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，但分子机制尚不清楚。miRNA的差异表达与糖尿病（DM）相关。该项目的长期目标是了解DM中miRNA的调控。炎症诱导肿瘤坏死因子α（TNF α）和基质金属蛋白酶-9（Mmp 9），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（Mef 2C：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（LC 3）、自噬相关基因3（Atg 3）和beclin-1（自噬标记物）被诱导。我们报道了Mmp 9损害心肌细胞的收缩性并增加纤维化，而Mmp 9的消融改善收缩性并上调miR-133。然而，Mmp 9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，TNF α被诱导，IL-10和miR-223被减弱。有趣的是，TNFa在肥大心肌细胞中是稳健的。为了研究Mmp 9介导的miR-133调控和自噬在糖尿病中的作用机制，我们通过删除糖尿病Ins 2 +/-秋田小鼠的Mmp 9基因来建立双敲除（DKO）模型。DKO（Ins 2 +/- /Mmp 9-/-）小鼠显示心脏功能改善、Mef 2C和miR-133的诱导以及LC 3和Atg 3（自噬）的抑制。miR- 133通过靶向Mmp 9的3 'UTR减轻糖尿病中的心肌Mmp 9。此外，用miR- 133和Mdivi-1（自噬阻断剂）治疗改善了糖尿病秋田患者的心脏功能。该提案的中心假设是炎症激活Mmp 9，Mmp 9抑制Mef 2c和miR-133，并诱导自噬，导致DM中的心功能障碍。我们将通过以下三个具体目标来检验该假设：目标#1：确定糖尿病中炎症是否诱导TNF α并减弱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中Mmp 9的激活以及Mef 2C和miR-133的抑制。假设：在糖尿病中，Mmp 9的激活减弱Mef 2c和miR-133，并诱导心脏纤维化。目的3：确定自噬是否会导致糖尿病患者的心功能障碍。假设：在糖尿病中，Mmp 9是强大的，它诱导自噬，导致心脏功能障碍。 这些研究将揭示炎症、miR-133和-223、Mmp 9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Mmp 9在糖尿病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.