长链非编码RNA MALAT1对早产儿高氧肺损伤保护作用机制的研究

Hyperoxia is the one of the important factors causing the occurrence and development of acute lung diseases in preterm infants. Whereas hyperoxia-induced lung injury remains a major health problem resulting in increased healthcare costs, prolonged hospital stays, and subsequent harmful effects on growth and neurodevelopment. However, the exact mechanism of hyperoxia-induced lung injury has not been demonstrated. In the previous reports, NF-E2 related factor 2 (Nrf2), a transcription factor for antioxidant response element (ARE)-mediated antioxidant and defense gene expression, is confirmed to play an important role in the pathogenesis of hyperoxia-induced lung injury..In recent years, increasing evidence suggests that noncoding RNAs play significant roles in the regulation of pathophysiological conditions. Besides small noncoding RNAs (eg, microRNAs), >200-nucleotide long transcripts, lncRNAs (long noncoding RNAs) have been confirmed to be a new class of regulators of cellular processes, such as cell growth, apoptosis. The metastasis-associated lung adenocarcinoma transcript 1, MALAT1, is a long non-coding RNA (lncRNA) that interacted with Nrf2 pathway. It has been demonstrated to be an important player in promoting tumor growth by cell cycle regulating. Up-regulation of long noncoding RNA MALAT1 has been functionally associated with cell proliferation and apoptosis inhibition. Therefore, MALAT1 might be a protective factor in hyperoxia-induced lung injury. .In this study, the expression of MALAT1, Nrf2/ARE signaling pathway related genes was investigated in the model of premature rats exposed to hyperoxia and hyperoxic alveolar epithelial cells type II (AEC Ⅱ) as well as the peripheral blood and bronchoalveolar lavage fluid samples of bronchopulmonary dysplasia (BPD) preterm infants, respectively, with qRT-PCR, immunohistochemistry, surface plasmon resonance and flow cytometry. Meanwhile, cell injury and apoptosis were also investigated in the process of occurance and development of hyperoxia-induced lung injury. Subsequently, to explore the role of MALAT1 involved in the process of occurance and development of hyperoxia-induced lung injury in preterm infants and its interaction with the Nrf2 pathway, expression of MALAT1 and Nrf2 were knockdown by transfection of siRNA in A549 cell (human lung adenocarcinoma cell line) with high oxygen exposure, and the differentiation of gene expression pattern was compared between two groups. .These results will contribute to looking for the protective factor, which can resistant to hyperoxia-induced lung injury in preterm infants.

高氧导致的急性肺损伤严重影响早产儿的肺发育，氧化应激是主要致病因素，但确切机制尚未阐明，更无有效的防治措施。前期研究表明以Nrf2/ARE 信号通路障碍为代表的氧化/抗氧化失衡起重要作用，而与Nrf2存在直接作用的长链非编码RNA MALAT1可促进细胞增殖、降低凋亡率，可能是机体的保护因子。本项目以高氧暴露早产鼠、AECII细胞和支气管肺发育不良早产儿外周血和肺泡灌洗液样本为研究对象，应用qRT-PCR、免疫组化、表面等离子体共振、流式细胞术等技术在不同水平阐述早产儿高氧肺损伤过程中MALAT1、Nrf2/ARE通路相关基因的表达变化及其对细胞损伤及凋亡的影响。随后，在高氧暴露人肺腺癌A549细胞中分别抑制Nrf2、MALAT1的表达，对比两组间相关基因表达的差异，探讨MALAT1在早产儿高氧肺损伤中的作用及其与Nrf2通路的相互调控机制。以期寻找早产儿高氧肺损伤的保护因子及防治新策略。

高浓度氧暴露导致的急性肺损伤（ALI）严重影响早产儿的肺发育，引起早产儿支气管肺发育不良（BPD），氧化应激是主要致病因素，但其确切机制尚未阐明，更无有效的防治措施。本项目通过以建立的早产新生SD大鼠高氧肺损伤动物模型、AECⅡ和A549细胞高氧暴露模型作为研究对象，探讨高氧暴露对早产鼠/早产儿机体内MALAT1、Nrf2/ARE通路相关基因表达的影响，明确早产儿高氧肺损伤过程中MALAT1、Nrf2/ARE 通路的表达变化及其对细胞损伤及凋亡的影响，以及开展MALAT1、Nrf2/ARE通路相关基因在早产儿高氧肺损伤过程中的相互作用及调控机制的研究。本项目得出重要结果为，（1）高氧组新生早产大鼠在第7、10、14天体重显著下降，但身长变化不明显。（2）高氧组新生早产大鼠肺组织失去正常规则形态，结构紊乱，肺泡间隔变薄，肺泡腔增大，肺泡数目减少。（3）与空气组相比，高氧组新生早产大鼠肺组织中MALAT1表达在第1天时显著减弱，4、7天明显增强，10天开始出现显著减弱，14天时下降到最低。（4）与空气组相比，高氧组新生早产大鼠肺组织Nrf2 mRNA 表达在第1天显著减弱，4、7、10天显著增强，第14天时表达减弱。（5）高氧组早产鼠肺组织中HO-1 mRNA相对表达量在第7天时低于空气组，第10、14天时表达显著高于空气组。高氧组早产鼠肺组织中GCLC mRNA表达量在第1、4、7天时显著低于空气组。（6）高氧暴露后早产新生大鼠肺组织中TNF-α、IL-6、Nrf2和miRNA-125b显著增加，且IL-6与miRNA-125b表达显著正相关。siRNA抑制A549细胞Nrf2表达，miRNA-125b同样受到抑制，同时细胞凋亡明显增加。（7）早产儿外周血标本验证结果表明，与对照组早产儿比较，MALAT1在高氧肺损伤早产儿外周血中表达显著上调。本项目得出重要结论为，高氧暴露导致早产鼠生长发育迟缓、肺发育阻滞，出现类似BPD病理改变。MALAT1、Nrf2、HO-1、GCLC、miRNA-125b、TNF-α、IL-6均参与高氧所致BPD发病过程，IL-6可能作为miRNA-125b关键因子发挥作用，miR-125b作为Nrf2的下游靶点参与BPD保护。lncRNA MALAT1可能通过抑制细胞凋亡，对BPD起保护作用，为临床防治BPD提供新策略。