MiR-409-3p Regulates Angiogensis, 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。然而，这些组织的适应性调节不良可能导致 胰岛素抵抗我们对血管生成如何影响脂肪组织的理解仍然存在关键差距 功能障碍和整体代谢。microRNAs（miRs）参与血管生成的调控， 对病理生理刺激的反应。miR在调节饮食诱导的血管生成反应中的作用 胰岛素抵抗知之甚少。 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）。miR-409- 3 p的过表达降低了ZEB 1和 MAP 4K 3在EC中的表达，而抑制则具有相反的效果。ZEB 1的siRNA敲低或 MAP 4K 3在EC中的表达表型模仿了miR-409- 3 p过表达的作用，并且显著地抑制了miR-409- 3 p的表达。 降低EC增殖和迁移。3 T3-L1细胞或人皮肤脂肪类器官与 从过表达miR-409- 3 p的EC收获的上清液具有棕色脂肪细胞表达降低的作用， 通过RT-qPCR和蛋白质印迹分析，从EC收获上清液， miR-409- 3 p缺陷增加棕色脂肪细胞标志物的表达。全身静脉给药 通过CD 31染色，给予DIO小鼠LNA-抗miR-409- 3 p抑制剂显著增加血管生成， 通过RT-qPCR、Western blot和免疫组织化学检测，BAT和sWAT中的UCP-1水平较高 分析，同时改善葡萄糖和胰岛素耐受性和整体代谢。因此，我们假设 miR-409- 3 p是脂肪组织中EC生长和血管生成的关键调节因子， 改善DIO中代谢功能障碍。为了探索这一点，我们首先在Aim 1中提出研究分子 miR-409- 3 p调控EC生长和血管生成的机制。在目标2中，我们将描述 EC中miR-409- 3 p调节脂肪组织中布朗宁的机制。最后，在Aim 3中，我们将 探讨miR-409- 3 p中和在脂肪组织血管和DIO发生中的作用 和小鼠的胰岛素抵抗。这些研究的成功完成将使人们对监管作用有更深入的了解。 miR-409- 3 p在饮食诱导的肥胖和脂肪组织功能障碍中血管生成受损之间的作用， 这一效应可用于肥胖诱导的胰岛素抵抗的治疗干预。

PROJECT SUMMARY/ABSTRACT White and brown adipose tissues are highly vascularized organs, capable of plasticity based on metabolic demands and energy expenditure. However maladaptive regulation of these tissues can lead to insulin resistance. 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. 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). Overexpression of miR-409-3p decreased ZEB1 and MAP4K3 expression in ECs, whereas inhibition had the opposite effect. 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 supernatant harvested from ECs overexpressing miR-409-3p had decreased expression of brown adipocyte markers (UCP1, Cidea) by RT-qPCR and Western blot analyses, whereas supernatant harvested from ECs deficient in miR-409-3p increased expression of brown adipocyte markers. Systemic intravenous delivery of LNA-anti-miR-409-3p inhibitor to DIO mice significantly increased angiogenesis by CD31 staining, accompanied by higher UCP-1 in BAT and sWAT by RT-qPCR, Western blot, and immunohistochemistry analyses, while improving glucose and insulin tolerance and overall metabolism. 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. To explore this, we first propose in Aim1 to 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.