PDCD4通过核内RNA/组蛋白调控甘油三酯代谢导致NAFLD/NASH早期肝脂肪变性发生的作用及其机制研究

NAFLD/NASH是严重危害人类健康的最主要慢性肝病，病因不明。我们前期发现PDCD4在动物模型和人脂肪肝肝细胞核中高表达；脂肪酸及过表达使其表达增高且入核、胞浆脂滴生成；质谱分析它与H2AX互作；RNAseq分析它调控4个TG代谢基因；生信分析PPARγ是其共同转录因子，前者可被HDAC3抑制。基于前期发现和PDCD4功能，推测PDCD4可能通过核内RNA/组蛋白调控TG代谢致早期肝脂肪变性发生。本项目拟用基因过表达/敲除或回补、激活/抑制AKT等处理脂肪酸诱导的脂质沉积细胞模型及PDCD4全身性/肝特异性敲除小鼠HFD-NASH模型，确定其调控的TG基因及入核机制；用SPR、RIP等明确其通过H2AX上调MGAT2等及结合RNA或募集eIFs下调ATGL的机制并在临床样本验证，阐明PDCD4通过结合RNA或H2AX调控TG代谢致早期肝脂肪变性发生的机制，为该病防治提供新药靶点和策略。

NAFLD/NASH is the leading cause of chronic liver disease which seriously endangers human health in the world, but its mechanism keep unclear. We previously found that PDCD4 was significantly highly expressed in the hepatocyte nucleus of HFD-NASH model and patients with fatty liver. And fatty acids could increase the expression of PDCD4 in CBRH hepatocytes and translocate it from cytoplasm to the nucleus of hepatocytes. We performed immunoprecipitation of nucleoproteins with PDCD4 antibody, and then MASS analysis found that histone H2AX was the most detected protein. This indicated that H2AX interacted with PDCD4 in nucleus. Overexpression of PDCD4 promotes the generation of lipid droplets, and knockout of PDCD4 can lead to the changes of multiple gene expression in different metabolic pathways and signaling pathways such as glucose and lipid metabolism, cancer and inflammation. Further analysis revealed that there are four differential genes enriched in the triglyceride metabolism pathway. Three of them, MGAT2, DGAT2 and SCD, are the key enzyme regulating anabolism of triglyceride, while the last gene, ATGL, is a key enzyme regulating catabolism of triglyceride. Bioinformation analysis found that PPARγ1 and PPARγ2 were the common transcription factors of these four candidate genes. Our previous experiment found that HDAC3 inhibited the expression of PPARγ. .As we all know, PPARγ regulates its target gene transcription through forming a dimer with RXR and then further forming a complex with histones and co-activators or co-inhibitors such as HDACs and NCOR1. Furthermore, it has been reported that PDCD4 inhibits the target gene translation through directly binding mRNA using its RNA binding domain or through binding eIFs to inhibit protein translation in cytosol or through binding nuclear eIFG to form the nuclear cap binding complex to cause the nonsense-mediated decay mechanism. Therefore, it is speculated that fatty acids activate PDCD4 and translocate it from cytoplasm to nucleus and may regulate triglyceride metabolism through nuclear RNA/histone H2AX and lead to early hepatic steatosis in NAFLD/NASH eventually. .In this project, fatty acid-induced lipid deposition (FFA-LD) cell model and PDCD4 whole-body/liver-specific knockout mouse HFD-NASH model were treated with gene overexpression/knockout (AAV8-mediated) or supplementation of genes, activation/inhibition of AKT, and so on, to determine the mechanism of regulating gene in triglyceride metabolism and nuclear translocation. The mechanism, of which PDCD4 of up-regulates MGAT2，DGAT2 and SCD through H2AX or down-regulates ATGL by binding RNA or recruiting eIFs, will be clarified using ChIP-PCR, Co-IP, SPR, RIP and cryo-EM, which will be verified with clinical samples. The mechanism will be finally clarified, which PDCD4 regulates triglyceride metabolism to cause early hepatic steatosis by binding intranuclear RNA or H2AX. This will provide new drug targets and strategies for the prevention and treatment for NAFLD/NASH.