| MicroRNAs(miRNAs) are a set of RNA molecules of18-23nt.They were processed from short stem-loop precursors that can be encoded in genomes of plants, animals and viruses. According to the current understanding, microRNA is firstly transcribed as long primary microRNA, which processed into60-70nt miRNA precursor(pre-miRNA) by nuclear III Drosha.Then the pre-miRNA is transported from nucleus to cytoplasm by Exportin-5and further cleaved into-22nt duplexes. miRNAs were first observed in C.elegans,and now they are known widespread in nature. Thus far, over3,500microRNAs have been identified, with greater than450founed in humans (MiRBase).The sequence so many miRNAs are conserved between distantly related organisms, suggesting that these molecules participate in essential processes. miRNA combines with several proteins, such as Argonaute, to form miRISC(miRNA induced silencing complex). Under the direction of miRNA, miRISC interacts with the target mRNA in an imperfect base pairing manner, which determines that a single miRNA can complement with numerous target mRNAs and regulate gene expression in the post-transcriptional level by pairing with30-untranslated regions (3’-UTRs) of the mRNAs. A computational analysis indicated that more than one-third of protein-encoding genes are regulated by miRNAs. They are involved in the regulation of avariety of biological processes including developmental timing, signaltransduction, apoptosis, cell proliferation and tumorigenesis. In one word, miRNAs play an important role in both physiological and pathological process.Researchs had shown that miRNA may be closely related with diseases occurred. miRNA expression and those regulation effects are different in the different types of diseases process. It has been demonstrated that miRNA are closely related to the development of cardiac hypertrophy, arrhythmia and other cardiovascular diseases. An animal study had shown that overexpression of miRNA-1in the ischemic myocardial tissue of the myocardial infarction rat model could inhibit cardiac connexin43(Cx43) and inward rectifier potassium channel2.1(Kir2.1) expression and triggered ventricular arrhythmia.Viral myocarditis (VMC), known as one of the most common cardiovascular diseases, is a serious threat to people’s lives and health, and causes a high rate of sudden unexpected death especially among young people. Problem is that some atypical death cases caused by VMC have no clear clinical symptoms and histopathological manifestation as well as comprehensive examination records, and thus an objective and detective indicator is necessary and demanding. we make the VMC models and through the production of primary cardiac cell culture, we practice the following four aspects studies in this study.Part I:Viral myocarditis Animal Model Set up[Objective] To replicate animal models of Viral myocarditis.[Methods] Sixty Balb/c mice were randomized into2groups they were groups of day7(n=30), day15(n=30). Ten mice selected randomly from each group were injected ip with Eagle’s minimal essential medium (EMEM) solution, and served as controls; the remaining of them were inoculated iP with diluted coxsackievirus B3(CVB3) EMEM solution to establish animal model of Viral myocarditis. Samples of heart tissue were processed for histological and morphological examination.[Results] Degeneration and necrosis of cardiomyocyte, inflammatory infiltration, collapse of cardlac muscle fibers and little fibrosis loeated around necrosis were observed in hearts of CVB3-infected mice on day7; less inflammatory infiltration and manifest interstitial fibrosis were seen in infected mice on day15.[Conclusion] Viral myocarditis animal models were sucessfully established with CVB3infection.Part II:Analysis Of MicroRNA-1Differential Expression Pattern In The Models Of VMC.[Objective] To investigate the expression of miRNA-1in the animal models of VMC and its putative regulate target.[Methods] In the research of the differential expression of miRNA in VMC tissuce,20were inoculated iP with diluted coxsackievirus B3(CVB3) EMEM solution were assigned into VMC group,10male mice were served as control group. The research of miRNA-1expression in VMC by fluorescent quantitative real time polymerase chain reation (RT-PCR). By using the soft of Targetscan, miRNAbase, we predicted the putative target.[Results] Using qRT-PCR to detect miRNA-1expression, we found the level of miRNA-1significantly increased higher in the VMC group. By using the Targetscan, miRNAbase, we considered Cx43as a putative target of miR-1.[Conclusion] We found the level of miRNA-1significantly increased200%fold higher in the animal models of VMC. And we verifed that the3’-UTR of Cx43contain nucleotides that are complementary to the5’end of miR-1, miR-1may be the putative target of Cx43.Part III:Analysis Of The Expression Of Cx43In The Models Of VMC[Objective] To investigate the expression of Cx43in the mice models of VMC and its putative regulate target.[Methods] In the research of the expression of Cx43in VMC group mice, Cx43was measured by Real-time PCR、Western blot and immunohistochemistry in myocardial tissue.[Results] Cx43protein expression was significantly reduced in VMC mice while there was no significant difference in the its mRNA levels in the study groups.[Conclusion] We found Cx43protein expression was significantly reduced in VMC mice. An inverse correlation of expression between miR-1and Cx43protein in VMC samples.Part IV:The Regulation Of Cx43By miR-1In Myocardial Cells[Objective] The regulation of Cx43by miR-1in myocardial cells.[Methods] To increase or decrese the miRNA level in normal myocardial cells, miRNA-1、inhibitor AMO-miR-1were transfected with lentivirus, respectively. Then we detected the level of protein Cx43.[Results] Overexpression of miR-1in myocardial cells was accompanied by a selective decrease in expression of the protein Cx43, but without changing its transcript level. miR-1suppresses Cx43expression at the post-transcriptional level.[Conclusion] miR-1suppresses Cx43expression at the post-transcriptional level. |
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