miR-200a在DEHP暴露致骨骼肌胰岛素抵抗过程中的调控机制研究

Di-(2-ethylhexyl) phthalate (DEHP) is one of the ubiquitous environmental endocrine disruptors, which is widely used as plasticizers in food packaging, children’s products, medical devices, household products and industrial plastic, etc. Human can be continuously and inevitably exposed to DEHP from multiple sources. Recently, growing evidence has indicated that exposure to DEHP is associated with an increased risk of insulin resistance, but the underlying molecular mechanisms are limited. MicroRNA (miRNA) is a group of single stranded small non-coding RNA, which has a critical role in posttranscriptional regulation of key genes involved in insulin signaling pathway, contributing to the development of insulin resistance. Skeletal muscle has been considered to be a major regulator of systemic glucose homeostasis. In our present study, we found that exposure to DEHP (2 or 20 mg/kg/day) induced insulin resistance and decreased the insulin-induced phosphorylation of AKT (S473/T308) in skeletal muscle in C57BL/6 mice. Moreover, expression of miR-200a was increased in the DEHP-exposed skeletal muscle. Bioinformatics-based studies suggested that 3’UTR of some key insulin signaling genes, such as Igf1r, Insr, Irs1 and pik3r1, are targets of miR-200a, so we hypothesized that miR-200a is a critical regulator of DEHP-induced insulin resistance in skeletal muscle. In the present study, we generated in-vivo mice model and in-vitro cell line model to test whether exposure to DEHP was able to induced insulin resistance in skeletal muscle, and if so, whether these adverse effects were mediated by impaired regulation of miR-200a-PI3K/Akt signaling pathway. We firstly tested the effects of exposure to DEHP on the whole-body glucose homeostasis and the components/modulators of PI3K/Akt insulin signaling pathway in skeletal muscle; and moreover, investigated the dose/time/response relationship. Then, we determined the expression pattern of miR-200a in DEHP-exposed skeletal muscle; screened and identified miR-200a regulated essential target genes in PI3K/Akt insulin signaling pathway network. After that, intervention experiments were performed to ensure the possibility that exposure to DEHP impaired insulin signaling in skeletal muscle by selectively regulation of miR-200a-PI3K/Akt interaction network. Taken together, this study is expected to address the mechanism of DEHP-induced insulin resistance at molecular levels, and furthermore, to evaluate the feasibility of miRNA as biomarkers.

DEHP暴露是胰岛素抵抗的风险因素之一，但作用机制研究报道较少。miRNA作为内源性小分子RNA，可通过调控胰岛素信号通路参与胰岛素抵抗的发生。骨骼肌是胰岛素抵抗发生的主要部位。前期研究发现，DEHP暴露导致小鼠骨骼肌中miR-200a表达及胰岛素刺激下Akt磷酸化水平显著变化。所以，本项目拟以C57BL/6小鼠和C2C12细胞株为研究对象，系统分析DEHP暴露致骨骼肌胰岛素抵抗发生的时程和量效规律；研究DEHP暴露对骨骼肌PI3K/Akt胰岛素信号通路的影响；探讨DEHP暴露诱导骨骼肌miR-200a表达变化的规律和特点；筛查DEHP暴露诱导骨骼肌PI3K/Akt信号通路转导阻滞过程中受miR-200a调控的靶基因并构建miR-200a-mRNA相互作用的分子调控网络；最终揭示DEHP暴露诱导骨骼肌胰岛素抵发生的分子机制。本项目有望找到DEHP暴露致胰岛素抵抗发生的潜在生物标志物。

近年来的研究显示，环境内分泌干扰物暴露也是导致人群糖尿病发生的一个不可忽视的危险因素。本项目选取了两种典型的内分泌干扰物邻苯二甲酸二(2-乙基)己酯(DEHP)和双酚A (BPA)作为研究对象，展开其致机体胰岛素抵抗、胰岛β细胞分泌功能异常及非酒精性脂肪肝发生中分子机制。本项目现已取得较为理想的研究成果：(1) 研究DEHP暴露对小鼠葡萄糖耐量、胰岛素耐量以及骨骼肌中Akt依赖的胰岛素信号通路和氧化应激通路关键基因和蛋白表达的影响。结果表明：DEHP暴露显著降低小鼠的葡萄糖耐量和胰岛素耐量，引起胰岛素抵抗的发生。鉴于骨骼肌在胰岛素抵抗中的关键作用，发现DEHP暴露抑制小鼠骨骼肌中Insr和Irs1的表达水平，干扰Akt依赖的胰岛素信号通路与骨骼肌中Keap1-Nrf2抗氧化信号通路障碍相关。抗氧化剂NAC处理，通过改善Keap1-Nrf2抗氧化信号通路，可恢复Insr和Irs1的表达水平，从而保护了DEHP诱导的胰岛素抵抗的发生。细胞水平研究发现，诱导分化后的C2C12细胞经DEHP暴露引起的胰岛素抵抗是与DEHP下调miR-17介导氧化应激，引起miR-200a表达上调，从而靶向抑制Insr和Irs1有关。(2) 探究miRNA在BPA暴露对小鼠胰岛细胞分泌功能紊乱中的作用。结果表明：miR-338可以作为胰岛素分泌的重要调节因子，miR-338通过与胰腺关键转录因子Pdx1相互作用，能够直接调节胰岛素分泌功能。短期BPA暴露导致原代培养的小鼠胰岛细胞中Pdx1表达升高，而长期BPA暴露导致原代培养的小鼠胰岛细胞中Pdx1表达显著受抑。短期BPA暴露可通过Gpr30下调miR-338的表达，促进胰岛素分泌；长期BPA暴露则通过Glp1r上调miR-338的表达，通过引起胰岛细胞凋亡发生，导致胰岛素分泌能力下降。(3) 探究BPA暴露如何调控SREBF1导致非酒精性脂肪肝发生。结果表明：BPA暴露通过干扰miRNA的生成机制抑制miR-192表达下降，miR-192可结合到SREBF1的3’UTR，通过促进SREBF1的表达引起肝脏脂质蓄积，导致非酒精性脂肪肝发生。项目已正式发表2篇SCI研究论文，1篇正在投稿中；培养硕士研究生2名。项目完成预定研究计划，达到预期的研究目标。