C/EBPα异构体p30在急性肝损伤修复中的作用及机制

C/EBPa-p42 is a putative proliferation suppressor. However, among the HCC cell lines, the Hep3B and Huh7 expressed high levels of C/EBPa, which were violating the progression-repression feature of C/EBPa-p42. Knocking-down its expression reduced target-gene expression, colony formation, and cell growth, associated with a decrease in cyclin A and CDK4 concentrations and E2F transcriptional activity. C/EBPa-p30 is an 30kDa isoform which retains the DNA-binding domain, while lacks the N-terminal transactivation domain and observed as dominant-negative over p42 for years. Recently, different functions were reported that p30 could act as a transcriptional activator of the PIN1 and miR-181a genes. Moreover, mouse models have shown that the increased expression of C/EBPa-p30 had induced leukemia transformation. These results were in good consistency with previous reported date that cebpa was up-regulated in a subset of human hepatocellular carcinomas (HCCs) compared with adjacent nontumor tissues. Thus the regulation and function of C/EBPa-p30 have necessity to be investigated. There're only a few studies reported p30, mainly in leukemia background, very few related with liver functions. As C/EBPa was a transcriptional factor,CCl4 induced acute liver injury model is suitable to clarify its role in liver auto-reparing process...Because epigenetic control of liver function has been previously implicated in the regulation of liver proliferation, we generated C/EBPa-p42 ＆ C/EBPa-p30 adenovirus to induce in vivo over-expression.The C/EBPa-p30 over-expressed mice model were made by tail-vein injection of adenovirus ADV-C/EBPA-p30, the ADV-C/EBPA-p42 and ADV-CTR were controlled groups. p30 over expression mice have altered liver morphology and altered liver function leading to changes of glucose metabolism and blood parameters. These animals have increased liver proliferation in response to carbon tetrachloride (CCl4)-mediated acute injury as well as liver surgery. Examination of the proliferative capacity of C/EBPa-p30 livers showed that livers of these mice have a higher rate of proliferation at the early stage of liver regeneration...The transcriptome analysis has been performed with RNA-sequencing technique from the liver specimens of 0h, 24h, 48h since CCl4-injection. Plenty of potential target genes, including coding and non-coding genes, has been screened out, and potential transcription binding site has been evaluated by the bioinformatic analysis PROMO and ENCORE. The function and mechanism studies would be finished in vitro, with AML-12 cell lines. C/EBPa expression was knocked-down by small interfering RNA, and stimulated by adenovirus. Then, the expression-level of target genes could be manipulated by genetic-altered plasmids. The regulation of C/EBPa-p30 would be studied by quantitative reverse transcription–polymerase chain reaction (qRT- PCR), Western blotting, immunohistochemistry, methylation-specific PCR, and chromatin immunoprecipitation assays. Functional assays included colony formation, methylthiotetrazole, bromodeoxyuridine incorporation, and luciferase-reporter assays may probably be adopted under necessity. ..The aims of this study were to elucidate mechanisms that are involved in the regulation and function of the C/EBPa-p30 for liver auto-reparing process after acute liver injury .

肝炎对人民身体健康造成了严重的损害，促进肝脏的自主修复是肝炎治疗的主要手段，而转录因子在肝脏损伤修复过程中发挥了重要的调控作用。转录因子C/EBPα具有多个异构体，此前认为其42kDa的p42异构体是唯一的功能蛋白，但近年研究发现其30kDa的异构体p30亦具有调控作用。前期我们发现急性肝功能衰竭患者的肝组织p30异常下调且p42高表达，与正常肝脏、硬化肝脏差异明显；通过小鼠急性肝损伤模型证实p30可减轻肝脏损伤并促进肝细胞增殖。为了阐明p30调控肝损伤修复的机制，本项目经转录组测序已确定多个差异表达基因，拟分析p30与初筛基因的结合位点，确定转录调控区域，明确其下游靶基因；采用分子细胞学方法结合实验动物模型，研究p30在肝损伤修复过程中的生物学功能及调控机制。通过本课题的实施有助于阐明肝再生过程中的一个重要环节，为后续转化医学研究奠定基础。

肝炎对人民身体健康造成了严重的损害，促进肝脏的自主修复是肝炎治疗的主要手段，而转录因子在肝脏损伤修复过程中发挥了重要的调控作用。转录因子C/EBPα具有多个异构体，此前认为其42kDa的p42异构体是唯一的功能蛋白，但近年研究发现其30kDa的异构体p30亦具有调控作用。通常认为C/EBPα具有驱动终末分化和抑制细胞增殖的能力，而将C/EBPα的Y285A或R297A进行点突变后，失去调控髓系血细胞及脂肪细胞分化的功能，p30恰恰缺失了这些N端氨基酸突变位点。体外实验模型验证了p30有促进肝细胞增殖的作用。小鼠四氯化碳急性肝损伤模型显示过表达C/EBPα-p30可减轻小鼠肝细胞凋亡、促进肝细胞再生。通过转录组测序分析，p30特异性地调控了数百个基因，通过对下游基因的通路富集分析，p30过表达的小鼠在细胞周期、代谢、生理功能等方面对下游基因的调控存在明显差异。肝脏修复早期，C/EBPα-p30可调控Cyclin B1、Cyclin E1、CDK1、CDK2、CDK4等细胞周期相关基因明显表达上调；在肝再生的第2-3天，p30可调控Cyclin E1、CDK1等基因明显表达上调，促进肝细胞增殖；在小鼠70%肝切除模型中，C/EBPα-p30过表达后肝脏再生速度明显增快。本课题的实施发现了p30的潜在临床应用价值，为急性肝损伤和急性肝功能衰竭的临床治疗提供了潜在靶点。