tRF-Gly在小鼠酒精性脂肪肝中的作用及其机制研究

Alcoholic liver disease（ALD）is caused by the long-term excessive alcohol consumption, however, the mechanism is not fully understood. Studies have indicated that tRNA-derived fragments (tRFs) are a class of novel small RNAs with important biological functions. By deep-sequencing of small RNAs, the result showed that the expression of tRF-Gly was higher in AFL liver tissues than that in normal liver tissues, and the expression of tRF-Gly in chronic model (6 weeks) was significantly higher than that in the chronic and binge model (NIAAA model) (2 weeks). Using anti-sense to inhibit tRF-Gly, ALT and AST were significantly decreased, and complement C3a was also significantly reduced; Oil red O staining showed that the fat droplets were significantly smaller, and the number was significantly reduced. Our hypothesis is that tRF-Gly plays an important role in the occurrence and development of AFL by the complement pathway. To test this hypothesis, the adenovirus overexpressed tRF-Gly or chemically synthesized anti-sense inhibitor will be used to study the effect on the lipid metabolism, complement and complement inhibition factor in vivo. Subsequently target genes screening by gene chip will be performed. The molecular mechanism and signal pathway of tRF-Gly in the occurrence and development of AFL will be further analyzed by using shRNA knockdown technology. These studies will further help understand the pathogenesis of ALD, and provide a new theoretical basis for the prevention and treatment of ALD.

酒精性肝病是长期过度饮酒引发的肝脏疾病，其机制尚不完全清楚。研究表明tRNA来源的小片段（tRF)是一类具有重要功能的小分子RNA。我们前期实验经小分子RNA测序发现，tRF-Gly在小鼠酒精性肝病肝组织中的表达比对照高，并且慢性模型（6周）中的表达比NIAAA模型(2周）有更显著升高。用反义链抑制体内tRF-Gly，ALT和AST明显下降，补体C3a也明显降低；油红O染色结果显示脂肪滴明显变小，数量也明显地减少。我们的假说是tRF-Gly通过补体途径在酒精性脂肪肝病发生发展中发挥重要作用。用腺病毒过表达tRF-Gly或化学合成反义链抑制剂进行小鼠体内实验，研究其对补体成分、补体调节蛋白、脂代谢相关基因等的影响，并寻找它的靶标基因。采用shRNA等方法，剖析tRF-Gly在酒精性脂肪肝发生发展过程中的机制及信号通路。这些研究有助于了解酒精性肝病的致病机制，为酒精性肝病的防治提供理论依据。

项目的背景.酒精性肝病（ALD）是长期过度饮酒引发的肝脏疾病，严重威胁人民群众健康，其发病机制尚不完全清楚，并缺乏有效地治疗靶标和临床治疗的药物。..主要研究内容.（1）tRF-Gly在ALD中的表达水平及与ALD的关系。取小鼠酒精性肝病肝组织和对照肝组织进行小分子RNA深度测序，筛选得到甘氨酸转运 RNA 衍生片段（tRF-Gly）；并用定量RT-PCR等验证tRF-Gly的表达。.（2）tRF-Gly在ALD中的作用。过表达tRF-Gly或合成反义链抑制剂进行小鼠体内实验，进行病理分析；检测肝功能、补体成分、细胞因子、氧化应激、脂肪代谢等。.（3）tRF-Gly在ALD中的作用机制。过表达tRF-Gly或合成反义链抑制剂进行小鼠体内实验,检测补体成分、脂肪代谢相关基因等，利用基因芯片筛查tRF-Gly的靶标基因并验证。..重要结果.经研究显示: 在酒精喂养的小鼠中， tRF-Gly的表达上调；当抑制体内 tRF-Gly 后，减轻酒精喂养导致的脂肪肝但对炎症没有作用。在 C3 缺失或 CR2-Crry 处理的小鼠中，tRF-Gly 的表达下调，而在 C5 缺陷的小鼠中则没有下调；C3缺失对酒精诱导的肝脏脂肪变性和炎症具有保护作用。补体 C3的靶向抑制剂，CR2-Crry，对酒精诱导野生小鼠的脂肪肝有保护作用。C3 激活产物通过调节 CYP2E1 会调控tRF-Gly表达。肝组织的转录组分析显示，抑制tRF-Gly会上调 Sirt1表达， 进而影响下游脂肪合成和β-氧化途径。 在机制上，tRF-Gly 与 AGO3 相互作用，序列互补性靶向 3′UTR 从而下调 Sirt1 表达。临床样本中，与健康人相比，ALD患者 C3d、CYP2E1 和 tRF-Gly 的表达都上调，而 Sirt1 的表达降低。总之，研究结果表明，C3 激活产物通过调控肝脏中 tRF-Gly表达， 促使肝细胞脂肪沉积。抑制补体 C3 激活或 tRF-Gly可能是潜在和精准的ALD治疗方法。..关键数据及其科学意义.研究诠释了ALD的补体依赖性机制，揭示了ALD新的发病机制并提出了靶向性补体调控防治ALD的新策略；抑制tRF-Gly有效减轻脂肪变但不影响炎性变，补体抑制剂能够减轻酒精引起的小鼠肝损伤。申请3项用于临床ALD精准防治的发明专利，已授权2项。