cells were lysed to determine the luciferase expression level.And quantitative analysis.6.Western blotThe microRNA overexpression plasmid was transfected into primary cardiomyocytes.The protein was extracted with RIPA lysate.The expression of target gene protein was detected by western blot.The membrane was electrophoresed on 100 v electrophoresis,transferred to the membrane by wet 75 V and 2h,and blocked with 5% milk for 2h.Incubate the primary antibody overnight at 4°C.Repeat washing three times with TBST for 15 min each,incubate the secondary antibody for 2 h in the dark,TBST wash three more times,develop with developing solution,scan protein bands in the Odyssey Infrared Laser Scanning Imaging System and analyze to further identify miRNA-148 a Can regulate MAP3K4 expression.7.Statistical analysisThe measurement data are expressed as mean ± standard deviation,with each experiment being repeated at least 3 times and using two independent samples for t-test.We default to P <0.05 as a criterion for significant differences.Results1.SD rat myocardial hypertrophy modelAbdominal aorta coarctation is one of the most common methods for the construction of rat heart failure models.In this experiment,24 rats survived successfully.In some experimental groups,SD rats showed signs of weight loss and sparse hair.The appearance of rat heart with 4 w of abdominal aortic coarctation was significantly larger than that of the control group;echocardiography of rats showed that after 4 weeks of ligation,the EF value of the experimental group was significantly decreased;in addition,the ANP and myh7 were significantly increased in the experimental group,while myh6 was significantly increased.The change was not obvious;HE hyperstained sections showed marked hypertrophy,indicating that the model was successfully constructed.2.Primary Cardiomyocyte Hypertrophy ModelThe isolated cardiomyocytes were stimulated with PE and AngII,respectively,and labeled with ?-actinin antibody for immunofluorescence staining.The cell diameter and surface area were increased under confocal microscopy,the cell morphology was full,and the results of quantitative PCR were verified.3.miRNA-148 a expression decreased in hypertrophic modelWe used quantitative PCR to detect the expression level of miRNA-148 a in myocardial tissue,PE,and AngII.The results showed that the expression of miRNA-148 a was decreased in rat cardiac hypertrophy and AngII-induced hypertrophy model,which was consistent with the results of previous studies..4.Overexpression of miR-148 a promotes hypertrophy of primary cardiomyocytesThe primary cardiomyocytes were transfected with miRNA-148 overexpressing adenovirus.The expression of miRNA-148 was detected by qRT-PCR after 48 hours.Compared with the control group,the results suggested a 13-fold increase,suggesting that the transfection was successful.The expression of ANP,myh6,and myh7 was detected.Among them,ANP and myh7 were significantly increased.There was no significant difference in myh6,indicating that overexpression of miR-148 a can promote cardiac hypertrophy.5.Knocking down miR-148 a can partially reverse cardiac myocyte hypertrophyMiR-148 a inhibitor was constructed and transfected into PE-induced hypertrophic cardiomyocytes.The expression of miR-148 a was detected by quantitative PCR.The results showed that the expression of miR-148 a was decreased by about 60%,and the expression of miR-148 a could be effectively inhibited;meanwhile,the expression level of ANP was higher than that of PE stimulation group.Compared with a significant decline,but still higher than the basal level;suggest that reducing the expression of miR-148 a can partially reverse myocardial cell hypertrophy.6.miRNA-148 a inhibits the expression of MAP3K4The use of TargetScan,microRNAorg and other database search predictions indicate that MAP3K4 may be a potential target gene of miR-148 a,so the dual luciferase reporter gene assay,further explore the potential mechanism of miR-148 a regulation of heart failure,the results showed that overexpression of miR-148 a can The luciferase expression in MAP3K4-3'UTR region was significantly inhibited,suggesting that MAP3K4 is the target gene of miR-148 a.Overexpression of miR-148 a overexpressing adenovirus was transfected into primary cardiomyocytes.Western blot results showed that the expression level of MAP3K4 protein was significantly reduced.This result also suggested that MAP3K4 is the target gene of miR-148 a.ConclusionIn the successfully constructed rat abdominal aorta constriction model and cardiomyocyte hypertrophy model,ANP,myh7 were significantly elevated,myh6 expression was decreased or there was no statistical difference,and miRNA-148 a expression was decreased.Overexpression of miRNA-148 a in cardiomyocytes can promote hypertrophy of cardiac muscle cells,and knockdown of miR-148 a in cardiomyocytes can partially reverse cellular hypertrophy.miRNA-148 a targets MAP3K4 and inhibits the expression of this gene,probably through this pathway to promote myocardial hypertrophy.