| Heart failure (HF) is the end-stage of various serious cardiovascular diseases. The five-year survival rate of patients diagnosed with HF is only50%. Therefore, HF is believed one of the major diseases that harm human health. Recent years domestic and foreign researchers generally agreed that HF was substantially a molecular-clinical syndrome, and the abnormal alternation and regulation of specific gene was closely related with the mechanism of HF. MicroRNA (miRNA) was a class of new found intrinsic non-coding small single-strand RNA molecule, whose function was mainly induction of gene silencing, thereby involving in post-transcript gene regulation. The expression pattern of specific miRNA depends largely on the patterns of tissue and cell type, conditions of meTab.olism and the states of diseases. Epigenetics data showed that miRNA was involved in multiple basic pathological processes of HF, such as cardiomyocyte apoptosis, myocardial fibrosis, myocardial hypertrophy and cardiac remodeling, etc. Due to the lack of standardized procedures and cases, and the high variation among cases, the study on miRNA of human failing heart was restricted to a great extent. Nevertheless, some researchers have compared the miRNA expression profile between human failing heart and experimental animal model heart, and found that most of the expression patterns of miRNAs showed relatively well consistency. Thus, simulation of different human HF using experimental animal HF model then performing miRNA expression profile analysis could provide valuable reference for the pathogenesis study of HF in human.The present study constructed post-MI HF model through the ligation of the anterior descending coronary artery in rats, and detected the miRNA expression profile between the model group and control group in left ventricular myocardial tissue within the scope of whole genome by solexa high-throughput sequencing, and analyzed the differentially expressed known miRNAs as well as predicting new miRNAs. Using RNAhybrid software we predicted the target genes of differentially expressed miRNAs, then GO annotation and KEGG pathway analysis based on the target genes was performed, which could help us to understand the mainly involved biological function and biochemical pathways and signal transduction pathways of the candidate target genes. To test the accuracy and reliability of sequencing, we performed the real-time quantitative PCR using the same sample as the sequencing. Additionally, several differentially expressed miRNAs (miR-31a-5p,199a-5p,122-5p and208a-3p) was tested in4weeks,8weeks and10weeks after operation, respectively, aiming to detect the expression patterns of different periods after myocardial infarction and understand their potential role in the development of heart failure. Finally, we preliminarily explored the effect of miR-31a-5p and miR-199a-5p on cardiomyocyte apoptosis induced by Angll through loss of function (with miRNA inhibitor) and gain of function (with miRNA mimics) for the two miRNAs.Results:1. We successfully induced rat myocardial infarction through thoracotomy anterior descending coronary artery ligation method. The echocardiography, cardiac hypertrophy index, the BNP levels and myocardial pathologic morphology analysis based on4weeks,8weeks and10weeks after operation showed that the cardiac function of model heart decreased significantly, and with time progression among the model hearts, the cardiac function declined progressively, the adverse cardiac remodeling gradually aggravated, and10weeks after MI operation has formed sTab.le heart failure.2. The miRNA expression profiles in left ventricular myocardial tissue between control group and HF group were obtained through high-throughput sequencing. There were225kinds of known miRNAs identified, of which107kinds of miRNAs whose expression level between the two groups have significant difference, i.e., compared with the control group,79kinds of miRNAs up-regulated significantly and28kinds of miRNAs down-regulated significantly; The fold change of above differentially expressed miRNAs higher than2between the two groups was found in18miRNAs, including16kinds of miRNAs significantly up-regulated in the heart failure group and2kinds of miRNAs down-regulated significantly in the heart failure group.3. Using RNAhybrid software to predict the potential target genes of the18differentially expressed miRNAs, we obtained a total of31401candidate target genes. GO and KEGG analysis based on these predicted target genes were performed, which suggested that compared with the reference genome, the candidate target genes mainly enriched in signaling pathways, cell communication, signal transduction, stress reaction, cell surface receptors, cell surface regulation and some other GO terms, and the significantly enriched36KEGG pathways were mainly involved in Wnt signaling pathways, VEGF signaling pathways, MAPK signal pathway, cell cycle, type II diabetes, Notch signal pathway, vascular smooth muscle contraction and other pathways associated with heart failure.4. Real-time quantitative PCR results were consistent with the sequencing results, which confirmed the accuracy of sequencing results.5. The detection of4differentially expressed miRNAs in different periods after myocardial infarction showed that the expression levels of specific miRNA in the development of heart failure after myocardial infarction was changed dynamically: the expression levels of miR-199a-5p and miR-31a-5p gradually increased with time progression after MI operation, which suggested that they might be involved in the pathogenesis of HF; There was not obvious change in the expression level of miR-122-5p at the early stage of post-MI, but its expression level was significantly up-regulated while forming sTab.le HF, which revealed that miR-122-5p might be the potential biomarker of HF; The expression of miR-208a-3p was up-regulated in the early stage of post-MI, however, its expression level gradually decreased with time going on, and its expression level was significantly lower than that of the control at10 weeks post-MI. These data suggested that specific miRNA possibly played a different role at different period of HF progression.6. Angiotensin II at a concentration of100nM could successfully induce cardiomyocyte apoptosis. The gain-of-function and loss-of-function experiment has found that miR-199a-5p can significantly promote AngⅡ-induced cardiomyocyte apoptosis, as well as induce cardiomyocyte apoptosis itself. On the other hand, miR-31a-5p could only slightly increase AngⅡ-induced cardiomyocyte apoptosis, and simply over-expresssion of miR-31a-5p yet has no obvious effect on cardiomyocyte apoptosis.Conclusion:1. The study has screened a total of107kinds of differentially expressed miRNAs, among which18miRNAs’expression levels changed more than2times between the control group and HF group. Our results have provided some evidence for the study of pathogenesis of heart failure.2. The dynamic expresseion patterns of miR-31a-5p,199a-5p,122-5p and208a-3p in different periods post-MI were detected, which suggested that the role of specific miRNA in different periods of HF might be different.3. MiR-199a-5p could induce cardiomoyocyte apoptosis, inhibition of miR-199a-5p may be the effective means to prevent cardiomoyocyte apoptosis induced by AngⅡ. |
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