长链非编码RNA-Neat1调控p53/BECN1参与移植肝损伤的分子机制研究

Long noncoding RNAs (lncRNAs) exhibit tissue/developmental-stage-specific expression and can act as microRNA sponge or regulate gene transcription, translation to play important roles in various diseases. However, their expression and roles in the development of livergraft injury are still unclear. We have shown that lncRNA-neat1 is downregulated via microarray screening and tissue redetection. Moreover, downregulation of neat1 in hepatic cell line can significantly attenuate cell injury in hypoxia and reoxygenation condition, increase miR-107 expression and reduce P53/BECN1 expression. Bioinformatic analysis indicated that neat1, acting as miR-107 sponge to regulate P53/BECN1 expression, plays roles in the development of livergraft injury. Nevertheless, the detailed mechanisms still need to be illuminated. This study will reveal regulatory relationships among lncRNA-neat1, miR-107 and P53/BECN1, and clarify the mechanism of neat1 serving as miR-107 sponge to regulate P53/BECN1 expression in vitro or in vivo. Meanwhile, we will evaluate their clinical significances in tissue and plasma levels. This study would provide novel insights into the prevention and treatment of livergraft injury.

lncRNA表达具有组织时序性，可发挥miRNA海绵或调控基因转录、翻译等功能，在多种疾病中发挥重要作用，但在移植肝损伤时的表达和作用机制尚不明确。前期芯片筛选和移植肝组织验证发现lncRNA-neat1呈高表达。下调neat1后，可抑制缺氧/复氧时的肝细胞损伤，同时发现miR-107表达上调和P53表达下调。生物信息学预测其可通过ceRNA方式结合miR-107调控P53/BECN1表达，但具体机制仍待探究。本课题拟通过体内、外实验揭示neat1、miR-107和P53/BECN1之间的调控作用，阐明neat1充当miR-107海绵调控P53/BECN1，从而影响移植肝损伤的分子机制。从组织和血浆水平，评价lncRNA-neat1/miR-107调控P53/BECN1/信号途径对移植肝损伤的临床意义，为其防治提供新思路。

供肝短缺问题导致边缘性供肝使用增多，而此类供肝对缺血再灌注（I/R）损伤十分敏感，直接影响移植肝功能和患者临床预后。本研究以lncRNA Neat1为分子靶标，分析了lncRNA Neat1在肝脏I/R损伤过程中的表达情况，并阐明了 lncRNA-Neat1 作为 miR-107 海绵调控下游Atg7分子，影响 H/R 条件下肝细胞活性和稳态的分子机制，明确了调控lncRNA-Neat1/miR-107/Atg7信号轴对减轻肝脏I/R损伤的临床意义。重要结果有：构建小鼠70%肝脏缺血再灌注（I/R）模型，组织H&E和肝功检测发现I/R-1h后出现明显肝损伤，TUNEL染色显示肝组织有明显细胞凋亡，炎性因子IL-6、IL-1β、CXCL10、TNF-α和lncRNA Neat1表达上调。mRNA和蛋白水平检测凋亡相关基因p53、Bax表达上调，Bcl-2表达量下调，自噬相关基因Atg7和自噬水平上调。利用AML12细胞建立氧糖剥夺模型（OGD/R）模拟肝脏I/R过程：OGD-6h组中Neat 1表达趋势与肝脏I/R过程一致。给予细胞不同氧糖恢复时间，发现OGD-6h/R-3h组细胞发生明显凋亡，且OGD-6h/R-3h组p53，Bax、Atg7表达上调，Bcl-2表达降低，细胞自噬水平提高。软件分析miRNA-107-3p与Neat 1存在1个7碱基，2个6碱基结合位点。构建7碱基突变体，荧光素酶报告基因检测结合能力，证实Neat1 和miR-107-3p可直接结合，在I/R和OGD-6h/R-3h模型中，miR-107-3p表达降低。而抑制Neat1能够增加miR-107-3p表达,，降低Atg7表达和细胞自噬水平， p53表达和细胞凋亡率降低，但不影响Bax、Bcl-2表达。核质分离揭示Neat1在细胞质和细胞核中均存在，FISH证明Neat1和miR-107-3p共定位于细胞质中。在OGD/R模型中，过表达miR-107-3p能够降低Neat1、Atg7的表达量和自噬水平。抑制miR-107-3p能够增加Neat1、Atg7的表达量和自噬水平。挽救实验表明：降低Neat1的同时加入miR-107-3p 抑制剂能够增加Atg7的表达及自噬水平，说明存在Neat1/miR-107-3p /Atg7轴参与调控OGD/R诱导的自噬过程。