心肌缺血再灌注损伤时长链非编码RNA(AK138945)-miRNA-内质网应激调控网络的机制研究

Myocardial ischemia-reperfusion injury (MIRI) is the most serious complication after myocardial reperfusion treatment in patients who suffered myocardial infarction. Long non-coding RNA (lncRNA), a new class of functional mRNA-like transcripts lacking significant open reading frames or protein-coding capacity, are emerging as an important layer in the gene regulatory network. Although lncRNA plays an important role in the process of heart disease, it still the beginning of the lncRNAs era in fundamental cardiovascular research, systematic studies on the roles of lncRNAs in cardiovascular disease are still lacking. Our previous study has shown that the expression of lncRNA-AK138945 is decreased significantly in the myocardial tissue (in vivo model) and primary cultured neonatal cardiomyocytes or hiPS-CM (in vitro model) suffered MIRI. Moreover silence of AK138945 could increase the expression of miR-1/miR-881 and reduce myocytes viability simultaneously. Further forecast by database has shown AK138945 can be regulated by its upstream transcription factors (TF) such as SP1, NFATC2, FOXO3 and SOX10. And AK138945 could also regulate miR-1/miR-881-induced endoplasmic reticulum (ER) stress. Therefore deficient of AK138945 is the key point in endoplasmic reticulum stress of MIRI. Based on these results and hypothesis, we will use variety of molecular biological techniques to carry out the following research: .(1) to determine a regulatory network of TF-AK138945-miR-1/miR-881-ER stress in MIRI..(2) to prevent and cure MIRI by targeting application of transgenic technology based on AK045950 at the phase of ischemic preconditioning, providing new ideas and new initiatives for the prevention of MIRI.

心肌缺血再灌注损伤（MIRI）是心肌梗死患者接受心肌血流复灌治疗后出现的最严重的并发症，长链非编码 RNA（lncRNA）在心脏疾病的进程中发挥着重要作用。本项目初步研究结果显示在小鼠MIRI模型和离体MIRI细胞模型中lncRNA-AK138945的表达水平显著降低，同时沉默AK138945后能够显著增加miR-1/miR-881的表达，并降低细胞活力。通过网络预测AK138945受上游 SP1等4 个转录因子的调控，同时AK138945也能调控其下游miR-1/miR-881诱导的内质网应激。因此AK138945表达缺失是MIRI时内质网应激的关键环节。本项目将应用分子生物学等手段阐明（1）MIRI时转录因子-AK138945- miR-1/miR-881-内质网应激的调控网络；（2）首次在缺血预适应阶段以AK138945为靶点进行基因预防和治疗，为MIRI的防治提供新思路和新措施。

心肌缺血再灌注损伤（MIRI）是心肌梗死患者接受心肌血流复灌治疗后出现的最严重的并发症，因此，发现调控MIRI的关键机制，寻找MIRI治疗的新策略十分必要。长链非编码RNA（lncRNA）在心脏疾病的进程中发挥着重要作用。在本项目的资助下，我们研究发现lncRNA-AK138945在小鼠MIRI模型和离体缺血再灌注损伤细胞中表达水平显著降低。在细胞水平，敲减lncRNA-AK138945后，小鼠原代心肌细胞活性降低，细胞凋亡增加。在分子水平，敲减lncRNA-AK138945后，促凋亡蛋白Caspase3、Caspase9以及Bad表达水平明显升高，抗凋亡蛋白Bcl2表达明显降低。通过生物信息学预测以及应用 Liuciferase assay检测，我们发现miR-1a-3p是lncRNA-AK138945的下游作用靶点。通过构建miR-1a-3p过表达的转基因小鼠，我们发现miR-1a-3p可以促进心肌缺血再灌注损伤。进一步机制研究显示GRP94是miR-1a-3p的直接作用靶点，抑制GRP94可以促进缺血再灌注损伤后的心肌细胞凋亡。Western blot和qRT-PCR实验检测结果显示，敲减lncRNA-AK138945显著抑制GRP94蛋白和mRNA水平。对lncRNA-AK138945调控缺血再灌注损伤的上游机制研究中，我们发现FOXO3与lncRNA-AK138945的启动子区具有多个结合位点。并且过表达FOXO3能够上调lncRNA-AK138945和GRP94的表达，抑制miR-1a-3p的表达。结论及科学意义：本研究首次发现心肌缺血再灌注损伤时lncRNA-AK138945表达降低，使得miR-1a-3p的表达升高，靶向抑制内质网应激相关蛋白GRP94的表达，诱发心肌细胞凋亡。本项目首次构建了转录因子FOXO3-lncRNA-AK138945-miR-1-GRP94内质网应激信号网络，为临床缺血再灌注损伤治疗提供新策略。