MiR-30c Participates In Diabetic Cardiomyopathy Via Regulation Of Autophagy

BackgroundDiabetes millitus (DM) has been a serious disease for peoples’lives. It is indicated that there are285million diabetes patients in the world, and the number of diabetes will almost doubles in2025. Cardiovascular complications are the leading cause of mortality in diabetic patients. Among them, diabetic cardiomyopathy exhibites abnormal cardiac structures and functions, which is independent of hypertension, heart valve abnormalities, coronary diseases and other heart diseases. Emerging researches have showed that lipotoxicity, glucotoxicity, impaired calcium homeostasis, altered metabolic substrates, oxidative stress, mitochondrial dysfunction, inflammation, and endoplasmic reticulum stress (ER stress) are all involved in pathogenic process of type2diabetic cardiomyopathy, but the exact molecular mechanism is still unclear. Recent studies have reported that microRNAs and autophagy are associated with insulin resistance in myocardium, which inspires us from a new perspective.MicroRNAs (miRNAs, miR) is a class of non-coding small RNAs widely existing in the organism, uaually22-23-nucleotide single-stranded. Through pair-binding with 3’-untranslated region (3’-UTR) of target mRNA, miRNAs regulate the expression of target genes in the post-transcription level. Many experiments have proved that miRNAs participate in serious biological processes; at the same time, dysfunctions of miRNAs are involed in a variety of diseases. Recent researches have shown that miRNAs, especially cardiac-abundant miRNAs, play important roles in the pathogenesis of many heart diseases. The researchers over-expressed these miRNAs in myocardial cells, and they became hypertrophy or volume was decreased, revealling the crucial roles of miRNAs in the pathological process of myocardial hypertrophy and ventricular remodeling. However, the role of miRNAs in diabetes and the cardiovascular complications is still unknown.Autophagy is a conserved procress which degradates intracellular components for reuse. By degradation of long-life proteins, impired proteins and damaged organelles, autophagy serves for cell survivals. Under basal circumstances, low level autophagy in the heart is a protect mechanism response to stress; however, excessive autophagy may lead to heart cell damages. Recent studies have pointed out that in the fructose-induced mice model, the activation of autophagy is associated with increased cardiac peroxidation products, fibrosis, and cell death, therefore participating in the formation of insulin resistance in the heart. It suggests autophagy has a key role in the pathogenic mechanism of diabetic cardiomyopathy. In addition, a recent report that miR-30a regulates autophagy in cancer cells attracts widely attentions, indicating miRNAs play an important role in the regulation of autophagy.MiR-30family is one of the cardiac high-expresed miRNAs. Earlier experiments showed that miR-30participated in the regulation of myocardial hypertrophy, ventricular remodeling, myocardial fibrosis and a series of cardiac dysfunctions. But the role of miR-30c in diabetic heart is unclear. The regululating relationship among MiR-30c, autophagy and diabetic cardiomyopathy inspires us to wonder:whether miR-30c is involved in the pathological process of type2diabetes? If this is the case, whether miR-30c participates in the pathogenic process of diabetic cardiomyopathy through regulation of autophagy? Focusing on these hypothesizes, we explore to find a novel therauputic way for diabetic cardiomyopathy from miRNAs.MethodsUse real-time PCR to test if there are differences in cardiac expression of miR-30c between type2diabetes db/db mice and normal controls C57BL/Ks mice.Western-blot was employ to detect in protein level that whether autophagy was differently regulated in myocardium of db/db mice compared with C57BL/Ks controls.H9c2cardiac cells were cultured in high concentrations of FFAs to test expression of miR-30c and autophagy in stimulation of FFAs.Use bioinformatic software to predict the potentcial targets of miR-30c. Then clone the3’-UTR of potential target mRNA to pMIR-Report vector, and verify the target gene of miR-30c by luciferase report assay, Western-blot was used to re-verify the target gene.Package miR-30c, anti-miR-30c and target mRNA respectively to cardiac-specific viral expression vector, recombinant adeno-associated virus vector9(rAAV9), and detect virus titer and transfection efficiency.Express the relative viruses above in myocardium of db/db mice by tail vein injection.Employ echocardiographic test and hemodynamic analysis to examine cardiac function of treated db/db mice.Western-blot and electron micrograghic analysis are used to test whether autophagy flux is regulated by differentially expressed miR-30c.Bioinformatic software is utilized to predict upstream regulator of miR-30c. Clone5’-promoter region of miR-30c to pGL3report vector, and testify the up-stream regulator by luciferase report assay. Re-verify the regulation by western-blot. Results1. Differential expression of miR-30c in type2diabetic myocardial tissue compared with controlThrough the real-time PCR detection, miR-30c expression in db/db mice cardium is confirmed to be decreased compared with normal controls.2. Enhanced Autophagy in myocardial tissue of type2diabetes.Beclin1and LC3-Ⅱ, autophagy associated proteins, are increased in db/db hearts compared with controls, suggesting autophagy flux is enhanced.3. Regulaltion of FFAs in vitroExpression of miR-30c was down-regulated while Beclin1and LC3-Ⅱ protein levels were increased in H9c2cultured in high concentration of FFAs compared with controls, suggesting FFAs stimulates autophagy in heart cells.4. Target gene of miR-30cBeclin1was predicted to be the most putative target gene of miR-30c by TARGETSCAN software. Luciferase report assay and western-blot comfired miR-30c could silenced expression of Beclin-1.5. Regulation of miR-30c in db/db heartEchocardiographic test and hemodynamic analysis showed overexpression of miR-30c resulted in alleviation of cardiac dysfunction in db/db mice, which could be encountered by co-overexpression of Beclin-1; while knockdown of miR-30c deteriorating the cardiac dysfunction. At the same time, western-blot and electron micrograghic analysis indicated that overexpression of miR-30c inhibited the beclin-1promoted autophagy in db/db hearts, but anti-miR-30c enhanced excessive autophagy. 6. Upstream regulator of miR-30cThrough bioinformatic prediction, NF-κB was identified as the putative regulator for5’-promoter region of miR-30c. Luciferase report assay and real-time PCR confirmed that NF-κB enhanced miR-30c expression by regulating the5’-promoter region of miR-30c.7. Repression of NF-κB in late stage of type2diabetesIn the heart of late stage of type2diabetic db/db, expression of NF-κB was reduced while its antagonist IκB-α was enhanced.ConclusionIn our study, cardiac tissues from type2diabetic model db/db mice and normal control C57BL/Ks were explored. Using molecular biology, cell biology and bioinformatics technology, the functions and mechanisms of miRNA-30c in diabetic heart were investigated in vivo and in vitro. We found that:1. Expression of miR-30c is decreased while autophagy is enhanced in the heart of db/db mice or FFAs treated H9c2cells;2. miR-30c can inhibit autophagy by repression of Beclin-1, therefore serves as a protective regulator in diabetic heart;3. NF-κB positively regulates expression of miR-30c, and down-regulated NF-κB contributes to loss of miR-30c in late stage of type2diabetic db/db mice.Conclusively, in type2diabetes, loss of miR-30c contributes to the process of diabetic cardiomyopathy through enhancement of Beclin-1promoted autophagy. This result may provide a novel therapeutic way for diabetic heart from a new point of view.