Primary Study Concerned On MiR-182 Regulating Diabetic Cardiomyopathy Via Targeting Rac1

Objective: To find the upper mechanism of Rac1 in regulating diabetic cardiomyopathy and the role of micro RNA in the development of diabetic cardiomyopathy. The ultimate purpose is to provide a novel clue for the insight into the pathogenesis and clinical treatment of diabetic cardiomyopat hy.Methods: Part 1: The candidates of Rac1 were predicted by applying bioinformatic, and then expression of all of mi RNAs candidates in hearts of diabetic mice were verifed by real time RT-PCR. Then the relationship between Rac1 and those candidate micro RN As in hearts of diabetic mice were investigated with Pearson relavant analysis, of which the negative strongest relationship with Rac1 was the object in this study.Part 2: Neonatal mice cardiomyocytes were cultured and randomly divided into 4 groups: normal glucose group, high glucose group, selected micro RN A mimics control group, high glucose plus selected micro RNA mimics control group. The level of selected mi RNA and Rac1 was detected by real-time RT PCR in cardiomyocyte exposed to high glucose condition. Furthermore, expression level of Rac1, β-MHC and α-SMA m RNA and protein were detected in cultured cardiomyocyte treated by high glucose for 48 hours after transfection of selected mi RN A mimics by real- time RT-PCR and Western blot.Part 3: Fourty 8-week-old C57 mice were randomly divided into 4 groups: normal control group(n = 5), selected micro RNA mimics control group(n = 5), type STZ diabetes group(n = 15) and type STZ diabetes treated by selected micro RNA mimics(n = 15). Type STZ diabetes mice were modeled by intraperitoneal injection of STZ. Eight weeks after modeling, the weight and blood glucose exchange of mice in each group were recorded and the cardiac function were evaluated by a high-resolution ultrasonic imaging system for small animals(Vevo 2100). The size of cardiomyocyte and the myocardial fibrosis were observed by HE staining and Masson staining respectively. The cardiac tissue of mice hearts were used to observe the ultrastructural changes by electron microscopy. Real-time RT-PCR was used to detect the expression levels o f β-MHC, α-MHC, ANP, Col I, Col III, selected micro RNA and Rac1 m RNA.Result: Part 1: A total of 6 mi RNAs candidates potentially targeting Rac1 were screened by bioinformatics,which were mi R-182、mi R-142-3p、mi R-140、mi R-101a、mi R-429、mi R-200 b. Among these candidates,mi R-182、mi R-142-3p downregulated remarkably in cardiac tissues in db /db mice compared with db/m controls(p<0.05). Mi R-182 showed the most significantly negative relevance with Rac1 by Pearson relevant analysis(r =-0.89102).Part 2: The downregulation of mi R-182 and upregulation of Rac1 were also validated in high glucose- induced cardiomyocyte. After transfecting of mi R-182 mimics, the morphology changes of high glucose-induced hypertrophic cardiomyocyte were released, and expression of Rac1 and β-MHC were significantly decreased in cardiomyocyte incubated with high glucose while the expression change of α-SMA level was not significant.Part 3: Mi R-182 mimics improved the cadiac function of type STZ diabetic mice with the increase of left ventric ular function and left ventricular short axis fractional shortening(p<0.05). Mi R-182 mimics significantly decreased the expression level of Rac1, ANP, Col I, Col III m RNA and the ratio of β/α-MHC in cardiac tissue(p<0.05). HE staining reveals that cardio myocytes in the normal control group and the mi R-182 control group arranged regularly with the size of nuclear being uniform, while in type STZ diabetic mice myocardial hypertrophy were significant and the nuclear was inregular and myofibril arranged irregularly. Masson staining reveals that cardiomyocytes were dark red and the collagen were dark blue; the collagen tissue in the normal control group and the mi R-182 control group arranged regularly and appropriately, while in type STZ diabetic mice the collagen tissue significantly increased and were arranged irregularly; in group mi R-182 mimics + type STZ diabetic mice, the collagen tissue were significantly decreased compared with group diabetic mice. Transmission electron microscopy revealed that cardiomyocytes in the normal control group and the mi R-182 control group arranged regularly, and the sarcomere and ligh dark band were clear, and the uniformly sized mitochondrial were arranged regularly around the cardiomyocyte with round or oval shape; but cardiomyocytes in group type STZ diabetic mice were significantaly changed with irregular arrangement of cardiomyocyte and swelling and accumulated inregular mitochondrial and increased lipid droplets, glycogenosome and autophagosome. Those changes were significantly released in group mi R-182 mimics + type STZ diabetic mice.Conclusion:(1) Mi R-182 was finally confirmed as the candidate micro-RNA targeting Rac1 after using bioinformatic method and animal model of diabetic cardiomyopathy and Pearson relationship analysis.(2) Mi R-182 might involve in the regulation of high glucose- induced cardiomyocyte hypertrophy via targeting Rac1.(3) Mi R-182 mimics downregulated Rac1 in cardiac tissue of diabetic mice, and improved the cardiac function of diabetic mice, and decreased cardiac hypertrophy and cardiac fibrosis, and released ultrastructural changes of cardiac tissue in diabetic mice.