circ-CYSLTR2.4与白三烯反馈通路在支气管肺发育不良中的作用及机制研究

Bronchopulmonary dysplasia (BPD) is the most common complication of prematurity with appreciable morbidity and mortality in infants. Our pilot experiment showed that leukotrienes (LTs) pathway activation was the important mechanism of inflammation injury and disturbance of lung development in BPD. However, the mechanism is not clear up to now. Our previous experiments found that the physiopathologic development of BPD was accociated with the regulation between circular RNA-CYSLTR2.4 and LTs. Firstly, we verified that circ-CYSLTR2.4 was upregulated whilst micro RNA-4287 (miR-4287) was downregulated in the rats lung tissue with BPD. Secondly, our investigation suggested that circ-CYSLTR2.4 was regulated by cysteinyl leukotriene receptor 2 (CysLTR2), and potential binding sites of miR-4287 were found within the circ-CYSLTR2.4 sequence. Finally, we demonstrated that CysLTR2 was the taget gene of miRNA-4287. Taken together, we speculate that high expressed circ-CYSLTR2.4 in BPD acting as the miRNA-4287 sponge enhanced the persistent activation of LTs pathway. circ-CYSLTR2.4 suppressed the expression of miRNA-4287 which elevated the expression of CysLTR2. Therefore, we put forward the hypothesis that the feedback loop between the inhibition of miRNA-4287 via circ-CYSLTR2.4 and CysLTR2 activation, sustain activate LTs pathway which excabate the inflammatory injury and arrested alveolarization in BPD. Consequently, we will investigate mechanisms based on experimental model of BPD and molecular mechanism to illuminate the mechanism of the feedback loop between circ-CYSLTR2.4 and LTs in BPD. Our findings also provide new evidences for the treatment of BPD in preterm infants.

支气管肺发育不良(BPD)是影响早产儿生存、预后的危重症之一。研究发现，白三烯通路活化可致BPD炎性损伤及肺泡发育异常，但机制不明。我们前期发现：1.BPD大鼠肺组织circ-CYSLTR2.4表达上调，而miR-4287表达下调；2.circ-CYSLTR2.4受白三烯受体2(CysLTR2)调控，并可吸附抑制miR-4287；3.CysLTR2为miR-4287靶基因。基于此我们提出假说：BPD肺组织高表达的circ-CYSLTR2.4通过海绵抑制效应，降低miR-4287表达，从而上调miR-4287靶基因CysLTR2表达，形成反馈通路，持续活化白三烯信号，导致肺炎性损伤、肺泡化异常及BPD产生。本课题利用芯片筛选、生物信息学分析、细胞功能验证等方法，从体内动物实验、体外细胞及分子水平阐明circ-CYSLTR2.4与白三烯反馈通路在BPD中的作用，为BPD治疗提供新思路。

BPD是早产儿致命性肺部疾病，系炎症和氧化应激等多个致病因素在肺发育不成熟的基础上共同作用的结果，但发病机制尚未完全阐明，且目前尚无针对性的预防或有效的治疗药物。现已知环状RNA（circRNA）在各种肺疾病中起着至关重要的作用，然而在BPD中的机制却不甚明了。通过芯片测序获得BPD患儿外周血中circRNA的表达谱，发现一个复杂的ceRNA调控网络。进一步生物信息学分析表明，circABCC4和白三烯通路中的PLA2G6共享miR-663a的MRE，提示存在circABCC4 / miR-663a / PLA2G6轴。利用生物信息学预测、RT-qPCR、RNA免疫沉淀（RIP）和萤光素酶报告确定circABCC4吸附miR-663a和下游靶基因PLA2G6。利用RNA干扰和慢病毒转染A549细胞系，检测炎症因子和氧化应激指标LTB4R、KL-6、PLA2G6和SOD的表达水平。通过萤光素酶报告，回复实验来确认circABCC4，miR-663a和PLA2G6之间的相互作用关系。检测 高氧诱导的 BPD新生大鼠模型肺组织中PLA2G6、LTB4R、KL-6和SOD的表达水平。在BPD患儿外周血，BPD大鼠肺组织和高氧暴露的A549细胞系中circABCC4的表达上调，而circABCC4的表达与BPD患儿的不良临床病理特征和不良预后相关。过表达circABCC4后可促进高氧暴露的A549细胞凋亡并抑制其增殖。circABCC4可以直接与miR-663a相互作用，然后充当miRNA的海绵来上调miR-663a靶基因PLA2G6的表达，从而促进BPD的发展。验证了circABCC4在BPD中表达水平上调，而circABCC4的高表达与BPD患者的不良临床病理特征和不良预后相关；circABCC4通过miR-663a / PLA2G6轴促进BPD的进展，有望成为治疗BPD患者的新的诊断标志物和靶标。本项目为应用白三烯受体拮抗剂、脂氧素及其类似物治疗新生儿支气管肺发育不良等肺部疾病提供新的诊疗途径，为开发国家一类创新化学药品提供实验依据并奠定理论基础。