Inflammation, miRNA and autophagy in diabetes

描述（由申请人提供）：尽管在了解糖尿病并发症方面取得了巨大的进步，但其分子机制尚不清楚。miRNA差异表达与糖尿病（DM）有关。该项目的长期目标是了解miRNA在DM中的调控作用。炎症诱导肿瘤坏死因子α (TNFa)和基质金属蛋白酶9 (Mmp9)，并减弱白细胞介素-10 （IL-10）和miR-223。此外，肌球蛋白增强因子-2c （Mef2C: miR-133（抗肥大和抗纤维化）的诱导剂）下调，微管相关蛋白1轻链3(LC3)，自噬相关基因3（Atg3）和beclin-1（自噬标志物）被诱导。我们报道了Mmp9损害心肌细胞的收缩性并增加纤维化，而消融Mmp9可改善收缩性并上调miR-133。然而，Mmp9和miR-133轴在自噬和DM中的作用尚不清楚。我们的初步研究表明，在糖尿病心脏中，TNFa被诱导，IL-10和miR-223被减弱。有趣的是，TNFa在肥厚型心肌细胞中表现强劲。为了研究Mmp9介导的miR-133调控和糖尿病自噬的机制，我们通过删除糖尿病in2 +/-秋田小鼠的Mmp9基因创建了双敲除（DKO）。DKO （Ins2+/- /Mmp9-/-）小鼠显示心功能改善，诱导Mef2C和miR-133，抑制LC3和Atg3（自噬）。miR- 133通过靶向Mmp9的3'UTR来减轻糖尿病患者的心肌Mmp9。此外，miR- 133和Mdivi-1（自噬阻滞剂）治疗可改善糖尿病秋田犬的心功能。该提案的中心假设是炎症激活抑制Mef2c和miR-133的Mmp9，并诱导自噬导致糖尿病患者心功能障碍。我们将通过以下三个具体目标来验证这一假设：目的1：确定炎症是否诱导TNFa并减弱糖尿病患者的miR-233和IL-10。假设：在糖尿病中，炎症诱导TNFa并下调miR-223和IL-10。目的2：确定心脏纤维化是否部分归因于糖尿病患者Mmp9的激活和Mef2C和miR-133的抑制。假设：在糖尿病中，Mmp9的激活减弱了Mef2c和miR-133，并诱导了心脏纤维化。目的3：确定自噬是否导致糖尿病患者心功能障碍。假设：在糖尿病中，Mmp9是强大的，它诱导自噬导致心功能障碍。这些研究将揭示炎症、miR-133和-223、Mmp9和自噬在糖尿病心功能障碍中的作用。这也将为评估miR- 133和Mmp9在糖尿病性心肌病中的治疗潜力提供动力。

DESCRIPTION (provided by applicant): Despite the gigantic stride made towards understanding of diabetic complications, the molecular mechanism is unclear. Differential expression of miRNA is associated with diabetes mellitus (DM). The long term goal of the project is to understand the regulation of miRNA in DM. Inflammation induces tumor necrosis factor alfa (TNFa) and matrix metalloproteinase-9 (Mmp9), and attenuates interleukin-10 (IL-10) and miR-223. Also, myosin enhancer factor -2c (Mef2C: an inducer of miR-133(anti-hypertrophy and anti-fibrosis)) is down regulated and microtubule-associated protein1 light chain3 (LC3), autophagy related gene3 (Atg3) and beclin-1 (markers of autophagy) are induced. We reported that Mmp9 impairs contractility of cardiomyocytes and increases fibrosis, whereas ablation of Mmp9 improves contractility and up regulates miR-133. However, the role of Mmp9 and miR-133 axis in autophagy and DM is unknown. Our preliminary studies show that TNFa is induced and IL-10 and miR-223 are attenuated in diabetic hearts. Interestingly, TNFa is robust in hypertrophic cardiomyocytes. To investigate the mechanism of Mmp9 mediated regulation of miR-133 and autophagy in diabetes, we created double knock out (DKO) by deleting Mmp9 gene from diabetic Ins2+/- Akita mice. DKO (Ins2+/- /Mmp9-/-) mice revealed improvement in cardiac function, induction of Mef2C and miR-133, and inhibition of LC3 and Atg3 (autophagy). The miR- 133 mitigates myocardial Mmp9 in diabetes by targeting 3'UTR of Mmp9. In addition, treatment with miR- 133 and Mdivi-1(blocker of autophagy) improved cardiac function in diabetic Akita. The central hypothesis of the proposal is that inflammation activates Mmp9 that inhibits Mef2c and miR-133, and induces autophagy resulting into cardiac dysfunction in DM. We will test the hypothesis by the following three specific aims: Aim#1: To determine whether the inflammation induces TNFa and attenuates miR-233 and IL-10 in diabetes. Hypothesis: In diabetes, inflammation induces TNFa and down regulates miR-223 and IL-10. Aim# 2: To determine whether the cardiac fibrosis is due, in part, to activation of Mmp9 and inhibition of Mef2C and miR-133 in diabetes. Hypothesis: In diabetes, activation of Mmp9 attenuates Mef2c and miR-133, and induces cardiac fibrosis. Aim # 3: To determine whether the autophagy causes cardiac dysfunction in diabetes. Hypothesis: In diabetes, Mmp9 is robust and it induces autophagy causing cardiac dysfunction. These studies will unravel the roles of inflammation, miR-133 and -223, Mmp9 and autophagy in cardiac dysfunction in diabetes. It will also provide impetus to assess the therapeutic potential of miR- 133 and Mmp9 in diabetic cardiomyopathy.