Regulation Of Nucleolin On MicroRNAs And Its Role In Myocardial Protection

Aims:To investigate the differentially expressed miRNAs between nucleolin transgenic mice and wild mice. To observe the role of nucleolin and miRNAs regulated by nucleolin in injured myocardium and test whether the myocardial protective role of nucleolin was related to microRNAs which regulated by nucleolin. To explore the potential mechanism by which nucleolin regulates miRNAs.Methods:Differentially expressed miRNAs between nucleolin transgenic mice and wild mice was investigated by microRNAs microarray and verified by qRT-PCR (real time-reverse transcription-polymerase chain reaction). The mice models of doxorubicin-induced myocardial injury and myocardial ischemia-reperfusion injury were applied. The expression of nucleolin and miRNAs which regulated by nucleolin, were determined by Western-blot and qRT-PCR. The roles of nucleolin and miRNAs regulated by nucleolin were examined by "gain-and loss of-function experiment" in myocardial cells treated with DOX/H2O2. The target mRNA of important miRNAs were predicted by bioinformatics software and the expression of target mRNAs was examined by Western-blot in myocardial cells. The miRNAs or protein, which may interact with nucleolin, were tested by RNA-immunoprecipitation and protein-immunoprecipitation, and co-localization of nucleolin and its interact protein was examined by confocal microscopy, aiming at revealing the mechanisms by which nucleolin regulates miRNA biogenesis.Results:(1) nucleolin could affect the biogenesis of many kinds of miRNAs as shown by miRNA microarray. Among them, sixteen miRNAs including miR-21were up-regulated and eight miRNAs including miR-1were down-regulated in nucleolin transgenic mice. Differential expression miRNAs in nucleolin transgenic mice and wild mice were verified by qRT-PCR, and miR-21, miR-320were found up-regulated and miR-133, miR-378were down-regulated in nucleolin transgenic mice.(2) The DOX-treated acute/chronic myocardial injury model was successfully constructed in mice. The expression of miR-21and nucleolin increased in the myocardium and rat primary myocardial cells treated with DOX. The cardiac functions were markedly depressed in the mice treated with DOX. Cell viability decreased, cell apoptosis and the release of LDH increased in DOX treated rat primary myocardial cells; Over-expression of nucleolin attenuated DOX-induced cell apoptosis and the release of LDH, whereas knock down of nucleolin increased DOX-induced cell apoptosis and the release of LDH. Similarly, over-expression of miR-21attenuated DOX-induced cell apoptosis and release of LDH, whereas knock down of miR-21increased DOX-induced cell apoptosis and release of LDH. The protective roles of nucleolin over-expressing was neutralized by miR-21inhibitor and the harmful roles of siRNA-nucleolin was compensated by miR-21mimic. BTG-2was predicted to be a target mRNA of miR-21by bioinformatics software and the expression of BTG2was down-regulated in DOX-treated myocardial cells.(3) A myocardial ischemia-reperfusion (I/R) injury model was successfully constructed in mice. The expression of miR-21increased both in I/R injury myocardium and H2O2-treated rat primary myocardial cells. The expression of nucleolin decreased in I/R injury myocardium and H2O2-treated rat primary myocardial cells, but the expression of phosphorylated nucleolin increased in I/R injury myocardium. The cell viability decreased, cell apoptosis and LDH release increased in H2O2-treated rat primary myocardial cells; Over-expression of nucleolin attenuated H2O2-induced cell apoptosis and LDH release, whereas knock down of nucleolin by siRNA increased H2O2-induced apoptosis and the release of LDH. Similarly, over-expression of miR-21 attenuated H2O2-treated H9c2cell apoptosis and release of LDH, whereas knock down miR-21increased H2O2-induced apoptosis and release of LHD. The protective roles of nucleolin over-expression was neutralized by miR-21inhibitor and the harmful effect of nucleolin down-regulation was compensated by miR-21mimic. Nucleolin not only interacted with AGO2, but also interacted with miR-21by protein-immunoprecipitation and RNA-immunoprecipitation. Enhanced interaction was examined between phosphorylated nucleolin and AG02by confocal microscopy in the condition of oxidative stress.Conclusions:Nucleolin could regulate biogenensis of miR-21.The expression of MiR-21increased in acute/chronic DOX-induced myocardial injury and myocardial I/R injury. Nucleolin increased the cell viability, and decreased cell apoptosis and the release of LDH in oxidative stressed myocardial cells through up-regulating of miR-21expression. Phosphorylated-nucleolin may up-regulate miR-21generation by interacting with AG02.