C1QTNF6介导的lncRNA H19/miRNA-29b通路对缺血性脑卒中血脑屏障破坏的机制研究

lncRNA/miRNA/mRNA is a precise regulatory network for gene transcription. The secretion of IL-1β by neutrophils is a key step in initiating the destruction of blood-brain barrier and neuronal apoptosis after cerebral ischemia. Our previous study found that ischemic stroke increased the expression of lncRNA H19, decreased the expression of miR-29b in peripheral blood neutrophils, and increased the expression of inflammatory factor IL-1β. Bioinformatics predicts that the interaction of H19 and miR-29b regulate the expression of C1QTNF6. Based on the literature and preliminary results, we hypothesized that the H19/miR-29b/C1QTNF6/IL-1β axis in neutrophil is involved in the disruption of the blood-brain barrier after ischemic stroke, which promotes the apoptosis of neurons. This study intends to clarify the effects of C1QTNF6 gene on blood-brain barrier disruption and neurological prognosis of ischemic stroke from levels in molecules, cells, animals and patients; explore the regulation mechanism of H19/miR-29b interaction on C1QTNF6; determine the action of C1QTNF6 on IL-1β and endothelial cell tight junction proteins Occludin and ZO-1. This study will investigate the mechanism of blood-brain barrier disruption after ischemic stroke from the new viewpoint of lncRNA/miRNA interaction regulation target gene, and provide new ideas for neuroprotection of ischemic stroke.

lncRNA/miRNA/mRNA是基因转录后的精细调控网络。中性粒细胞分泌IL-1β是启动脑缺血后血脑屏障破坏、神经元凋亡的关键环节。我们发现：缺血性脑卒中患者中性粒细胞lncRNA H19表达升高，miR-29b表达降低，伴随IL-1β表达升高。生物信息学预测H19和miR-29b可相互作用共同调控C1QTNF6表达。本课题拟从患者、动物及细胞多层次验证假说：中性粒细胞H19/miR-29b/C1QTNF6/IL-1β通路参与缺血性脑卒中血脑屏障破坏，加速神经元凋亡。明确C1QTNF6对缺血性脑卒中神经功能预后的影响；探讨H19/miR-29b相互作用对C1QTNF6的调控机制；明确C1QTNF6对IL-1β及紧密连接蛋白Occludin和ZO-1的作用机制。本研究从lncRNA/miRNA相互作用调控靶基因的角度探讨缺血性脑卒中后血脑屏障破坏的发生机制，为卒中后的神经保护提供新思路。

目的：白细胞诱导的炎症过程在急性缺血性卒中的病理生理过程中发挥关键作用。本研究旨在阐明长非编码RNA（lncRNA）H19在缺血性脑卒中期间通过靶向白细胞中的miR-29b介导C1q肿瘤坏死因子6（C1QTNF6）调节的炎症机制。.方法：采用实时聚合酶链反应检测缺血性脑卒中患者和大脑中动脉闭塞大鼠白细胞中H19和miR-29b的表达水平。H19 siRNA和miR-29b antagomir分别用于敲除H19和miR-29b的表达水平进行研究。我们进行了体内和体外实验，以确定H19和miR-29b对缺血脑损伤后白细胞中C1QTNF6表达的影响；我们通过Western blot法和流式细胞法检测相应蛋白的表达水平和相应细胞的活性；通过冰冻切片的方法检测炎性反应对神经元活性的影响。.结果：在缺血性脑卒中患者的白细胞中检测到H19和C1QTNF6表达上调，而miR-29b表达下调。此外，miR-29b可以结合C1QTNF6 mRNA并抑制其表达水平，而H19可以发挥miR-29b海绵的方式以维持C1QTNF5的表达。C1QTNF6过表达促进白细胞中炎性因子IL-1β和TNF-α的释放，进一步加剧血脑屏障破坏，加重脑缺血损伤。MiR-29b表达升高可通过C1QTNF6抑制降低IL-1β表达，减轻血脑屏障（BBB）的破坏和白细胞浸润的浸润。此外，miR-29b在HL-60细胞中的过表达下调了OGD诱导的hCMEC/D3细胞凋亡，并增加了血脑屏障中的ZO-1和Occludin的表达水平。.结论：我们的研究结果证实，H19通过靶向脑缺血损伤中的miR-29b/C1QTNF6轴促进白细胞炎症。白细胞miR-29b通过抑制C1QTNF6的表达，保护血脑屏障的完整性来减轻炎症反应，这表明了一种基于miR-29b的新型缺血性脑卒中的治疗方法。