钙敏感受体介导的中性粒细胞外泌体LncRNA ENSRNOT00000039868在急性心肌梗死中的作用和机制研究

Neutrophil (PMN) is involved in the occurrence and development of acute myocardial infarction (AMI), however, the mechanism remains unclear. Our team has discovered that calcium-sensing receptor (CaSR) in the peripheral blood lymphocytes were involved in AMI, but whether the CaSR in PMN to participate in is not reported. In our pre-experiments, we have found that the activation CaSR in peripheral blood PMN promoted hypoxia/reoxygenation cardiomyocytes apoptosis, at the same time, the expression of LncRNA ENSRNOT00000039868 in exosomes from neutrophils increased; in addition, we detected that the expression of CaSR in peripheral blood PMNs decreased. So, we hypothesized that "CaSR in neutrophils may be involved in the development and progression of AMI by modulating LncRNA in exosomes". In order to confirm the hypothesis, we will use techniques such as gene transfection to verify the target gene of this LncRNA and related signaling transduction pathway; to confirm the role of PMN-derived exosomes and this LncRNA in AMI by remodeling rat ischemia-reperfusion model; to observe the pathway proteins and function indicators of cardiomyocytes by co-culturing neonatal rat cardiomyocytes under hypoxia-reoxygenation condition with exosomes secreted by PMN silenced or over-expressed with CaSR or this LncRNA. It will clarify the relationship between AMI and CaSR in PMN and LncRNA in exosomes, and find a new target for the prevention and treatment of AMI.

中性粒细胞(PMN)参与急性心肌梗死(AMI)的发生发展，机制尚不明确，本课题组发现淋巴细胞钙敏感受体(CaSR)也参与AMI的发生，而PMN的CaSR是否参与尚无报道。我们预实验发现PMN的CaSR活化促进缺氧复氧心肌细胞的凋亡，同时使PMN外泌体中LncRNAENSRNOT00000039868的含量增加；另检测到AMI大鼠外周血PMN的CaSR表达降低。由此推测“中性粒细胞CaSR通过调节外泌体LncRNA影响AMI的发生发展”。为证实该假说，本课题拟采用基因转染等技术验证此LncRNA的靶基因及其相关信号传导通路；利用大鼠心肌缺血再灌注模型，明确PMN外泌体及此LncRNA的作用；将缺氧复氧心肌细胞与沉默或过表达CaSR和此LncRNA的中性粒细胞分泌的外泌体共培养，检测通路蛋白及心肌细胞功能指标，从而阐明AMI与PMN中CaSR及外泌体LncRNA的关系，寻找AMI防治新靶点。

中性粒细胞(PMN)及其分泌物在急性心肌梗死(AMI)发生发展中的作用及其机制尚不明确。本课题在确定中性粒细胞上钙敏感受体（CaSR）对细胞因子的分泌和生物活性有调节作用的基础上，采用基因转染等技术首先从细胞层面验证了CaSR活化的中性粒细胞外泌体含有丰富的lncRNA ENSRNOT39868，可进入心肌细胞并显著降低缺氧复氧心肌细胞的凋亡及抑制ROS的产生；沉默lncRNA ENSRNOT39868可降低其下游靶蛋白血小板衍生生长因子（PDGFD）的表达并增强心肌细胞缺氧复氧损伤，此作用与AKT信号通路有关。利用动物模型进一步证实，急性心肌梗死及缺血再灌注大鼠外周血中性粒细胞上CaSR的表达量增多；CaSR活化的中性粒细胞分泌的外泌体，可减少缺血再灌注损伤心肌的梗死面积。进而，我们用核质分离技术对lncRNA ENSRNOT00000039868的细胞质中定位进行了检测，并通过miRDB和Targetscan等数据库查询到和验证了与lncRNA ENSRNOT00000039868及其靶蛋白竞争性结合的miRNA-207，证实miR-207具有miRNA反应元件（miRNA response element, MRE），lncRNA ENSRNOT00000039868可作为其竞争性内源RNA（competitive endogenous RNA, ceRNA），与miRNA-207的表达呈负相关；另外，还观察到miRNA-207 inhibitor转染的心肌细胞中促凋亡蛋白Bax含量减低，抑制凋亡蛋白BCL2含量升高，PDGFD mRNA和蛋白含量均升高，心肌细胞凋亡率减少；将心肌细胞转染miRNA-207 mimics后，获得相反结果。因此我们证实了CaSR可以影响外周血中性粒细胞源性外泌体中lncRNA ENSRNOT00000039868的含量，其进入心肌细胞胞质后，作为ceRNA与miRNA-207竞争性结合，进而降低心肌中PDGFD的表达，减轻心肌梗死及缺血再灌注损伤，这一作用与AKT通道有关。以上结果阐明了AMI与PMN中CaSR及外泌体LncRNA的关系及其作用机制，对AMI的防治有一定的指导意义。