miR-449a调控Notch-1/NF-κB信号通路在AGEs促皮肤角质形成细胞MMP-9表达中的作用机制研究

It is well accepted that increased levels of matrix metalloproteinase-9 (MMP-9) could be a contributing factor to poor wound healing in diabetic ulcers. We recently found that advanced glycation end products (AGEs) stimulated MMP-9 expression in keratinocytes via the RAGE, MAPKs pathways and NF-κB. However, the phosphorylation of these MAPKs was not associated with NF-κB activity. These results prompted us to determine in detail the mechanism of NF-κB and MMP-9 activity induced by AGEs in keratinocytes. Our preliminary experiments found that Notch-1 signaling was significantly activated in AGEs-cultured keratinocytes and inhibition of Notch-1 signaling using pharmacological modulators (DAPT) abrogated NF-κB and MMP-9 activation. In addition, the microRNA (miRNA) microarray, which analyzed the differentially expressed miRNA profiles during wound healing in diabetic and normal rats, revealed the miR-449a expression was downregulated in the diabetic wound. Bioinformatic analyses of the predicted target genes combined with our preliminary study showed that the miR-449a was likely to play a role in the regulation of Notch-1. In this project, we will further investigate the role of Notch-1 in activation of NF-κB using adenovirus vector system and siRNA methods, determine the targeted regulation of miR-449a to Notch-1 by luciferase assay and miR-449a lentivirus plasmid system, clarify the role of Notch-1 signaling and miR-449a in the AGEs-induced MMP-9 expression in keratinocytes. In addition, we will observe the effects of Notch-1 signaling and miR-449a on wound healing in diabetic rats. These studies will provide new ideas and strategies for the treatment of clinical diabetic wound healing.

基质金属蛋白酶-9（MMP-9）过表达与糖尿病皮肤创面难愈密切相关，我们已证明晚期糖基化终末产物（AGEs）通过MAPKs和NF-κB信号通路促进皮肤角质形成细胞MMP-9表达，而MAPKs不参与NF-κB活化。NF-κB和MMP-9活化机制如何？预实验发现阻断Notch信号通路后NF-κB活性下降，microRNA芯片技术显示糖尿病大鼠皮肤伤口愈合过程中miR-449a显著下调。本项目拟在前期研究基础上，通过腺病毒过表达技术和siRNA方法获得Notch-1在NF-κB活化中的作用；采用荧光素酶和慢病毒载体构建方法明确miR-449a对Notch-1的靶向调节作用，阐明Notch-1/NF-κB信号通路和miR-449a在AGEs促角质形成细胞MMP-9表达中的作用；并观察干预Notch-1信号通路和miR-449a对糖尿病大鼠皮肤伤口愈合的影响，为临床糖尿病难愈创面的治疗提供新思路。

目的：探讨miR-449a 调控 Notch-1/NF-κB 信号通路在 AGEs 促皮肤角质形成细胞MMP-9 表达中的作用机制。研究设计和方法：（1）免疫组化检测正常和糖尿病患者皮肤组织中Notch-1和MMP-9表达，了解其表达异常在糖尿病皮肤创面难愈过程中的作用。（2）通过实时定量PCR、Western方法检测AGEs干预后Notch-1/NF-κB和MMP-9表达；使用Notch-1 siRNA或Notch-1抑制剂预处理后检测NF-κB和MMP-9表达，探讨Notch-1在AGEs/MMP-9表达中的作用。（3）构建Notch-1荧光素酶报告基因载体，检测miR-449a对Notch-1的靶向调控。将携带有miR-449a基因的慢病毒感染细胞，检测Notch-1和MMP-9蛋白表达，进一步明确miR-449a调控Notch-1对MMP-9的影响。（4）制备糖尿病皮肤病变动物模型，检测不同时间点局部组织中蛋白的表达，阐明miR-449a和Notch-1在体内参与了AGEs促MMP-9表达。结果：（1）与非糖尿病皮肤相比，糖尿病患者皮肤创面中MMP-9和Notch-1表达水平显著升高。（2）在一定浓度范围内（50-300ug/ml）AGEs呈剂量依赖性的促进Notch-1和MMP-9表达。（3）Notch-1 siRNA显著抑制Notch-1表达，AGEs刺激后与对照组相比，NF-κB的p65核转移能力下降，MMP-9分泌减少；与对照组相比，DAPT预处理细胞30min后，NF-κB p65表达减少，MMP-9活性下调。（4）AGEs能够促进Notch-1蛋白表达和MMP-9活性增加，但同剂量（50ug/ml）AGEs干预后，Lenti-GFP组MMP-9活性和Notch-1蛋白水平均高于Lenti-GFP-miR-449a组（p<0.05）。（5）糖尿病组皮肤创面愈合过程中Notch-1和MMP-9表达显著升高，miR-449a的表达明显减少。DAPT干预后，不同时间点皮肤组织中Notch-1蛋白表达均明显降低，同时抑制NF-κB荧光素酶活性和MMP-9表达。结论：AGEs通过抑制miR-449a表达来调控Notch-1/NF-κB活性，进而促进细胞MMP-9过表达，干预Notch-1/NF-κB信号通路和miR-449a对糖尿病皮肤创面具有促愈作用。