MiR-21通过调控PTEN 参与TIMP-1抗糖尿病皮肤细胞凋亡的作用及机制研究

We recently found that the decreased level of tissue inhibitors of metalloproteinase-1（TIMP-1) could be a contributing factor to poor wound healing in diabetic ulcers. The local application of TIMP-1 containing hydrogels was effective to accelerate the rates of wound closure for diabetic rats.In addition，TIMP-1 protects human fibroblast from AGEs-induced apoptosis.These results prompted usto determine in detail the antiapoptotic mechanism of TIMP-1 in diabetic ulcers. The microRNA (miRNA) microarray, which analyzed the differentially expressed miRNA profiles during wound healing in diabetic and normal rats, revealed the miR-21 expression was downregulated in the diabetic wound. Our preliminary experiments also found that miR-21 was significantly activated by TIMP-1.Bioinformatic analyses of the predicted target genes combined with our preliminary study showed that the miR-21 may play a role in the regulation of PTEN.In this project, we will further investigate the role of TIMP-1 in activation of miR-21 using adenovirus vector system and siRNA methods, determine the targeted regulation of miR-21to PTEN by luciferase assay and miR-21 lentivirus plasmid system, and clarify the antiapoptotic role of TIMP-1/miR-21/PTEN in diatetic ulcers.These studies will provide new ideas and strategies for the treatment of clinical diabetic wound healing.

我们最近研究发现，组织金属蛋白酶抑制剂1（TIMP-1）通过抗凋亡作用促进糖尿病创面愈合，但其发挥抗凋亡作用的机制尚不明确。已知microRNA与细胞凋亡密切相关。我们在前期预实验中，通过microRNA芯片技术检测糖尿病大鼠皮肤伤口愈合过程中miRNA的变化，发现miR-21显著下调。体外预实验发现miR-21的表达受TIMP-1调节。进一步结合生物信息学分析miR-21的靶基因中包含PTEN。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TIMP-1促进miR-21表达的证据；采用荧光素酶和慢病毒载体构建方法明确miR-21 对PTEN的靶向调节作用；阐明TIMP-1/ miR-21/PTEN在抗糖尿病皮肤细胞凋亡中的作用，并观察干预TIMP-1/ miR-21/PTEN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。

新近研究发现组织金属蛋白酶抑制剂1（TIMP-1）通过抗凋亡作用促进糖尿病创面愈合，但其发挥抗凋亡作用的机制尚不明确。已知microRNA与细胞凋亡密切相关。我们在前期预实验中，通过microRNA芯片技术检测糖尿病大鼠皮肤伤口愈合过程中miRNA的变化，发现miR-21显著下调。体外预实验发现miR-21的表达受TIMP-1调节。进一步结合生物信息学分析miR-21的靶基因中包含PTEN。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TIMP-1促进miR-21表达的证据；采用荧光素酶和慢病毒载体构建方法明确miR-21 对PTEN的靶向调节作用；阐明TIMP-1/ miR-21/PTEN在抗糖尿病皮肤细胞凋亡中的作用，并观察干预TIMP-1/ miR-21/PTEN对糖尿病大鼠皮肤伤口愈合的影响。研究结果发现：①糖尿病患者皮肤创面细胞凋亡明显增加，TIMP-1、miR-21表达下降，PTEN表达增加。以糖基化终末产物（AGEs）模拟糖尿病状态，发现AGEs抑制人皮肤成纤维细胞TIMP-1表达。②外源补充rhTIMP-1 和通过腺病毒介导内源性TIMP-1过表达对AGEs诱导的人皮肤成纤维细胞凋亡具有保护作用。③腺病毒介导TIMP-1过表达,减少细胞凋亡,促进糖尿病大鼠伤口愈合。④Rh-TIMP-1和腺病毒介导TIMP-1过表达促进人原代皮肤成纤维细胞miR-21 表达，抑制PTEN表达。⑤双荧光素酶报告基因载体检测证实PTEN为miR-21的靶基因。以上研究结果为进一步确立TIMP-1/miR-21/PTEN作为糖尿病难愈创面治疗的新靶点提供科学依据。