Regulatory Mechanism Of MicroRNA-30a Targeting Beclin-1 On Autophagy And Apoptosis In Murine Cardiac Myocytes

Background and purposeIschemic heart disease(ischemic cardiomyopathy,ICM),Coronary artery stenosis is mainly caused by coronary atherosclerosis,which further leads to long-term hypoxia of myocardial cells at the corresponding blood supply site,resulting in dystrophy,atrophy,apoptosis and even myocardial cell necrosis of the corresponding myocardial tissue,and eventually leads to myocardial fibrillation.ICM is also called myocardial fibrosis or myocardial sclerosis.According to the China Cardiovascular Disease Report 2016,which has become the first fatal disease among urban and rural residents.The investment of national medical resources has increased significantly,which is a major public health problem in China.Although PCI technology has made significant progress,significantly reducing the mortality rate of acute myocardial infarction,but after acute myocardial infarction,infarcted myocardial cells can not be repaired through regeneration,which will inevitably lead to ventricular remodeling and eventually lead to heart failure.Previous studies have found that when myocardial infarction occurs,both autophagy and apoptosis of myocardial cells in the ischemic region can occur.Autophagy is a process in which cells use lysosomes to degrade damaged organelles and macromolecule substances to produce amino acids and other small molecule substances to be reused or generate energy to maintain basic cell life activities.It is also a unique life phenomenon of eukaryotes.It is involved in regulating the metabolic balance between degradation,synthesis and reuse of cellular substances.Previous studies have shown that autophagy of myocardial cells can significantly reduce the size of myocardial infarction in the center of myocardial infarction.Apoptosis is a form of cell death,and it is an autonomous and programmed cell death regulated by genes.It has been reported that inhibiting cardiomyocyte apoptosis can significantly save the loss of cardiomyocyte in the occurrence of myocardial infarction,thereby improving the cardiac function after myocardial infarction,and thus benefit patients.Autophagy and apoptosis play an important role in the development,differentiation and homeostasis of mammals.Therefore,it is very important to maintain the cardiac function of patients by properly regulating the autophagy and apoptosis of myocardial cells during the occurrence of myocardial infarction.MicroRNAs(miRNAs)are a class of highly conserved,endogenous,non-coding small RNAs with a length of about 18-22 ribonucleic acids that regulate gene expression at the post-transcriptional level.MicroRNAs are involved in almost all pathophysiological processes of the heart.MicroRNAs are closely involved in the regulation of cardiac development,cardiac hypertrophy,heart failure and reperfusion injury,and play an important role in the differentiation,growth,proliferation and apoptosis of cardiomyocytes,myocardial fibrosis and angiogenesis.And may be one of the key regulatory factors,[1-4].At present,microRNA-1,microRNA-21,microRNA-23a,microRNA-133,microRNA-208 and microRNA-320 are expressed in myocardial tissues,and the functions and targets of other microRNAs involved in myocardial physiological and pathological processes need to be further confirmed.The microRNA-30 family,located on chromosome 6q13,consists of five members:a,b,c,d and e.Its precursor(pre-microRNA)consists of 70 nucleotides that can b e spliced into mature microRNAs by nuclease in the cytoplasm.In recent years,how microRNA-30a causes autophagy and apoptosis of myocardial cells in the process of myocardial infarction has attracted much attention.The specific role of microRNA-30a in the process of autophagy and apoptosis of myocardial cells is not yet clear.Whether microRNA-30a participates in the regulation of autophagy and apoptosis of myocardial cells in the process of myocardial infarction needs further study.Whether microRNA-30a is involved in the regulation of myocardial autophagy and apoptosis during myocardial infarction is needed.We further study.Therefore,this study explores the possible mechanism of microRNA-30 autophagy and apoptosis in myocardial infarction,which provides new clues for clinical diagnosis and treatment of myocardial infarction,and lays a molecular biological foundation for new therapeutic targets of myocardial infarction.We found that when myocardial cells were starved by Earle's balanced salt solution,autophagy and apoptosis were observed,and the expression of microRNA-30a was down-regulated.These results suggest that microRNA-30a may be involved in the regulation of autophagy and apoptosis of myocardial cells treated with EBSS starvation.(1)To further study the changes of autophagy and apoptosis of cardiomyocytes by overexpressing microRNA-30a and knocking down the expression of microRNA-30a.(2)constructing a myocardial infarction model in mice to study the regulation of MicroRNA-30a on autophagy and apoptosis in vivo.(3)to study the potential target genes of MicroRNA-30a induced autophagy and apoptosis in cardiomyocytes.Research methods1.Isolation and culture of mouse cardiomyocytesAfter ultraviolet disinfection of cells and super-clean table,the newborn mice were killed by neck-leading for 24 hours,then the surface of the mice was disinfected with 75%alcohol.The heart of the mice was taken out and the atrial structure of the mice was removed.The ventricles of the mice were cut into 1 mm~3 tissue blocks by ophthalmic bending scissors.The mice were digested overnight in 4 C ice with 5 ml1%collagenase I for about 20 hours.After completing the above steps,the cells were digested in the cell incubator for 5-10 minutes,and the tissues were blown until they were completely digested.Then the digestion was terminated with DMEM culture medium.After centrifugation,D-hanks solution was re-suspended and washed.After centrifugation,the myocardial cells needed for the experiment were separated by differential attachment.Myocardial cells were cultured in DMEM medium of 0.1 mm BrdU and cultured in Earle's equilibrium salt solution to establish the starvation model of myocardial cells.2.Real time fluorescence quantitative PCR(qRT-PCR)Trizol method was used to extract the total RNA of mouse cardiomyocytes and the reverse transcriptase was cDNA.In strict accordance with SYBR Green I operation to complete qRT-PCR,U6 is set as internal reference.3.Construction of adenovirus and transfection of mouse cardiomyocytesThe adenovirus titer was determined strictly according to the cytopathy(CPE)method.We constructed the over-expression adenovirus of microRNA-30a according to the specific method of AdEasy adenovirus packaging system.MicroRNA-30a inhibitor was packaged in Lipofectamine 2000 and then transferred into the cardiomyocytes obtained in the previous experiment.We set the Control inhibitor transfected cardiomyocytes as negative control.Real-time fluorescence quantitative PCR(qRT-PCR)was used to determine the transfection efficiency of microRNA-30a inhibitor and miroRNA-30a overexpressed adenovirus.4.Western blot assay(Western blot)The expression levels of GAPDH protein,Beclin-1 protein,P62 protein and LC3protein in mouse cardiomyocytes were detected by Western blot.The myocardial cells of mice were lysed with RIPA lysate containing PMSF.The protein was extracted,the protein concentration was determined and the samples were balanced.Then routine SDS-PAGE electrophoresis was carried out,and the cells were wetted for 1 hour.At room temperature,5%skimmed milk was used to seal it up.The first antibody was incubated at 4 C and overnight.The second antibody was incubated at room temperature for about 1 hour.Finally,the strip was analyzed by software and our experimental results were obtained.5.Immunohistochemical staining and cellular immunofluorescenceImmunohistochemical staining was used to stain the heart of mice.Myocardial tissue of mice was immobilized with 4%polyformaldehyde for 24 hours.Paraffin was embedded and sectioned.After dewaxing,paraffin sections were hydrated and sealed with 5%goat serum for about 10 minutes.Anti-P62 and anti-LC3 antibodies were incubated at 40C for overnight.At last,we added second antibodies at 40C for about 1 hour.At last,the color reagent was developed.Color and seal.After treating myocardial cells according to different experimental designs,the myocardial cells were fixed with 4%paraformaldehyde for 10 minutes at room temperature,then permeated with 0.1 Triton X-100 for 10 minutes,then sealed with5%goat serum for about 30 minutes.Finally,anti-LC3 antibody was added to incubate myocardial cells at room temperature for about 1 hour,then washed with PBST for 3 times and then added with anti-rabbit IgG antibody.After incubation at room temperature for about 30 minutes,0.1%DAPI was dyed and sealed immediately.Laser confocal microscopy was used to observe and photograph it.6.Successful establishment of myocardial infarction model in miceAfter successful anesthesia in BALB/c mice,tracheal intubation was performed and animal ventilator was connected.The respiratory frequency was set to 150 times per minute,the tidal volume was set to 3 m1,and the respiratory ratio was set to 1:2.Then the ECG simulator was connected.After routine disinfection,a scarf was laid on the left chest of BALB/c mice and an incision of about LCM length was cut with ophthalmic scissors;the subcutaneous muscles of BALB/c mice were separated bluntly by separating forceps,so that the ribs were fully exposed,the intercostal muscles were cut along the fourth rib space,the ribs were opened with a chest distractor,the thoracic cavity was fully exposed,the pericardium of the mice was cut along the bottom of the heart with ophthalmic scissors,and the left atrial appendage was explored 2-3 mm below./Three places were punctured with 9-0 needled ophthalmic surgical suture,and the needles were knotted from the right lateral edge of left atrial appendage.After ligation,the local color of the anterior wall of the heart turned pale.The electrocardiogram of BALB/c mice showed that the ST segment of the arch was raised upward,indicating that the model of myocardial infarction in mice was successfully constructed.7.Detection of flow cytometryMyocardial cells were treated according to the experimental design,digested with trypsinase without EDTA,and collected the cells needed for the experiment.After centrifugation,the supernatant was discarded and added with 1 Xbindingbuffer500ul,the cells were suspended.Again,add Annexin V-FITC or Annexin V-APC5ul and shake them to mix evenly.They are incubated at room temperature and in a dark environment for about 15 minutes.Add PI3ml shake to mix,incubate in dark environment,incubation time is about 5 minutes.Finally,the cells obtained from the above steps will be transferred to the sampling tube for analysis and instrumental detection.8.MASSON staining and TUNEL methodMasson staining was used to detect myocardial fibrosis.The specific procedure was strictly followed by the instructions of Masson staining kit.TUNEL staining was used to detect apoptosis of cardiac myocytes.The specific procedures were carried out in strict accordance with the instructions of the apoptosis detection kit.The total number of myocardial cells and the number of TUNEL positive cells were counted randomly under 10 high power microscopy.The apoptotic rate of cardiomyocytes is the ratio of TUNEL positive cells to the total number of cardiomyocytes.9.Double luciferase reporter gene methodLuciferase plasmids containing mutant and wild Beclin-1 gene 3'UTR were co-transfected into 293 T cells with microRNA-30a analogues,respectively.After 48hours of transfection,293 T cells were lysed.Then the luciferase activity of sea kidney and the luciferase activity of firefly were detected,and the ratio of them was calculated.Finally,the ratio was analyzed statistically.10.Statistical methodWe used GraphPad Prism to statistically analyze the data obtained from the experiment.Two independent sample t-tests were used to compare the data between the control group and the experimental group.The measurements were expressed as mean standard deviation(X±s).The difference was statistically significant with P<0.05.Results1.Starvation induces autophagy in cardiac myocytesAfter starvation with Earle's equilibrium salt solution for 4 hours,the autophagy ability of mouse cardiomyocytes increased significantly.(1)Western blot results showed that the ratio of LC3-II/LC3-I was significantly higher than that of LC3-II/LC3-I in normal culture group,but the expression level of P62 was significantly lower than that of P62 in normal culture group.(2)Immunofluorescence assay showed that LC3 red fluorescent dots accumulated in the cytoplasm of myocardial cells after starvation for 4 hours in Earle's balanced salt solution,and fluorescent dots could be regarded as a marker of autophagy.These results suggest that starvation with Earle's equilibrium salt solution can successfully induce autophagy in cardiomyocytes.(3)Real-time quantitative polymerase chain reaction(qRT-PCR)results showed that the expression of microRNA-30a in mouse cardiomyocytes starved with Earle's equilibrium salt solution was significantly down-regulated compared with that in normal culture group,and microRNA-30a was most significantly down-regulated at4h of starvation treatment.2.MicroRNA-30a can promote the autophagy of cardiomyocytes induced by starvation(1)MicroRNA-30a overexpression was achieved by transfection of microRNA-30a overexpression adenovirus into cardiomyocytes.The results of qRT-PCR showed that the expression of microRNA-30a in cardiomyocytes transfected with microRNA-30a adenovirus increased 15 times,which indicated that the microRNA-30a adenovirus was highly effective.(2)Two days after adenovirus transfection,autophagy was induced by starvation in Earle's balanced salt solution.Western blot analysis showed that the ratio of LC3-II/LC3-I in neonatal rat cardiomyocytes increased significantly after overexpression of microRNA-30a,while the expression level of P62 decreased significantly.Similarly,the results of immunofluorescence assay showed that the number of red fluorescent spots in cardiomyocytes of mice increased significantly after overexpression of microRNA-30a.All these results suggest that microRNA-30a overexpression can significantly promote the autophagy of starvation-induced cardiomyocytes in mice.(3)In the model of myocardial infarction in mice,the over-expression of microRNA-30a in myocardial cells showed a higher level of LC3 expression,but the expression of P62 decreased significantly.This result indicated that the autophagy level of myocardial cells increased significantly after the over-expression of microRNA-30a.(4)MicroRNA-30a inhibitor was used to treat cardiomyocytes in order to further test the effect of microRNA-30a on autophagy.QRT-PCR showed that the expression level of MicroRNA-30a was significantly down regulated after MicroRNA-30a inhibitor transfection.Western blot results showed that the down-regulation of microRNA-30a expression could significantly reduce the LC3-II/LC3-I ratio and increase the level of P62.Using immunofluorescence assay,we also found that the red fluorescent spots in myocardial cells decreased significantly after microRNA-30a was down-regulated.All these results suggest that MicroRNA-30a can enhance the autophagy activity of starved cardiomyocytes.3.MicroRNA-30a inhibited starvation induced cardiomyocyte apoptosis.(1)After myocardial cells were cultured in Earle's balanced salt solution,there were obvious apoptosis as well as autophagy.Under the same starvation conditions,the flow cytometry showed that compared with the control group,mice overexpressing microRNA-30a cardiomyocytes had lower apoptosis level and lower apoptosis rate.(2)In order to further study the effect of endogenous microRNA-30a on the apoptosis of cardiomyocytes in mice,we set the control inhibitor as negative control.We knocked down the expression of MicroRNA-30a in cardiomyocytes by using microRNA-30a inhibitor.The results showed that under the same starvation condition,when the expression level of microRNA-30a was down-regulated,the cardiomyocytes of mice showed more obvious apoptosis and higher apoptosis rate.(3)In the model of myocardial infarction in mice,over-expressed microRNA-30a myocardial cells showed fewer TUNEL-positive apoptotic cells compared with the control group,which indicated that over-expression of microRNA-30a could inhibit ischemia and thus reduce apoptosis of myocardial cells.All these results suggest that microRNA-30a can inhibit starvation-induced cardiomyocyte apoptosis.4.MicroRNA-30a regulates autophagy and apoptosis of mouse cardiomyocytes by inhibiting the expression of Beclin-1.(1)Potential target genes of microRNA-30a in cardiomyocytes were screened out in the bioinformatics database.It was found that the potential target gene of microRNA-30a was Beclin-1.Further searches revealed that Beclin-1 3'UTR might contain MicroRNA-30a binding sites.(2)We first used a dual luciferase reporting system to verify our hypothesis that the target gene for microRNA-30a was Beclin-1.Our results showed that up-regulation of the expression of microRNA-30a significantly inhibited the activity of luciferase in the vector carrying wild-type Beclin-13'-UTR sequence,but up-regulation of the expression of microRNA-30a did not affect the activity of luciferase in the vector carrying the mutation sequence of Beclin-13'-UTR.This result confirms that the direct target gene of MicroRNA-30a is Beclin-1.(3)Western blot was used to verify the hypothesis that the expression level of Beclin-1 protein increased significantly when the expression level of microRNA-30a was lowered.On the contrary,the expression level of Beclin-1 protein could be significantly inhibited when the expression level of microRNA-30a was up-regulated.These results suggest that microRNA-30a may regulate autophagy and apoptosis of cardiac myocytes by targeting Beclin-1.Conclusion1.After starvation with Earle's balanced salt solution,autophagy was successfully induced in isolated mouse cardiomyocytes.2.In the starvation model of cardiomyocytes,the expression of microRNA-30a in cardiomyocytes decreased significantly,and the most significant decrease was observed in Earle's balanced salt solution starvation for 4 hours.3.Overexpression of microRNA-30a can significantly promote the autophagy of starvation-induced cardiomyocytes,while knocking down microRNA-30a can significantly inhibit the autophagy of cardiomyocytes.4.MicroRNA-30a overexpression could significantly inhibit starvation-induced cardiomyocyte apoptosis,while knocking down microRNA-30a could promote cardiomyocyte apoptosis.5.Beclin-1 is the direct target of microRNA-30a,and microRNA-30a can directly inhibit the expression of Beclin-1,which may promote the autophagy of starvation-induced cardiomyocytes and inhibit the apoptosis of starvation-induced cardiomyocytes.