Hub Targets Analysis of miRNA-mRNA-TF Network in Diabetic Cardiomyopathy

Background : The pathogenic mechanism and development of the diabetic 17 cardiomyopathy(DCM) has been generally explained, and it is clear that the 18 microRNAs(miRNAs), mRNAs and transcription factors(TFs) participate in the 19 process of the DCM disease. Yet, the hub targets of the disease progression are not 20 clear. Methods : To figure out the problem, we downloaded data sets from the Gene 21 Expression Omnibus(GEO) database (GSE44179 and GSE4745). The targeted 22 mRNAs of miRNAs were downloaded from TargetScan, miRBD and microT-CDS 23 database. Gene Ontology (GO) enrichment of miRNAs and mRNAs were analysed in 24 DAVID.R studio software was used to visualize the results of screened targets and 25 GO enrichment. Cytoscape software was used to visualize the miRNA-mRNA-TF 26 interaction network and calculate the hub targets. Results : We filtered eight miRNAs, 27 nine mRNAs and ten transcription factors(TFs) by bioinformatics analysis, and 28 constructed a miRNA-mRNA-TF network . The top ten degrees of nodes in the 29 network are rno-miR-7a, Hnf4a, rno-miR-17, rno-miR-21, rno-miR-122, rno-miR- 200c, Med1, Mlxipl, SP1 and rno-miR-34a, which were closely related to the process 31 of DCM. Conclusion : This study revealed that rno-miR-7a, Hnf4a, rno-miR-17and 32 rno-miR-21 may play vital role in the progress of diabetic cardiomyopathy.


Introduction
Diabetic cardiomyopathy(DCM) is a specific form of heart disease, induced by insulin 36 resistance in heart tissue, hyperinsulinemia and hyperglycaemia, which are 37 independent of other cardiac risk factors, including coronary artery disease and 38 hypertension. These metabolic disturbances promote cardiac remodeling, fibrotic 39 diastolic dysfunction and decreased ejection fraction in the DCM patients [1] .The 40 pathophysiology changes of diabetic cardiomyopathy were well explained, which 41 contained cardiac hypertrophy, fibrosis and cardiac functional changes such as 42 systolic and diastolic dysfunction [2] .Recent evidences indicated that several 43 microRNAs (miRNA) played critical role in the pathogenesis of DCM, and 44 contributed to regulating genes related to cardiomyocyte hypertrophy, oxidative 45 stress, cardiac fibrosis and apoptosis [3] .Certain mRNAs and TFs were proved 46 contribute to the pathology of DCM, as well [4] .Whereas, the regulation relationship 47 norvegicus models. The mRNA expression profile data were a part of GSE4745, 60 which performed on Plat-form GPL85 and contained ventricle myocardium tissue 61 from four healthy and four diabetic cardiomyopathy rattus norvegicus models after 42 62 days feeding. We got the differentially expressed miRNAs and mRNAs on GEO by 63 analysis with GEO2R.We filtered the specific expression sequences by volcano plot 64 and heat map of miRNAs and mRNAs in R studio (version 1.4.1717).The threshold 65 value of log FC is >1.5 or <-1.5 and p<0.05.  The data sets of mRNA and miRNA which we obtained in GEO    The specific expressed mRNAs of DCM. 134 In volcano map, the cutoff value is ±1.5. We screened upregulated and downregulated   148 We obtained the targeted mRNAs of miRNAs in Targetscan, miRBD and microT-CDS 149 databases on line. We took intersection of these three sets and miRNAs we screened   171 We analysed GO enrichment of the eight mRNAs we identified previously in DAVID DNA-templated. In addition, the mRNA COL1A1 was related to every BP pathway.

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The CC pathway, intracellular organelle, was related to all these eight mRNAs we 180 screened. In MF pathways, the most involved pathway was protein binding.

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Furthermore, the mRNAs NR4A3 and SP1 were involved in every MF pathway we The top one expressed sequence was rno-miR-7a.MicroRNA miR-7,an evolutionarily 239 ancient miRNA, played a crucial role in disease process of heart, brain, endocrine 240 pancreas, skin and cancer, in human and mice [9] .Several researches demonstrated that 241 miR-7,the most sensitive regulator, was over expressed in both myocardial infarction 242 and stage heart failure mouse models, which implies that it involved in pathology of 243 myocardial infarction and stage heart failure [10] .Evidence demonstrated that miR-7 244 was downregulated in end-stage dilated cardiomyopathy and they also showed that 245 over expression of miR-7 reduced expression of ERBB2,which was critical for 246 prevention of dilated cardiomyopathy [11] .Moreover,miR-7 also showed highly 247 conserved expression in insulin-producing cells of the animal kingdom. In mouse β-248 cells, miR-7 inhibited glucose-stimulated insulin secretion [12] . compared to wild-type mice models [13] .Moreover,miR-17 promoted cardiomyocyte 256 hypertrophy, proliferation, and survival. Evidence proved that miR-17 contributes to exercise-induced cardiac growth and prevented ventricular remodeling, including 258 attenuating cardiac apoptosis, decreasing fibrosis, and preserving cardiac 259 function [14] .Expression of miR-17 also suppressed mouse cardiac senescence [15] .

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In mammal organ systems, miR-21 is universally expressed, such as the heart.

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Cardiac hypertrophy is a common pathological response to cardiovascular diseases, 263 including endocrine disorder, hypertension, ischemic heart disease and vascular 264 disease. It proved that miR-21 was significantly upregulated in hypertrophic animal 265 hearts. Cardiac fibrosis is a pathological feature of cardiac hypertrophy and heart 266 failure,while,miR-21 was proved that significantly upregulated in cardiac 267 fibroblasts [16] .There was also strong evidence for the role of miR-21 in cardiac 268 fibrosis [17] .Transfection of primary cardiac fibroblasts with a synthetic miR-21 269 precursor gave rise to an increase in fibroblast growth factor 2 (FGF2) secretion.

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Concretely speaking, genes of encoding collagens and extracellular matrix molecules 271 were highly upregulated in cardiac fibrosis, but they were reduced after specific 272 inhibition of miR-21 [18] . was regulated by transcription factors HNF1A [21] .MicroRNA miR-122 was the most 286 abundant liver miRNA with exquisite tissue specificity, and it was vital in the maintenance of lipid and glucose homeostasis [22] .Evidence proved that miR-122 signaling pathway [24] .

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In a DCM rat model, the expression of miR-34a was upregulated.While,miR-34a 302 mimic induced H9c2 cell apoptosis in HG condition [25] . Evidence also demonstrated 303 that miR-34a reduced type I collagen production, cell viability, and migration and 304 increased apoptosis of CFs by targeting Pin-1 signaling, so that it could attenuate 305 myocardial fibrosis [26] .