MiR-409-3p regulates angiogenesis, brown fat adiposity, and insulin resistance

项目总结/摘要 白色和棕色脂肪组织是高度血管化的器官，能够基于 代谢需求和能量消耗。在我们对血管生成 影响脂肪组织功能障碍和整体代谢。microRNAs（miRs）参与了 血管生成对病理生理刺激的反应。然而，miRs在调节 饮食诱导的胰岛素抵抗中的血管生成反应知之甚少。 使用miRNA-Seq方法，我们确定miR-409- 3 p表达在人乳腺癌中显著增加， 饮食诱导肥胖（DIO）小鼠和人类糖尿病患者棕色脂肪组织（BAT）的内皮细胞（EC） 血浆样品与非糖尿病患者相比。miR-409- 3 p过表达可显著抑制EC 生长和迁移，而miR-409- 3 p抑制具有相反的效果。初步研究表明， miR-409- 3 p靶向锌指E-box结合同源异型盒1（ZEB 1）的3 'UTR和丝裂原活化的 蛋白激酶3（MAP 4K 3）。siRNA敲低EC中的ZEB 1或MAP 4K 3表达 表型模拟了miR-409- 3 p过表达的作用，并显著降低了EC增殖， 迁移与来自EC的条件培养基共培养的3 T3-L1细胞或人皮肤脂肪类器官 过表达miR-409- 3 p降低了BAT标记物（UCP 1，Cidea）的表达，而条件表达则降低了BAT标记物（UCP 1，Cidea）的表达。 来自miR-409- 3 p缺陷的EC的培养基显著增加BAT基因表达的表达。 从机制上讲，胎盘生长因子（PLGF）响应于miR-409- 3 p抑制而从EC分泌 促进3 T3-L1向棕色样脂肪细胞分化，如通过增加的UCP 1表达所测量的。 将LNA-抗-miR-409- 3 p抑制剂全身递送至DIO小鼠显著增加血管生成， 通过CD 31染色测量BAT和皮下白色脂肪组织（sWAT）中的UCP-1表达，而 改善葡萄糖和胰岛素耐受性和能量消耗。因此，我们假设miR-409- 3 p 作为脂肪组织中EC生长和血管生成的关键调节剂， DIO中的功能障碍。因此，在Aim 1中，我们将研究miR-409- 3 p在细胞内表达的分子机制。 调节EC生长和血管生成。在Aim 2中，我们将描述miR-409- 3 p在细胞内表达的机制。 EC调节脂肪组织中的布朗宁。最后，在Aim 3中，我们将探讨miR-409- 3 p对细胞凋亡的影响。 在脂肪组织的脉管系统中中和以及在小鼠中DIO和胰岛素抗性的发展。 这些研究的成功完成将有助于深入了解miR-409- 3 p在以下两个方面的调节作用： 在饮食诱导的肥胖和脂肪组织功能障碍中血管生成受损， 用于治疗肥胖引起的胰岛素抵抗。 !

PROJECT SUMMARY/ABSTRACT White and brown adipose tissues are highly vascularized organs, capable of plasticity based on metabolic demands and energy expenditure. Critical gaps remain in our understanding of how angiogenesis impacts adipose tissue dysfunction and overall metabolism. MicroRNAs (miRs) are implicated in the regulation of the angiogenic response to pathophysiological stimuli. However, the role of miRs in regulating the angiogenic response in diet-induced insulin resistance is poorly understood. Using a miRNA-Seq approach, we identified that miR-409-3p expression was significantly increased in endothelial cells (ECs) of brown adipose tissue (BAT) of diet-induced obese (DIO) mice and in human diabetic plasma samples compared to non-diabetic patients. Overexpression of miR-409-3p markedly inhibited EC growth and migration, whereas miR-409-3p inhibition had the opposite effects. Preliminary studies indicate that miR-409-3p targets the 3'UTRs of Zinc Finger E-box binding Homeobox 1 (ZEB1) and Mitogen-activated protein kinase kinase kinase kinase 3 (MAP4K3). SiRNA knockdown of ZEB1 or MAP4K3 expression in ECs phenocopied the effects of miR-409-3p overexpression and significantly decreased EC proliferation and migration. 3T3-L1 cells or human skin fat organoids co-cultured with conditioned media from ECs overexpressing miR-409-3p had decreased expression of BAT markers (UCP1, Cidea), whereas conditioned media from ECs deficient in miR-409-3p markedly increased expression of BAT gene expression. Mechanistically, Placental Growth Factor (PLGF), secreted from ECs in response to miR-409-3p inhibition promoted 3T3-L1 differentiation towards brown-like adipocytes as measured by increased UCP1 expression. Systemic delivery of LNA-anti-miR-409-3p inhibitor to DIO mice significantly increased angiogenesis as measured by CD31 staining, UCP-1 expression in BAT and subcutaneous white adipose tissue (sWAT), while improving glucose and insulin tolerance and energy expenditure. Therefore, we hypothesize that miR-409-3p serves as a critical regulator of EC growth and angiogenesis in adipose tissue and may improve metabolic dysfunction in DIO. Thus, in Aim1 we will investigate the molecular mechanisms by which miR-409-3p regulates EC growth and angiogenesis. In Aim2, we will delineate the mechanisms by which miR-409-3p in ECs regulates browning in adipose tissues. Finally, in Aim3, we will explore the effect of miR-409-3p neutralization in the vasculature of adipose tissues and development of DIO and insulin resistance in mice. Successful completion of these studies will shed insights on the regulatory role of miR-409-3p between impaired angiogenesis in diet-induced obesity and adipose tissue dysfunction, an effect that could be exploited for therapeutic intervention in obesity-induced insulin resistance. !