长链非编码RNA MIPRL在急性心肌梗塞中的作用及分子机制

RP11-64B16.4, newly discovered in 2017, is a human-mouse homologous long non-coding RNA. Up to now, its role in acute myocardial infarction(AMI) has not been studied or reported. According to our preliminary results, we named it Myocardial Ischemic Protection Related LncRNA (MIPRL). The purpose of this project is to explore the role of MIPRL in AMI and its corresponding molecular mechanisms. Our central scientific hypothesis is that MIPRL up-regulates in acute myocardial ischemic and anoxic injury and PI3-kinase/Akt signaling pathway is involved in its up-regulation process. MIPRL inhibits the development of hypoxic-ischemic cardiomyocyte injury (apoptosis) with the aid of its downstream target gene heat shock protein B8 (HSPB8). . This scientific hypothesis is strongly supported by our previous experimental results. In the hypoxic model of human cardiomyocytes and primary mouse cardiomyocytes, we detected a significant elevation of MIPRL. And in AMI mouse, the MIPRL in the infarcted area was also significantly increased. By transfecting with recombinant adenovirus in vitro and in vivo to control the expression of MIPRL, we confirmed that MIPRL could alleviate hypoxia-induced myocardial apoptosis both in cultured cells and AMI mouse. Subsequently, we found that the inhibition of PI3-kinase/Akt pathway can down-regulate the expression of MIPRL. And up-regulating MIPRL can positively regulate HSPB8，which indicates that PI3-Kinase/Akt may be involved in the upstream regulation of MIPRL and MIPRL can ameliorate ischemia and hypoxia-induced myocardial injury by modulating its target gene HSPB8.. We will conduct further studies through the following four aspects: 1) To clarify the role of MIPRL in hypoxic-induced cardiomyocyte apoptosis in cultured human and mouse cardiomyocytes. 2) To determine the role of MIPRL in acute myocardial infarction in mouse AMI models. 3) To explore the molecular mechanisms of MIPRL in acute myocardial infarction in cultured cardiomyocytes and mouse AMI models. 4) To further confirm the role of MIPRL in AMI and its molecular mechanisms in vitro and in vivo by constructing MIPRL knock-out mice. The completion of this project may not only provide a new molecular mechanism for the pathogenesis of AMI，but also provide a new therapeutic target for the treatment of AMI.

RP11-64B16.4是2017年新发现的人与小鼠同源的长链非编码RNA，但其在急性心肌梗塞(AMI)中的作用未见报道。据初步研究结果,我们将其命名为心肌缺血保护相关LncRNA(MIPRL)。本项目研究目的：探讨MIPRL在AMI中的作用及分子机制。科学假说是：MIPRL在心肌缺血缺氧时显著上调，PI3-kinase/Akt信号转导途径参与其上调过程。MIPRL可通过其下游靶基因HSPB8发挥心肌保护(抗凋亡)作用。我们前期实验结果强力支持该假说。我们拟下述方案进一步探索：①在人及小鼠心肌细胞缺氧模型中明确MIPRL在缺氧诱导心肌细胞凋亡中的作用；②在小鼠AMI模型中明确MIPRL在心肌梗死中的作用；③在心肌细胞及小鼠AMI模型中探索MIPRL心肌保护作用的分子机制；④采用MIPRL基因敲除小鼠，进一步测定其在AMI中的作用及其分子机制。本项目有望为AMI提供新的分子机制及治疗靶点。

急性心肌梗塞（AMI）目前机制未明，探讨其发病的新分子机制仍是国际心血管界的研究热点。我们前期研究发现长链非编码RNA MIPRL（心肌缺血保护相关LncRNA）在AMI中高表达，详细机制不清。. 本课题采用野生型及MIPRL基因敲除鼠制备AMI模型，并通过心肌原位注射MIPRL过表达腺病毒回复心肌MIPRL表达。结果显示，MIPRL敲除可扩大心梗面积，加重心功能受损程度，加重心肌纤维化。回复MIPRL表达可逆转上述现象，证实MIPRL在AMI中发挥心肌保护作用。. 随后进行转录组学及功能富集分析，发现凋亡信号通路被显著富集，因此将MIPRL的调控机制锚定于细胞凋亡。在AMI动物及细胞实验中发现，与野生型小鼠相比，MIPRL基因敲除鼠心肌凋亡相关蛋白表达进一步升高，TUNEL阳性细胞数明显增多，回复心肌MIPRL表达可逆转细胞凋亡程度，提示MIPRL通过调控细胞凋亡发挥心肌保护作用。. MIPRL是反义链LncRNA，由热休克蛋白8（HSPB8）反向转录而成，推测HSPB8可能参与了MIPRL介导的急性心肌梗死后心脏的保护作用。为此，我们从体内、体外两个水平证实了MIPRL对HSPB mRNA及蛋白水平均存在同向调控作用。随后构建HSPB8敲减腺病毒，在小鼠AMI模型及心肌细胞缺氧模型中验证HSPB8对细胞凋亡的调控作用。最后，采用MIPRL过表达腺病毒及HSPB8敲减腺病毒联合调控，结果显示，在AMI动物及细胞缺氧模型中，当HSPB8表达受阻时，MIPRL无法有效改善心肌细胞凋亡程度，从而未能有效改善心功能、减少心肌纤维化程度。因此，证实心肌梗塞中MIPRL通过调控HSPB8，抑制细胞凋亡，发挥心肌保护作用。. 最后，采用RNA-Fish技术明确MIPRL定位于细胞胞浆，且在缺氧损伤时在胞浆浓聚。RNA稳定实验结果显示，随放线菌素D处理时间延长，HSPB8 mRNA呈不断下降趋势，而MIPRL基因敲减后HSPB8 mRNA衰减趋势明显增强，提示MIPRL增加了HSPB8 mRNA稳定性。. 本研究证实急性心肌梗死中MIPRL通过增加HSPB8 mRNA稳定性，上调HSPB8表达，从而减轻心肌细胞凋亡，发挥心肌细胞保护作用。这一结论是对急性心肌梗死致病机制的有效补充，为临床治疗急性心肌梗死提供新的靶点和理论。

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