长链非编码核糖核酸ANRIL调控YY1/NF-kB在老年心肌缺血后处理钝化作用中的机制研究

Antisense Noncoding RNA in the INK4 Locus (ANRIL) is the well-defined genetic risk locus associated with coronary artery disease (CAD), while little is known regarding its role in the pathological processes of CAD. Our earlier research found that ischemia postconditioning (IPost) showed protective effects on ischemia/reperfusion (I/R) injury in rats. However, the cardioprotection of Ipost in aged hearts is impaired and its mechanism has not yet been fully elucidated. Our previous research found that ANRIL expression was reduced in aged hearts of rats, Ipost could inhibit the NF-κB nuclear translocation without the alleviation of myocardial I/R injury. We proposed that the reduction of myocardial ANRIL expression could be responsible for the impaired cardioprotection of Ipost in aged rats, ANRIL participated the regulation of NF - kB signaling pathways and RNA binding protein YY1 combined with ANRIL may be involved in the regulation of the downstream target genes. Our study intends to examine the cardioprotection of IPost in aged rats using I/R injury model in vivo and in vitro. Further, we intend to explore the potential regulation mechanisms including the correlation of ANRIL expression and I/R injury, the participation of ANRIL in NF-κB singaling pathway and the interaction of ANRIL and YY1 for the expression of a subset of pro-inflammatory genes controlled by NF-κB signaling. Our research would explain the decrease in tolerance for I/R injury and sensitivity for treatment in aged hearts and provide important theoretical evidences for clinical new treatment strategies.

长链非编码核糖核酸ANRIL是冠心病的遗传易感位点，有关ANRIL与心肌老化及缺血再灌注（I/R）损伤的研究相对匮乏。缺血后处理（IPost）具有心肌保护作用，但我们前期研究发现IPost对老年心肌保护作用钝化，进一步研究发现，老年大鼠心肌ANRIL表达降低，Ipost可抑制NF-κB核内移却未减轻I/R损伤。因此，我们提出假说：老年心肌ANRIL表达下降导致IPost保护作用钝化，ANRIL可能通过调控NF-kB信号通路介导IPost钝化，RNA结合蛋白YY1可与ANRIL作用参与下游靶基因的调控。我们拟应用在体及体外I/R损伤模型研究IPost对老年大鼠心肌ANRIL表达以及I/R损伤预后的影响；ANRIL参与IPost对老年大鼠心肌YY1/NF-κB信号通路的调控，以揭示ANRIL在老年心肌IPost保护作用的钝化机制。本课题将为临床老年患者心肌I/R损伤治疗提供新思路及实验依据。

缺血后处理可通过激活内源性保护通路减轻组织缺血再灌注（I/R）损伤，但是其在老年患者中是否仍存在保护作用尚存争议。长链非编码RNA（lncRNA）H19在缺血性疾病中发挥着重要作用，但是其在老化心肌中的功能尚不明确。因此本研究将探讨H19在老化心肌缺氧后处理中的作用机制。本课题通过体外培养老化心肌细胞建立缺氧复氧（H/R） 损伤模型，运用分子生物学等方法证明老化心肌细胞缺氧后处理不能改善缺氧复氧损伤,右美托咪定缺氧后处理具有抗缺氧复氧损伤的作用，探索LncRNA H19在老年大鼠缺血后处理中发挥的具体作用机制以及ALKBH5对lncRNA H19表达的调控机制。结果显示，H19在老化心肌细胞缺氧后处理时表达下降。敲低H19时会增加老化心肌细胞缺氧后处理损伤。H19可通过miR-29b-3p/cIAPl通路在老化心肌缺氧后处理中发挥作用。老化心肌缺氧复氧和缺氧后处理时细胞总体RNA m6A甲基化水平增加，去甲基化酶ALKBH5表达下降，而右美托咪定后处理可降低缺氧复氧引起的RNA m6A甲基化的增加，升高ALKBH5的表达。敲低AKLBH5后，H19的m6A的修饰程度增加，表达下降，老化心肌缺氧后处理损伤增加。而右美托咪定缺氧后处理会减轻敲低ALKBH5后引起的老化心肌细胞缺氧复氧损伤。本研究力图阐明老年心肌对于I/R 损伤的耐受性下降以及对于治疗手段的敏感性下降的原因机制，为临床老年患者心肌缺血再灌注损伤的治疗选择及应用提供新的思路以及提供有力的科学理论依据。