HuR保护XBP-1 mRNA调控内质网应激与肝再生的分子机制研究

Human antigen R (HuR) is a RNA binding protein, which plays an important role in the regulation of cell proliferation, differentiation, signal transduction and other physiological and biochemical process. X box binding protein 1 (XBP-1) is the endoplasmic reticulum stress related genes, which enters the nucleus and activation of target genes (such as XBP-1 and GRP78) expression, enhanced cell the survival and adaptive response; XBP-1 mRNA 3' UTR has HuR binding sites. Partial hepatectomy (PH) propels HuR transport from nucleus to the cytoplasm of liver.cells, and increases the expression of GRP78. This research study the role of HuR on the regulation of liver regeneration by application of liver cell specific HuR knockout mice (Hur-LKO) PH model; We regulate the expression of HuR in Huh7 cell line, then to expounds the regulatory mechanism of HuR on XBP-1 by mutagenesis and luciferase reporter gene system. What’s more, our group overexpress XBP-1u and XBP-1s in different HuR level both in vivo and in vitro, to detect the expression of GPR78, and to observe the liver regeneration capacity of mice and the survival condition. Finally, to analysis the regulation of HuR-XBP-1 pathway on liver regeneration, and to provide an effective target for prevention and treatment liver regeneration disorder after PH.

人抗原R（HuR）是一种RNA结合蛋白，在调节细胞增殖、分化，信号转导等生理生化过程中发挥重要作用。X盒结合蛋白1（ XBP-1）是内质网应激相关基因，其进入细胞核，激活靶基因（比如XBP-1与GRP78）表达，增强细胞的存活和适应性反应；XBP-1 mRNA 3’UTR上存在HuR结合位点。肝部分切除（PH）的应激使HuR由核质转运至胞浆，且肝细胞GRP78表达升高。本研究应用肝细胞特异性敲除Hur小鼠（Hur-LKO）PH模型研究HuR对肝再生的调节作用；在Huh7细胞系调控HuR表达，利用点诱变技术及荧光酶报告基因系统阐述HuR对XBP-1的调控机制；通过体内和体外实验，不同HuR水平下高表达XBP-1u和XBP-1s，检测GPR78表达，观察小鼠肝再生能力及生存情况。综合分析HuR-XBP-1旁路对肝再生的调控作用，为PH术后肝再生障碍的预防和治疗提供有效可行的作用。

肝部分切除术是多种良恶性肝脏疾病的首选方案，但部分患者在术后会出现肝脏再生缓慢或再生功能障碍，手术预后差。目前临床上针对肝再生障碍的治疗方法有限。在肝再生过程中，大量未折叠蛋白激活IRE1α-XBP1旁路以对抗内质网应激。XBP1 mRNA 3’UTR上存在HuR结合位点，HuR是维持细胞生长和增殖的重要基因，通过结合mRNA对目的基因表达进行转录后调控。本研究通过建立HuR敲降的Huh7稳转细胞系，探究HuR和内质网相关基因XBP1的关系。建立肝细胞特异性HuR敲除小鼠，PH诱发内质网应激，探究肝再生过程中，HuR和内质网应激的联系。研究发现1、Huh7细胞发生内质网应激时HuR出核入胞。2、相较于对照组，Sh-HuR Huh7细胞在发生内质网应激时，XBP1和GRP78表达下降。表明HuR可能调控内质网应激相关蛋白XBP1和GRP78的表达。3、HuR对XBP1 mRNA 3’UTR有调控作用。预测有3处HuR可能的结合位点，且site3和miR-33a-5p结合位点重合。表明HuR和miR-33a-5p存在协同影响XBP1表达的可能。4、PH前后，实验组小鼠GRP78表达量均低于对照组，表明HuR可能通过调节内质网应激影响肝再生。5、高脂喂养实验表明HuR敲除小鼠肝脏的脂质代谢能力可能下降。