【作者】 孙瑜；

【导师】 郑凌；

【作者基本信息】 武汉大学 ， 细胞生物学， 2020， 博士

【摘要】 全球肥胖的患病率正持续增加,肥胖作为肝细胞癌(hepatocellular carcinoma,HCC)等疾病的主要危险因素正威胁着人类的健康。目前,大约有三分之一的育龄妇女受肥胖的影响,且肥胖母亲的后代在成年后易患肥胖及相关代谢疾病。但是,母系肥胖与子代肝癌发生发展之间的关系以及其调控机制尚有待研究。本论文应用二乙基亚硝胺(diethylnitrosamine,DEN)在多代高脂喂养的肥胖母鼠后代中诱导HCC,探索母系肥胖与子代肝癌发生发展之间的关系以及其调控机制。实验发现,随着母鼠高脂喂养代数的增加,其后代小鼠患HCC的严重程度逐代加剧。通过对小鼠肝脏肿瘤进行RNA-seq测序发现miR-27a-3p及其靶基因Acsl1和Aldh2的表达量都受母系高脂喂养代数的影响出现传代性变化。在体外实验和裸鼠皮下成瘤实验中,发现高脂生长环境中,下调Acsl1和Aldh2能够协同促进细胞增殖和肿瘤生长。同时,在裸鼠皮下成瘤实验中,向瘤内注射miR-27a-3p激动剂(agomir),能够通过下调Acsl1和Aldh2而加剧肿瘤生长;相反的,向瘤内注射miR-27a-3p拮抗剂(antagomir),能够通过上调Acsl1和Aldh2而抑制肿瘤生长。更重要的是,在多代高脂食物喂养下,母鼠血清中miR-27a-3p的水平也随着高脂喂养代数的增加而逐代升高。向孕鼠体内注射miR-27a-3p agomir,发现其子代小鼠包括胎鼠、幼鼠和成年小鼠的肝脏中miR-27a-3p显著性升高,而Acsl1和Aldh2的表达量显著性下调;对这些幼鼠进行DEN刺激,发现孕鼠体内注射miR-27a-3p agomir会加重子代小鼠HCC的发生发展。进一步通过TCGA分析显示,HCC病人中Acsl1和Aldh2显著性下调,且Acsl1和Aldh2的下调以及miR-27a-3p的上调与HCC病人生存率之间存在显著相关性。并且,通过对临床HCC病人肝脏切片进行免疫组化和原位杂交实验,发现在HCC病人脂肪肝和非脂肪肝的肿瘤和非肿瘤组织中,miR-27a-3p与Acsl1或Aldh2的表达量呈负相关。综上所述,本论文表明,在母系肥胖中,miR-27a-3p-Acsl1/Aldh2调控轴可调控代间代谢变化,影响子代肝癌发生发展。更多还原

【Abstract】 The prevalence of obesity is increased globally and is threatening human health.Obesity is a major risk factor for multiple diseases such as hepatocellular carcinoma(HCC).At present,about one third of women of childbearing age are affected by obesity,and the offspring of obese mothers are prone to develop obesity and related metabolic diseases in adulthood.However,the relationship between maternal obesity and the development of HCC in offspring and its underlying mechanism remain unclear.In this study,diethylnitrosamine(DEN)was used to induce HCC in the mouse offspring of multigenerational high-fat diet(HFD)feeding obese mothers to explore the relationship between maternal obesity and the development of HCC in offspring.It was found that with the increase of generation of HFD-fed in mother mice,the severity of HCC in their offspring increased over generations.Through RNA-seq in mouse liver tumors,it was found that the expression levels of miR-27a-3p and its target genes,Acsl1 and Aldh2,were influenced by generation of maternal HFD feeding and presented transgenerational changes.In vitro experiment and the tumor xenografts experiment both demonstrated that down-regulation of Acsl1 and Aldh2 could synergistically promote cell proliferation and tumor growth in hyperlipidmia growth environment.Meanwhile,in the tumor xenografts experiment,the injection of miR-27a-3p agomir into the subcutaneous tumor could aggravate the tumor growth by down-regulating Acsl1 and Aldh2 levels.Contrastly,the subcutaneous injection of miR-27a-3p antagomir could inhibited tumor growth by up-regulating Acsl1 and Aldh2 levels.More importantly,the serum levels of miR-27a-3p in female mice stimulated by multigenerational high-fat feeding also increased with generation.When miR-27a-3p agomir was injected into pregnant mice,miR-27a-3p was significantly increased in the liver of its offspring,including fetal mice,young mice and adult mice,while the expression levels of Acsl1 and Aldh2 were significantly down-regulated.Aggravation of the development of HCC was found in the offspring of miR-27a-3p agomir injected pregnant mice.Further TCGA analysis showed that Acsl1 and Aldh2 were significantly down-regulated in HCC patients,and there was significant correlation between the down-regulation of Acsl1 and Aldh2,the up-regulation of miR-27a-3p with the survival rate of HCC patients.In addition,immunohistochemistry and in situ hybridization experiments were performed on liver sections of HCC patients,and miR-27a-3p levels was found to be negatively correlated with the expression levels of Acsl1 or Aldh2 in tumor and non-tumor tissues of fatty liver and non-fatty liver of HCC patients.In summary,this study indicates that the miR-27a-3p-Acsl1/Aldh2 regulatory axis can regulate intergenerational metabolic changes and affect the development of HCC in the offspring of maternal obesity.更多还原