lncRNA-malat1调控bcl-2/rheb参与边缘性供肝IRI的分子机制及临床意义

The expression of long non-coding RNA (lncRNA) is characterized by tissue/developmental-stage-specificity, which can affect the stability of mRNA, competitively bind miRNA, regulate gene transcription and translation, and participate in various pathophysiological processes. However, the expression and function of lncRNA in marginal donor liver ischemia-reperfusion injury (IRI) are still unclear. Through the study of gene sequencing, histology and cellular function, we have revealed the downregulation of malat1 in IRI marginal donor liver of rat, and in human liver grafts. And, we also observe increased cellular apoptosis and autophagy in all liver tissues. Overexpression of malat1 can alleviate hypoxia-reoxygenation injury of hepatocytes, resulting in miR-16 downregulation, coupled with bcl-2 and rheb upregulation. Bioinformatic analysis not only indicates malat1 can bind to miR-16, but also hints that bcl-2 and rheb could be downstream target genes of miR-16. Based on in vitro and in vivo study, we’re going to unravel potential regulation networks among malat1, miR-16 and bcl-2 or rheb, i.e., malat1 competitively binds to miR-16, impacting bcl-2 or rheb expression in IRI marginal donor livers. Moreover, we will assess the clinical value of these regulatory networks as molecular targets of marginal donor liver IRI prevention or treatment.

长链非编码RNA（lncRNA）表达具有时序性和组织特异性，可影响mRNA稳定、竞争结合miRNA、调控基因转录翻译等，参与各类病理生理过程。边缘供肝缺血再灌注损伤（IRI）时，组织中lncRNA表达和功能不明。通过基因测序、组织学、细胞功能学研究，我们发现大鼠脂肪供肝IRI及临床肝移植时，组织中malat1的表达显著降低；且细胞凋亡增多、自噬活性增强。过表达malat1可抑制缺氧/复氧时肝细胞损伤，并发现miR-16下调和bcl-2、rheb表达上调。生信预测malat1与miR-16有结合位点，bcl-2和rheb为下游靶基因。本项目拟从体内、外水平揭示malat1、miR-16和bcl-2、rheb之间的调控关系，阐明malat1竞争结合miR-16调控bcl-2、rheb，参与边缘供肝IRI的分子机制。从临床组织、血浆水平，系统评价该调控网络作为边缘供肝IRI防治靶标的应用价值。

供肝短缺问题导致世界范围内扩大标准供体的使用率提高，此类供体肝脏对IRI相对敏感，常导致肝移植患者预后不良。因此，IRI是边缘供肝的核心问题及未来的主要研究方向。lncRNA表达具有时序性和组织特异性，可影响mRNA稳定、调控基因转录翻译等，参与多种病理生理过程。本项目通过基因测序，筛选出lncRNA malat1作为研究目标，重点分析了肝脏IRI过程中malat1的异常表达趋势，阐明了干预malat1表达对缺氧/复氧条件下肝细胞活性和生理功能的影响，并确认了malat1通过竞争性结合miR-16-3p来调控ATG9a，从而影响缺氧/复氧条件下肝细胞的活性和稳态。在此基础上，通过体内干预实验进一步确认了malat1/miR-16-3p调控ATG9a对肝脏IRI的影响。本研究阐明了调控lncRNA-malat1/miR-16-3p/ATG9a信号途径对减轻肝脏IRI的临床应用价值。重要结果包括：建立70%小鼠肝脏缺血再灌注损伤模型，并确立缺血再灌注时间，明确了肝组织损伤程度和炎性细胞浸润、炎性因子表达情况；检测了组织中malat1和miR-16-3p的表达趋势，并明确了相关性；通过检测小鼠HIRI组织中分子调控网络，我们发现自噬、凋亡相关蛋白参与其中，如Rheb、ATG9a、LC3-II/LC3-I、p62、Bcl-2等。进一步通过建立小鼠肝实质细胞AML12的氧糖剥夺/复氧（OGD/R）模型，明确了malat1和miR 16-3p均在细胞质中定位，并以ceRNA的方式，调控下游靶基因ATG9a的表达，从而发挥生理调节功能。本研究阐明了lncRNA-malat1/miR-16-3p/ATG9a信号轴调控肝脏IRI过程中肝细胞凋亡、自噬的发生发展，进而影响肝损伤结果。