| Diabetes mellitus has reached an epidemic level worldwide, with a prevalence of 4% in 1995 and an anticipated prevalence of 5.4% in 2025, corresponding to 365 million people suffered from diabetes in 2011 and this number is expected to rise up to 552 million by 2030. Diabetic cardiomyopathy is responsible for higher incidence of sudden cardiac death and represents the leading cause of morbidity and mortality in the diabetic population. Diabetic cardiomyopathy is attributed to myocyte hypertrophy and the changes in the composition of the extrtacellular matrix with enhanced cardiac fibrosis. This serious and chronic complication has an asymptomatic onset and is characterized by impaired contractility and relaxation of the left ventricle independent of coronary artery disease or hypertension. Despite intensive investigations, the molecular mechanisms underlying diabetic cardiomyopathy remain poorly understood. Micro RNAs(mi RNAs, mi Rs) are an evolutionarily conserved class of small noncoding RNAs of 18-25 nucleotides in length, that act as post-transcriptional regulators of gene expression by binding to the 3’-untranslated region(3’-UTR), finally induce m RNA degradation and/or translational repression in diverse biological processes.Methods: 1. Rat Diabetic cardiomyopathy model establishment The 30 healthy SD rats(8 weeks old) were randomly divided into two groups: the DCM group(n = 15) and the control group(n = 15). The DCM group were treated by an intraperitoneal injection of streptozotocin(streptozotocin, STZ, 60 mg / kg) while the control group were treated by the injection of the corresponding volume of citrate buffer, then feed with conventional diet for 8 weeks. Measure the Glucose levels to confirm successful modeling of 1-DM and observe the myocardial pathological changes to confirm successful modeling of DCM. The 40 healthy C57BL/6 rats(6 weeks old) were randomly divided into two groups: The DCM group(n = 20) and the control group(n = 20). The DCM group were treated by an intraperitoneal injection of streptozotocin(streptozotocin, STZ, 150 mg / kg) while the control group were treated by the injection of thecorresponding volume of citrate buffer, then also feed with conventional diet for 8 weeks. Measure the Glucose levels to confirm successful modeling of 1-DM and observe the myocardial pathological changes to confirm successful modeling of DCM. 2. Neonatal rat cardiomyocytes culture and high glucose treatment Neonatal rat cardiomyocytes were isolated and cultured from the ventricles of 2-day- old Sprague-Dawley rats. In brief, the ventricles were excised, washed and cut into 1mm3 small pieces in D-Hanks’ balanced salt solution, then digested with 1% collagenase(Sigma, collagenase typeⅠ) in a carbon dioxide incubator to keep the reaction temperature at 37 °C. After dissociation, the cells were subjected to centrifugation, followed by differential preplating to enrich cardiomyocytes. And then, cells were plated in DMEM with 20% fetal bovine serum at a density of 3 to 5×105 cells /ml. Having been cultured for 2 hours(h), the medium was replaced with a new DMEM with 20% fetal bovine serum. Having been cultured for 48 hours(h), the medium was replaced with a serum free maintenance medium and incubated for another 12 h, then following the treatment with high glucose medium(25m M) for another 24 h. 3. Detection of gene-expression level Genome Microarrays:The TRIzol agent was used to extract the total RNA of tissues and cells. The sample RNA was marked after quality Inspection and Agilent mi RNAs microarray system was used to detect the abnormal mi RNA expression. q RT-PCR: The RNA extracted from cardiomyocytes was subject to reverse transcription with special stem-loop primer for mi RNAs. The oligod T and Random primers were used by the coding genes to synthesize the c DNA. Samples were normalized by evaluating GAPDH expression and mi RNA by RNU6 b. 4. Overexpression of mi R-144 or down regulate mi R-144 Mi RNA-144 mimic was a short double-stranded RNAs, in which one single-strand RNA identical to mature mi RNAs while another single-strand antisense RNA against mi RNAs with 2’-O-Me modification. Mi R-144 antagomir was a single-strand RNA which is made as the antisence complementary sequences to mature mi RNA-144 with 2’-O-Me modification Use mimic(25 p M) and inhabitor(25 p M) to up-regulate or down-regulate the mi R-144 in cardiomyocytes. 5. The role of mi R-144 in the high glycose-reducing cardiac myocyte apoptosisSeed the neonatal rat cardiomyocytes to the 12-wells plate. High glucose medium was used as a treatment accompanying with the up-regulation or down-regulation of mi R-144. Reactive oxygen species within the fluorescent probe was used to detect the ROS(Reactive oxygen species, ROS) alteration and the flow cytometry was used to detect the cardiac myocyte apoptosis. 6. Screening and confirmation the target gene of mi R-144 Bioinformatics: Several mi RNAs target prediction algorithms were employed, including Target Scan, Bibiserve, Pictar that incorporates sequence complementarity and m RNA target site accessibility. Luciferase Assays: Portions of the 3′ UTR of several potential targets were cloned into the 3′ UTR of luciferase in PGL3 plasmid, which were transfected into 293 T cells with p Super vector and mi RNAs, and the ability of mi RNAs to affect luciferase expression was determined in cardiomyocytes. Western blot and q RT-PCR: Cardiomyocytes were transfected with mi RNAs mimic/inhibitor which can overexpress or inhibit mi RNAs,and the protein of target genes was detected to analyze the effects of mi RNAs on potential target genes with Western blots, and the amount of target genes were normalized to the amount of endogenous control(GAPDH). 7. The effect on DCM caused by mi R-144 The 100 healthy C57BL/6 rats(6 weeks old) were randomly divided into four groups: The control group(n = 25), the mi R-144 group(n = 25), the DCM group(n = 25) and the DCM/mi R-144 group(n = 25). The control group were treated by the injection of the corresponding volume of citrate buffer and the DCM group were treated by an intraperitoneal injection of streptozotocin(streptozotocin, STZ, 150 mg / kg). The mi R-144 group was treated by the injection of mi R-144 antagomir(20 mg/kg) every 2 weeks on the basis of the control one while the DCM/mi R-144 group was also given an extra mi R-144 antagomir(20 mg/kg)injection compared to the DCM group. Then feed with conventional diet for 8 weeks. After confirming the successful modeling of 1-DM and DCM. Different detection means such as HE staining, Masson staining, TUNEL and Elisa were used to detect both the alteration of the intracellular environment and the tissue pathological changes. And the western blot and q RT-PCR were also used to measure the bio-markers.Results: 1. It is showed by the genome microarrays that there are disorders happened in theDCM mice serums and cardiac tissues. As the most significant sign, the expression of mi R-29 b was 37 times higher while the expression of other mi RNA in the serum also surged a lot than the control group. While in the cardiac tissues, the expression of mi R-144 nearly got a 75 percent down-regulation than the control group. QRT-PCR results showed that mi R-144 expression in mice cardiac tissue decreased significantly almost 60% to the control group, the expression in myocardial cells with a high glucose-treatment, also decreased to 40%. 2. After a high glycose treatment, there are more ROS produced in the interacellular environment and the m RNA of the atrial natriuretic factor could be increased. Been transfected with mi R-144 inhibitor can reduce the high glucose-induced oxidative stress and cardiac myocyte apoptosis and reduce high glucose-induced cardiomyocyte hypertrophy and inhibit the expression of ANF. It is be predicted by some bioinformatic issues that the mi R-144 binding sites which located in the 3’UTR areas exist in many genes. We have found in the luciferase assays that the overexpression of mi R-144 can significantly inhibited luciferase activity with Nrf2 gene 3’UTR sequence without affecting the target containing the mutant luciferase activity. It has been found by the western-blot that the protein of Nrf2 was down-regulated after the overexpression of mi R-144 in cardiomyocytes. 3. After a successful DCM model establishment by given a STZ treatment, we also gave a mi R-144 antagomir injection through tail vein to down-regulate the expression of mi R-144. Eight weeks later, it was found in the DCM group there is an obvious hypertrophy and high ROS level observed by the HE staining, the expression of the Nrf2 protein in DCM group was down-regulated while the m RNA expression of cardiac ANF, β-myosin heavy chain(β-myosin heavy chain, β-MHC), cardiac fibrosis-related genes transforming growth factor β(Transforming growth factor β, TGF-β) and collagen α1 chain(Col1A1) all up-regulated significantly; the MDA(malondialdehyde, MDA) level; the caspase-3 splice variant level, the number of apoptotic cells in cardiac tissue observed by TUNEL staining and the relative weight of the left ventricle all significantly increased. The cardiac function related indicators such as fractional shortening(Fractional shortening, FS), ejection fraction(Ejection fraction, EF), inotropic index +d F / dt max and-d F / dt min all decreased significantly. But when the group was given by a mi R-144 antagomir, all effect could be released and the mi R-144 antagomir could reverse all corresponding regulation. Conclusion:Inhibiting mi RNA-144 abated oxidative stress, reduced apoptosis, and improved cardiac function in STZ-induced diabetic mice, possibly via enhancing Nrf2 expression. The mi RNA-144 could thus be a novel target for treatment of type 1diabetic cardiomyopathy. |
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