NF-κB/GAS5/miR-26a/ROCK1信号轴参与调控心肌缺血再灌注损伤的机制研究

Long noncoding RNA (lncRNA)-GAS5 was found to be upregulated in myocardial ischemia/reperfusion (I/R) injury in vitro by RNA Sequencing and downregulating the expression of GAS5 alleviated myocardial I/R injury, but the mechanism is still unknown. We predicted that NF-κB, a transcription factor, could bind to the promoter region of GAS5 gene by bioinformatics analysis and further found inhibition of NF-κB led to downregulating the expression of GAS5. Moreover, GAS5 was localized predominantly in the cytoplasm. We also predicted that GAS5 could bind with miR-26a and found that downregulation of GAS5 led to upregulating the expression of miR-26a. Our previous study has shown that miR-26a inhibited the expression and activity of Rho-associated kinase 1 (ROCK1) through binding with its the three-prime untranslated region (3'-UTR), and then led to activation of PI3K/AKT signal, subsequently increased the phosphorylation of GSK-3β, thus inhibited the opening of mitochondrial permeability transition pore (MPTP) and cell apoptosis. Furthermore, we also found that downregulation of GAS5 led to decreasing the expression and activity of ROCK1. Therefore, we hypothesize that NF-κB binds with the promoter region of GAS5 gene and promotes its transcriptional activity, and then lncRNA-GAS5 could bind with miR-26a and regulate ROCK1, the target gene, acting as a competitive endogenous RNA (ceRNA), and subsequently modulated the opening of MPTP through PI3K/AKT/GSK-3β signal pathway. The aim of this study is to demonstrate this hypothesis and illuminate the mechanism that GAS5 regulates myocardial I/R injury. This study will discover a new target and lay the foundation for the therapy of myocardial I/R injury.

我们发现LncRNA-GAS5在心肌缺血/再灌注（I/R）损伤中表达上调，而下调GAS5减轻心肌I/R损伤。生物信息学预测到GAS5基因启动子区与NF-κB存在结合，并发现抑制NF-κB可下调GAS5。我们还预测到GAS5与miR-26a存在结合, 并发现miR-26a靶向抑制ROCK1，通过PI3K/AKT/GSK-3β信号通路，抑制线粒体渗透性开放孔道（MPTP）开放。预实验证实，下调GAS5增加miR-26a表达，抑制ROCK1表达及活性。因此，我们推测NF-κB与GAS5基因启动子区结合并增强其转录，GAS5进一步与miR-26a结合，作为竞争性内源 RNA（ceRNA）调控ROCK1，并通过PI3K/AKT/GSK-3β信号通路来调节MPTP开放，从而调控心肌I/R损伤。本课题旨在证明这一科学假说，以期阐明GAS5参与调控心肌I/R损伤的机制，为防治心肌I/R损伤提供新的靶点。

心肌缺血再灌注损伤(MIRI)是长期以来困扰心肌再灌注治疗的难题，其机制不清，缺乏有效治疗手段。本课题从长链非编码RNA(LncRNA)这一研究热点为切入，阐明LncRNA-GAS5参与调控MIRI过程的上下游分子机制。我们发现，GAS5在MIRI过程中表达量出现显著升高，其表达受到转录因子NF-κB的正性调控。下调GAS5表达可以在体外和在体器官水平上显著减轻缺血再灌注诱导的心肌细胞坏死和凋亡。此外，GAS5可以与miR-335特异性结合，通过竞争性内源性RNA（ceRNA）机制调控miR-335的靶基因ROCK1,并通过影响PTEN/AKT/GSK-3β信号轴调控线粒体渗透性开放孔道（MPTP）开放。综上所述，本课题阐明了NF-κB/GAS5/miR-335/ROCK1信号轴参与调控MIRI的机制，丰富人们对于LncRNA调控MIRI发生机制的认识，为防治MIRI提供新的靶点和理论支持。

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