TGF-β1 mRNA作为ceRNA调控Nrf2表达在脑出血后炎性损伤中的机制研究

The incidence, morbidity, and mortality of intracerebral hemorrhage remain high, yet the existing medical and surgical treatment methods are unable to prevent perifocal damage. The perihematoma inflammatory injury after intracerebral hemorrhage is complicated, and indicated the prognosis. The latest study suggested that TGF-β1 treatment for intracerebral hemorrhage in rats could inhibit the inflammatory response, but the mechanism was still unclear. Our study found that the expression of miR-93 in the perihematoma tissue of rats with intracerebral hemorrhage was significantly higher than that in the control group. Moreover, we confirmed that the expression of TGF-β1 could be directly regulated by miR-93 post-transcriptional control, and the down-regulation of TGF-β1 can lead to the significant increase of miR-93 expression. Our preliminary data showed that Nrf2-ARE signaling pathway could inhibit the expression of inflammatory factors after intracerebral hemorrhage, but the transcriptional regulation mechanism of Nrf2-ARE needs to be further study. Bioinformatics analysis found both TGF-β1 and Nrf2 mRNA could combine to miR-93. Therefore, we suggest that TGF-β1 mRNA could competitively inhibit Nrf2 mRNA binding to miR-93 after intracerebral hemorrhage and up-regulate Nrf2 expression to alleviate the inflammation response. The aim of this project is to define the expression characteristics and correlation of miR-93, TGF-β1 and Nrf2 at different time points after intracerebral hemorrhage in rats, and clarify whether TGF-β1 to exert anti-inflammatory effect by regulating Nrf2 expression, then illustrate whether TGF-β1 mRNA as ceRNA to be involved in the molecular mechanism of Nrf2 transcriptional regulation. This project may provide the new therapeutic targets for treatment of intracerebral hemorrhage.

脑出血患者灶周炎性损伤机制复杂并决定预后。最新研究提示针对大鼠脑出血的TGF-β1治疗可抑制炎症反应，但作用机理不明。前期预实验提示脑出血大鼠灶周组织中miR-93表达较对照组明显升高；并证实其可直接通过转录后水平调控TGF-β1，且下调TGF-β1可引起miR-93表达增加；我们已证实脑出血后Nrf2-ARE信号通路可抑制炎性因子的表达，但该通路的转录调控机制亟待深入研究。生物信息学分析TGF-β1与Nrf2mRNA均可与miR-93结合。故假设：脑出血后TGF-β1mRNA竞争性抑制Nrf2mRNA与miR-93结合，上调Nrf2表达以降低炎症。拟明确大鼠脑出血后不同时间点miR-93、TGF-β1、Nrf2的表达特征及其相关性；确定TGF-β1是否通过调控Nrf2表达发挥抗炎作用；阐明TGF-β1 mRNA作为ceRNA参与Nrf2转录后调控的分子机制；寻找新的脑出血干预靶点。

脑出血（ICH）在所有卒中亚型中死亡率最高，但有效的治疗方法尚未在临床上实施。据报道，转化生长因子-β1 (TGF-β1) 可调节 ICH 后小胶质细胞介导的神经炎症并促进功能恢复；然而，潜在的机制仍不清楚。非编码 RNA，如 microRNA (miRNA) 和竞争性内源性 RNA (ceRNA) 已成为人类疾病的关键调节因子。已知的 miR-93 靶点，核转录因子E2相关因子2 (Nrf2)，已被证明在 ICH 后具有神经保护作用。依据已有的研究和我们前期研究基础，我们假设TGF-β1 mRNA作为一种 ceRNA 竞争性抑制Nrf2 mRNA与miR-93-5p结合，上调Nrf2表达发挥抗炎作用，从而改善ICH 继发性损伤。我们使用短干扰RNA（siRNA）敲低TGF-β1，并使用药理学操纵的miR-93表达来阐明ICH（凝血酶处理的人小胶质细胞HMO6细胞）体外模型中miR-93，Nrf2和TGF-β1之间的机制联系。生物信息学预测分析表明，miR-93-5p可以与TGF-β1和Nrf2结合。我们发现这些HMO6细胞中的神经元miR-93显着降低，并且在ICH患者的新鲜脑组织中观察到类似的变化。最重要的是，我们使用荧光素酶报告基因测定来证明miR-93直接靶向Nrf2以抑制其表达，并且添加TGF-β1 3'UTR恢复了Nrf2的水平。此外，miR-93抑制剂增加了TGF-β1和Nrf2的表达并减少了细胞凋亡。上述结果证实了TGF-β1作为ceRNA的新功能，该ceRNA可以海绵miR-93以增加神经保护性Nrf2的表达并减少ICH后的细胞死亡。我们的研究结果支持miR-93作为治疗ICH的潜在治疗靶点提供了证据。