P53/miR-34a/SIRT1 正反馈弧参与大鼠肝再生终止的分子机制研究

The marked difference between Liver regeneration and liver tumor is that liver regeneration can be terminated promptly and liver size can be controlled accurately, but its control mechanism is still obscure. MicroRNAs(MiRNAs)，a non-coding RNA species, are known to play an important role in cell proliferation, differentiation and apoptosis through regulating the expression of their target genes at the post-transcriptional level. Only a few studies have yet addressed the relationship between miRNAs and liver regeneration，and the studies involved in roles of miRNAs in the termination of liver regeneration has not been seen. Then we carried out genomewide miRNA microarray studies, and first discovered that miR-34a is significantly up-regulated (up to six times) in the termination phase of rat liver regeneration. and our research shows that miR-34a has a negative role through targeting c-Met and INHBB in the termination of liver regeneration, but its high expression mechanism is not clear. Research suggests p53--miR-34a--SIRT1 can form positive feedback loop to amplify the negative effect , and which may be restricted by bile acid-FXR/SHP pathway. This suggests that demand for liver metabolic capacity may be correlated with justment of liver size. Under this assumption, we will carry out a study for mainly probing into the molecular mechanism of expression regulation of miR-34a in the termination of liver regeneration, using rat liver regeneration and liver cell model ,hepatic tumor and genetic intervention etc. This study will contribute to understand the molecular mechanism of liver regeneration, also to provide new ideas to explore the pathogenesis and treatment of acute liver failure, small-for-size liver transplantations or liver tumor.

肝再生与肝肿瘤显著差别是：肝再生能及时终止并精确控制肝大小。但对其中涉及机制认识很少。miRNAs与细胞增殖、分化和凋亡等密切相关，但尚未见miRNAs与肝再生终止和肝质量控制相关研究。先期我们用miRNAs芯片首次筛到在大鼠肝再生终止阶段miR-34a上调达6倍，并证实miR-34a通过靶向下调INHBB和c-Met参与肝脏再生终止。研究提示P53/miR-34a/SIRT1之间可形成正反馈弧而放大miR-34a的负调作用,该放大作用可能受到胆汁酸/FXR/SHP通路制约，这提示机体对肝代谢需求与肝大小调节之间是密切关联的。为此，本项目利用大鼠肝再生模型、肝细胞系、肝肿瘤标本，以及基因干预技术等，试图阐明上述两个通路通过协调miR-34a表达而参与调节大鼠肝再生终止和肝质量控制机制。本研究有助于完善对肝再生分子机制认识，为探索急性肝衰竭、小供肝移植和肝脏肿瘤发生和治疗提供启示

肝再生与肝肿瘤显著差别是：肝再生能及时终止并精确控制肝大小。但对其中涉及机制认识很少，先期研究提示P53/miR-34a/Sirt1之间可形成正反馈弧放大miR-34a负调作用,该放大作用可能受到胆汁酸/FXR/SHP通路制约。本课题试图阐明上述两个通路通过协调miR-34a表达而参与调节大鼠肝再生终止和肝质量控制机制。通过肝细胞，小鼠肝再生模型，基因干预和代谢组学等研究发现：.（1）小鼠肝再生模型显示P53/ miR-34a/ Sirt1正反馈弧在肝再生后期明显被激活，主要表现为p53、乙酰化p53和miR-34a 水平明显升高，而Sirt1表达明显降低，肝细胞增殖能力明显下降而凋亡水平明显升高；.（2）体内和体外过表达p53和miR-34a或下调Sirt1能明显激活P53/ miR-34a/ SIRT1正反馈弧，能明显抑制肝脏细胞增殖，降低肝再生末期小鼠肝体积比例；.（3）代谢组学显示小鼠肝脏再生过程中多种胆汁酸发生明显改变，其中T-β-MCA在小鼠肝再生后期明显升高，细胞水平和在体研究揭示T-β-MCA能明显抑制FXR/SHP通路，增强P53/miR-34a/Sirt1正反馈弧作用，从而抑制肝细胞增殖和促细胞凋亡功能，在肝再生后期终止中发挥了重要作用。这表明肝脏代谢功能成熟过程与肝再生终止过程密切相关的；.（4）肝癌细胞和肝癌组织标本研究发现P53/ miR-34a/ SIRT1正反馈弧紊乱可能与肿瘤发生、发展，以及肝癌患者的预后密切相关。研究结果提示表达野生型p53肝细胞癌患者的预后优于p53缺失或突变肝细胞癌患者，p53突变型和缺失型肝癌细胞内的P53/ miR-34a/ Sirt1正反馈弧明显紊乱，这可能与该类肝细胞癌患者预后较差密切相关。而干扰FXR/SHP通路和P53/ miR-34a/ Sirt1正反馈弧可以抑制肝癌细胞生长，这可能是肝癌治疗的重要靶点之一。.本研究结果进一步完善对肝再生分子机制认识，为探索急性肝衰竭、小供肝移植和肝脏肿瘤发生和治疗提供了重要启示和策略。