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的差异表达与糖尿病(DM)有关。该项目的长期目标是了解miRNA在糖尿病中的调节。炎症可诱导肿瘤坏死因子α(TNFa)和基质金属蛋白酶-9(MMP9)，降低白细胞介素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)的诱导物)表达下调，并诱导微管相关蛋白1轻链3(Lc3)、自噬相关基因3(Atg3)和Beclin-1(自噬标记)。我们报道，MMP9损害心肌细胞的收缩能力，增加纤维化，而消融MMP9改善心肌细胞的收缩能力，上调miR-133的表达。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在肥大的心肌细胞中是强大的。为了探讨Mmp9调控miR-133和自噬在糖尿病中的作用机制，我们从糖尿病Ins2+/-Akita小鼠中删除Mmp9基因，建立了双基因敲除(DKO)。DKO(Ins2+/-/Mmp9-/-)小鼠表现出心功能的改善，MEF2C和miR-133的诱导，以及Lc3和Atg3(自噬)的抑制。MiR-133通过靶向MMP9的3‘非编码区，减轻糖尿病患者的心肌MMP9。此外，用miR-133和Mdivi-1(自噬阻滞剂)治疗可以改善秋田糖尿病患者的心功能。该方案的中心假设是炎症激活了MMP9，而MMP9抑制了MEF2C和miR-133，并诱导了自噬，从而导致糖尿病患者的心功能障碍。我们将通过以下三个具体目标来检验这一假说：目的1：确定炎症是否在糖尿病中诱导了TNFa并减弱了miR-233和IL-10。假设：在糖尿病中，炎症诱导TNFa，下调miR-223和IL-10。目的#2：确定糖尿病心肌纤维化是否部分是由于MMP9的激活和MEF2C和miR-133的抑制所致。假设：在糖尿病中，MMP9的激活可以减少MEF2C和miR-133，并诱导心脏纤维化。目的#3：确定自噬是否导致糖尿病患者的心功能不全。假设：在糖尿病患者中，MMP9是强健的，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、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.