基于microRNA探讨双酚A致肝脏糖脂代谢紊乱的分子机制及父系传代效应

Bisphenol A (BPA)， a well-known environmental endocrine disrupting chemical, is used extensively in daily life. Humans can be exposed to BPA present in commonly used products. BPA exposure is now clearly recognized as a serious public health problem. Recently, growing evidence has indicated that exposure to BPA is associated with an increased risk of common metabolic diseases including insulin resistance, fatty liver disease，type 2 diabetes and etc., but the underlying molecular mechanisms are limited. In previous study, we found that exposure to BPA down-regulated miR-192 in the liver of male C57BL/6 mice, thereby contributing to the hepatic steatosis. So we suggest that miRNA can be a key regulator of lipid and glucose metabolism. In this study, we will firstly screen the potential miRNAs involved in the modulation of glucose and lipid metabolism in the liver of BPA-exposed male mice, respectively. And then, essential target genes and pathway network regulated by miRNA will be identified by performing modern molecular biology experiments. After that, intervention experiments will be performed to ensure the possibility that disorders of lipid and glucose metabolism induced by BPA are selectively mediated via the miRNA-mRNA interaction network. In addition, we intend to clarify the causal relationship between BPA exposure induced excessive hepatic lipid accumulation and insulin resistance. Furthermore, considering that paternal environmental exposure can influence disease risk in offspring, we will also investigate whether paternal BPA exposure disrupt the lipid and glucose homeostasis in the mice offspring, and whether these paternal effects are mediated via epigenetic marks in male germ cells. Taken together, this study is expected to address the mechanism of BPA-induced impairment of glucose and lipid homeostasis at molecular level, and furthermore, to create new insights into the role of environmental pollutants on chronic metabolic disease.

BPA暴露是一个不容忽视的公共卫生问题。研究指出，BPA可通过干扰机体的糖脂代谢稳态，诱导胰岛素抵抗、脂肪肝、2型糖尿病等疾病的发生发展，但机制尚不明确。项目前期研究发现，过表达miR-192有效干预了BPA暴露引起的雄性C57BL/6小鼠肝脏脂质蓄积，提示miRNA可能在BPA暴露致糖脂代谢紊乱的过程中发挥关键调控作用。所以，本项目拟筛查BPA暴露的雄性小鼠肝脏中介导糖代谢和脂代谢的主效miRNA，结合生物信息学分析，研究miRNA-mRNA的相互作用，构建主效miRNA的靶基因调控网络及主导分子信号通路，阐明BPA暴露致肝脏脂质蓄积与胰岛素抵抗之间的相互联系。以此为基础，项目还拟建立父系BPA暴露的动物模型，通过检测父本精子的表观遗传学改变探讨BPA暴露致父本肝脏糖脂代谢紊乱的传代效应及miRNA介导的分子机制。项目有望为环境化合物暴露致慢性代谢性疾病的发病机制研究提供新的思路与线索。

环境内分泌干扰物可通过干扰机体的能量代谢过程，诱导肥胖、胰岛素抵抗、脂肪肝、2型糖尿病等慢性代谢性疾病的发生发展，但机制尚不明确。本项目以miRNA-mRNA互作调控为切入点，分别筛查了典型环境内分泌干扰物BPA和DEHP诱导机体脂代谢紊乱和糖代谢异常病理过程中的主效miRNA，阐明其与靶基因的相互作用并构建主导分子调控信号通路，获得的研究结果较为理想且具创新性：（1）BPA可抑制肝脏中的核酸内切酶DROSHA，干扰miR-192的初级转录本转变为前体，影响miR-192的生物合成过程，最终导致成熟miR-192表达下调；miR-192靶向作用于SREBF1的3'UTR，特异调控BPA诱导的肝脏脂质蓄积；恢复miR-192表达虽然改善了肝脏脂质蓄积，但并没有阻断BPA暴露引起的高胰岛素血症和胰岛素抵抗，由此首次提出BPA暴露导致的胰岛素抵抗不依赖于肝脏脂质蓄积。（2）miR-338作为胰岛素分泌的重要调节因子，靶向调控胰腺关键转录因子Pdx1，调节胰岛细胞的胰岛素分泌功能。短期BPA暴露通过Gpr30下调miR-338，Pdx1表达升高，促进胰岛素分泌；长期BPA暴露则通过Glp1r上调miR-338，抑制Pdx1并诱导细胞凋亡，导致胰岛素分泌能力下降。BPA诱导外周胰岛素抵抗与胰岛细胞miR338-Pdx1信号通路动态调控胰岛素分泌由代偿增加向失代偿衰竭转变相关。（3）Dnmt3a依赖的启动子甲基化和lncRNA Malat1的“分子海绵功能”功能在DEHP暴露的骨骼肌细胞中协同下调miR-17。miR-17直接作用于Keap1和Txnip的3'UTR，影响骨骼肌的氧化还原平衡，使细胞产生氧化应激。氧化应激进一步上调了miR-200a，miR-200a靶向抑制Insr和Irs1，导致Akt介导的胰岛素信号通路转导障碍和骨骼肌葡萄糖摄取能力降低，最终促进胰岛素抵抗发生。AAV9介导的骨骼肌miR-17特异过表达和慢病毒介导的miR-200a沉默显著改善了DEHP暴露导致的胰岛素抵抗。综上，项目研究成果为环境化合物暴露致胰岛素抵抗、脂肪肝、2型糖尿病等慢性代谢性疾病的发病机制研究提供了新的思路与线索，并有助于寻找相关诊断标志物和治疗靶点。项目发表论文8篇，培养博士研究生2名，2名主要参与者由中级职称晋升为高级职称，超额完成研究计划，达到预期的研究目标。