The Study Of MiR-30b Regulating Diabetic Cardiomyopathy By Influencing Cardiomyocyte Apoptosis

Background and objectiveDiabetes is a kind of abnormal metabolic disease,which can be caused by different reasons.With the continuous improvement of living standards,increasing aging and continuous improvement of diagnostic methods,diabetes has become the third non-communicable disease,after cardiovascular diseases and malignant tumors.On the advanced stage of diabetes,easy to combine with some other organ tissue damage and not easy to cure.Frequent diabetic complications include diabetic foot,diabetic heart disease,diabetic nephropathy,diabetic retinopathy,respiratory tract infection,urinary tract infection,etc.The damage of diabetes to the heart is manifested in many aspects,for coronary atherosclerotic heart disease,diabetes is a major independent risk factor of coronary artery disease.Compared with patients without diabetes,coronary heart disease patients with diabetes onset time earlier,especially in a kidney disease coexist;Atypical angina is more common;Myocardial infarction mortality is higher.Diabetic cardiomyopathy(DCM)is a specific disease of diabetic heart complications.Most patients have a history of diabetes for more than 5 years,and myocardial injury caused by hypertension and coronary atherosclerotic heart disease has been excluded in the diagnosis process.When DCM occurs,the general pathological features of cardiomyocytes are as follows:cardiomyocyte hypertrophy,necrosis,apoptosis,decreased muscle fibers,collagen deposition,diffuse fibrosis of myocardial interstitium,hypertrophy of microvascular basement membrane,increase of matrix,and microvascular damage in myocardium.Electron microscope examination showed that the structure of myocardial cells was obviously damaged:mitochondria increased around the nucleus,mitochondria swelling,glycogen and lipid droplet accumulation around the mitochondria,etc.A number of physiological or metabolic abnormalities associated with elevated blood glucose include decreased glucose oxidation in cardiomyocytes,abnormalities in lipid metabolism,oxidative stress,activation of the renin-angiotensin-aldosterone system(RAAS)autonomic neuropathy,microcirculation dysfunction,abnormalities in calcium homeostasis,and increased accumulation of advanced glycation end products(AGEs),etc.The abnormal cardiac structure and function caused by the above pathophysiological changes had no symptoms at the initial stage,but gradually developed heart failure.With the development of the disease,gradually appear heart failure,first for the diastolic heart failure,its ejection fraction normal,and then for systolic heart failure,its ejection fraction decreased.Diabetes combined with heart disease has become one of the most dangerous disease combinations in contemporary medicine.At the same time,the incidence and mortality of diabetic cardiomyopathy are rising rapidly,which requires further research specifically for DCM patients.urrent research focuses on the pathophysiological abnormalities of myocardial metabolism and receptor signaling pathways.Abnormalities in myocardial metabolism include increased oxidative stress response(OS)and the formation of AGEs,etc.Activated by sugar toxic product many kinds of cell signaling pathways including protein kinase C(PKC)pathway,silk crack the original activated protein kinase(MAPK)pathways and inflammatory signaling pathways,etc.In addition,the apoptosis of cardiac structure and function of injury is an important part of the development of diabetic cardiomyopathy occurs.Apoptosis is a kind of programmed cell death.Under the action of various internal and external factors,specific intracellular programs are initiated through multiple signal transduction,and finally endogenous DNA endonuclides are activated,leading to natural cell death.Currently,Cytochrome C(Cyt C).P53,Bcl-2 and Caspase-3 are found to be related to the mechanism of apoptosis,among which Bcl-2 is the inhibitor of apoptosis and Caspase-3 is the promoter of apoptosis.MicroRNA(miRNA)is a kind of non-coding long chain DNA with length of 22-23 basic groups.It can combine with 3’-UTR of the target mRNA to inhibit mRNA translation or degrade mRNA directly.So,miRNA can control the expression level of target mRNA after transcription.Many studies have found that miRNA take part in many kinds of life activities in the body,including cell proliferation,proliferation.apoptosis and tissue development.Another study found that the increase in miRNAs expression of myocardial cells may participate in the ventricular remodeling,myocardial hypertrophy and some other diseases.Further,some scholars pointed out that micro RNA appeared in the process of cardiac lesions during insulin resistance.From the above,it can be concluded that microRNA may play an important role in the development of diabetic cardiomyopathy.The miR-30 family is one of the miRNAs highly expressed in cardiomyocytes.Studies have shown that miR-30c influence the process of diabetic cardiomyopathy by regulating Beclin protein expression.MiR-30b,a member of the miR-30 family,is located on human chromosome 8 and plays an important regulatory role in tumorigenesis,mitochondrial division and apoptosis,but the role of miR-30b in diabetic cardiomyopathy has not been reported.This give us hints,miR-30 b in DCM myocardial cells express?If the expression is abnormal,is it involved in the pathogenesis of diabetic cardiomyopathy?In this paper,we aimed to explore the potential role of miR-30b in DCM.The whole dissertation can be divided into four parts.In the first part,we constructed rat DCM models and the control,to screen differential expressed miRNAs by using microRNAs array with high throughput and qRT-PCR was used to verify.In the second part,we constructed high sugar models,and determined the influence of high sugar on myocardial apoptosis,expression levels of apoptosis factors and cell apoptosis inhibition factors and the mRNA levels of miR-30b.In the third part,transfection technology was used to interfere with the expression of miR-30b,and observed the influence of miR-30b interference on myocardial apoptosis,expression levels of apoptosis factors and cell apoptosis inhibition factors.In the fourth part,using microRNA database Target Scan predicted miRNA-30b potential target genes.Then,in the myocardial cell which was transfected with miRNA-30b mimic,we observed target genes expression on mRNA level and protein level,Further,using luciferase reporter technique verified the target gene of miR-30b.Part Ⅰ Clustering analysis of microRNA in cardiac tissue of rats with diabetic cardiomyopathyObjective:1.To explore the basic information and morphologic change of rats with DCM.2.To explore the differential expressed miRNA and verify.Methods:1.Experimental rats were divided into DCM group and the control group.Ultrasound cardiogram and HE staining were used to observe whether DCM rat models were constructed successfully.2.MiRNA expression profiles in DCM group and the control group were analyzed to screen differential expressed miRNAs.3.QRT-PCR was used to verify the expression of miR-30b in myocardial tissues of rats in DCM group and the control group.Results:1.A value,EDT,EDT’,IVRT and IVRT’ values in DCM group were higher than the control.E value,EF,E/A in DCM group were lower than the contro 1.Compared with the control group,in DCM group myocardial cells arranged disordered and loosened,size of nuclei irregularly sized,a small quantity of necrotic foci could be seen in interstitial structure and cardiomyocyte hypertrophy occurred.2.Compared with the control group,182 differential expressed miRNAs were found in DCM group,and the most upregulted miRNA was miR-30b.3.The expression of miR-30b in myocardial tissues of rats in DCM group was observably increased than the control group.Conclusions:MiR-30b expression in DCM group was upregulated,which indicated miR-30b involved in the occurrence and development of DCM.Part Ⅱ High concentration of sugar induced upregulation of microRNA-30b and cell apoptosisObjective:1.To explore the influence of high glucose on myocardial apoptosis and the mechanism.2.To explore the influence of high glucose on the expression of miRNA-30b.Methods:1.Experimental myocardial cells were divided into the control and high glucose group.Flow cytometry was used to analyze the myocardial apoptosis in the control and high glucose group.2.Western blotting was used to determine the protein expression of cell apoptosis related factor Caspase-3 and PARP,and cell apoptosis inhibition factor Bcl-2 in the two groups.3.QRT-PCR was used to determine the mRNA level of miRNA-30b in myocardial cell H9c2 in the two groups.Results:1.In high glucose group,the early apoptosis rate of myocardial cells increased with the increase of high glucose treating time,while the rate in the control group had no significant difference.2.In high glucose group,the protein expression of Caspase-3 and PARP increased and expression of Bcl-2 decreased dramaticlly with the increase of high glucose treating time,while the expression in the control group had no significant difference.3.In high glucose group,the mRNA level of miRNA-30b increased markedly with the increase of high glucose treating time,while the level in the control group had no significant difference.Conclusions:High glucose could promote myocardial apoptosis by increasing the expression of cell apoptosis related factor and decreasing the expression of cell apoptosis inhibition factor,and increasing the expression of miRNA-30b.Part Ⅲ Inhibition of miRNA-30b expression decreased apoptosis of rat cardiomyocytesObjective:To explore the effect of miRNA-30b expression on rat myocardial cells apoptosis.Methods:1.Experimental myocardial cells were divided into 4 groups:Normal group,High glucose group,High glucose+miRNA-30b inhibitor group and High glucose+inhibitor NC group.The apoptotic rate of myocardial cells in each group was determined.2.Western blotting was used to determine the protein expression of cell apoptosis related factor Caspase-3 and PARP,and cell apoptosis inhibition factor Bcl-2 in the four groups.3.Results:1.The apoptotic rate of myocardial cells in high glucose group was significantly higher than the control group,while the rate in high glucose+miRNA-30b inhibitor group was markedly lower than the high glucose+inhibitor NC group.2.The expression of Caspase-3 and PARP in high glucose group was significantly higher than the control group,while the expression in high glucose+miRNA-30b inhibitor group was markedly lower than the high glucose+inhibitor NC group.The expression of Bcl-2 in high glucose group was observably lower than the control group,while the expression in high glucose+miRNA-30b inhibitor group was markedly higher than the high glucose+inhibitor NC group.Conclusions:High glucose could promote myocardial apoptosis by increasing the expression of cell apoptosis related factor and decreasing the expression of cell apoptosis inhibition factor.After transfection with miRNA-30b inhibitor to decrease the expression of miRNA-30b,the apoptotic rate was reduced.Part Ⅳ MiRNA-30b negatively regulates the expression of Bcl-2Objective:To explore whether Bcl-2 is the target gene of miRNA-30b.Methods:1.Use bioinformatics was used to predict the potential target genes of miRNA-30,and luciferase gene reporting technology was used to verify the target;2.QRT-PCR and western blotting were used to determine the effect of miRNA-30b mimic transfection on the mRNA and protein expression of Bcl-2.3.luciferase reporter technology were used to verify the relationship between miRNA-30b and target genesResults:1.Target Scan(http://www.targetscan.org/)predicted apoptosis inhibitor bcl-2 was the potential target gene of miRNA-30b.2.After transfection with miRNA-30b mimic.luciferase activity of Bcl-2 3’-UTR wt decreased.3.Transfection of miRNA-30b mimic decreased the mRNA level and protein expression of Bcl-2.Conclusions:Bcl-2 is the target gene of miRNA-30b,and miRNA-30b can play its role in DCM by negatively regulating the expression of Bcl-2.Comprehensive conclusion:In diabetic cardiomyopathy.the expression of miRNA-30b is up-regulated,which promotes the apoptosis of cardiomyocytes through the negative regulation of its target gene Bcl-2.