Study On The Role Of MiR-27a In Mediating The Anti-apoptosis Effect Of GLP-1 Receptor Agonist On Cardiomyocytes

ObjectiveDiabetes(Diabetes mellitus, DM) is a metabolic disease characterized by increased chronic blood sugar levels which is made by a variety of factors including genetic and environmental factors. DM cardiovascular disease is one of the major complications of DM. Recent researches demonstrated that diabetic cardiomyopathy(DCM) is closely related to the high morbidity and high mortality of DM cardiovascular diseases. The myocardial cell apoptosis is one of the main pathogenesis of DCM. The liraglutide(LR) is a glucagon like-1(GLP-l) agonist. It can not only reduce blood gllucose level but also has the effect of cardiovascular system protection. Micro RNA(miRNA) is a kind of conservative endogenous non-coding single small RNA which plays an important role in the occurrence and development of cardiovascular disease(CVD). miR-27 a is one of miRNAs which has closely related to cell apoptosis. And our previous studies found that GLP-1 can inhibite the expression of miR-27 a myocardial cell by activating AMPK. Therefore this study is to investigate whether GLP-1 agonists LR can play the role of improving myocardial cell apoptosis by inhibiting the expression of miR-27 a, thus provides an theoretical basis for prevention and treatment of patients with DCM and reduces the mortality of DM cardiovascular disease.Methods1. H9c2 rat cardiomyocytes were cultured in normal sugar environment. Using the final concentration of 150 pmol and 300 pmol miR-27 a mimics and inhibitor transfect cells for 48 hours respectively, and set up the normal control group. Each group's miR-27 a expression level was detected by real time PCR, thus to detcte transfection efficiency.2. H9c2 rat cardiomyocytes were cultured in normal sugar environment. They were divided into 6 groups: the normal control group, lipo2000 group, low concentration of miR-27 a mimics group, high concentration of miR-27 a mimics group, low concentration of miR-27 a mimics N.C group, high concentration of miR-27 a mimics N.C group. The cell apoptosis was detected with TUNEL after transfecting 48 hours.3. H9c2 rat cardiomyocytes were cultured to divide into normal sugar group(LG group), high glucose group(HG group), HG+miR-27 a mimics group, HG+miR-27 a inhibitor group, HG+miR-27 a mimics N.C group, HG+miR-27 a inhibitor N.C group,HG+lipo2000 group, High osmosis group(HO group). The final transfection concentration is 300 pmol. The miR-27 a expression level was detected by real time PCR. The level of apoptosis-related protein Bax?Bcl-2?caspase-3?c IAP-1?Apaf-1 was detected by Western Blotting. The cell apoptosis was detected by TUNEL.4. H9c2 rat cardiomyocytes were cultured in high glucose environment. They were divided into 6 groups: the normal control group, Liraglutide group(LR group), miR-27 a mimics group, LR+miR-27 a mimics group, LR+miR-27 a mimics N.C group and lipo2000 group. The intervention concentration of liraglutide was 1000 nmol/L. The final transfection concentration is 300 pmol. The expression level of miR-27 a was detected by real time PCR and the cell apoptosis was detected by TUNEL after transfecting 48 hours.Results1. Compared with the control group, the expression level of the miR-27 a were elevated in low concentration and high concentration of miR-27 a mimics transfection groups(P<0.05). And the expression level of the miR-27 a in high concentration of miR-27 a mimics group was higher than low concentration of miR-27 a mimics group(P<0.05). When the transfection concentration of miR-27 a inhibitor was 150 pmol, miR-27 a expression was significantly inhibited(P<0.05).When the transfection concentration was 300 pmol,miR-27 a express was almost completely suppressed(P<0.05).2. Compared with the control group,the myocardial cell apoptosis index showed a concentration dependent increase(P<0.05). While there is no significant difference between control group, lipo2000 group, low concentration of miR-27 a mimics N.C group and high concentration of miR-27 a mimics N.C group(P>0.05).3. In the condition of high glucose environment, the expression level of miR-27 a and apoptosis index were significantly increased in miR-27 a mimics group(P<0.05). The expression of miR-27 a was significantly suppressed and the apoptosis index decreased significantly in miR-27 a inhibitor group(P<0.05). While lipo2000 group,mimics N.C group and inhibitor N.C group compared with the normal sugar control group did not cause the change of miR-27 a expression level and cell apoptosis index. And the contents of miR-27 a has no significant difference between HO group and normal sugar control group(P>0.05).4. Western Blotting experiment results showed that the expression level of Bax, caspase-3 and Apaf-1 protein in miR-27 a mimics group was increased,while it was reduced in miR-27 a inhibitor group,and the variance between these two groups had significant difference(P<0.05).At the same time, overexpression miR-27 a caused Bcl-2 and c IAP-1 protein levels decreased, while the expression levels increased in group that transfected with miR-27 a inhibitor, and the variance between these two groups had significant difference(P<0.05).5. Compared with control group, the miR-27 a expression level and myocardial apoptosis index decreased significantly in liraglutide group(P<0.05). And, the miR-27 a expression level and myocardial apoptosis index in miR-27 a mimics group and LR+miR-27 a mimics group were significantly higher than the control group(P<0.05). And the myocardial cell apoptosis index in LR+miR-27 a mimics group was lower than miR-27 a mimics group(P<0.05).Conclusion1. miR-27 a can promote the apoptosis of myocardial cells in a concentration dependent manner.2. In the condition of high glucose, miR-27 a can promote the apoptosis of myocardial cells by increasing the expression of Apaf-1?caspase-3 and Bax protein and inhibiting the expression of c IAP-1and Bcl-2 protein.3. GLP-1 agonists play the role of anti-myocardial cell apoptosis by inhibiting the expression of miR-27 a.