反义LncRNA调控角蛋白14在糖尿病皮肤病变机理中的研究

Diabetic dermopathy is one of the important factors leading to the difficulty of diabetic wound healing. In our previous study, we found that the level of protein KRT14 was dramatically down-regulated in skin tissue of diabetic rats while up-regulating KRT14 would accelerate the diabetic wound healing. As we all know, the protein KRT14, one of the major structural proteins of keratinocyte, possesses the properties of mechanical impact resistance and preventing chemical stimulation. It is the basis to maintain the normal structure of skin and also plays an important role in skin regeneration. And it has been proved that its down-regulation was one of the critical factors resulting to diabetic skin lesions. We have applied LncRNAs microarray technology to detect and screen a LncRNA KRT-AS located around KRT14, which was significantly down-regulated in diabetic skin tissues. Based on these informations and results, we aim to prove the mechanism that LncRNA KRT-AS regulates expression of KRT14 via in vivo and in vitro tests:KRT-AS helps to prevent KRT14 from degradation of nuclease so as to enhance KRT14 mRNA stability, thus improving the expression of KRT14 in skin and finally promoting the chronic wound healing. This research may helps to review the new mechanism that AGEs may regulate the low expression of KRT14 through LncRNA KRT-AS in the formation of diabetic dermopathy and provide scientific basis for LncRNA acting as novel targets for the therapy of diabetic skin to improve diabetic wound healing.

糖尿病皮肤病变是糖尿病足部慢性创面难以愈合的重要因素。课题组前期研究表明：糖尿病大鼠皮肤组织角蛋白14（KRT14）明显下降，而升高KRT14后糖尿病伤口愈合速度提高。KRT14是皮肤角质形成细胞的结构蛋白，具有抗机械、抗化学刺激等能力，是维持皮肤正常结构及影响再生能力的关键蛋白，其降低是导致糖尿病皮肤病变的关键因素。通过LncRNAs芯片技术筛选出位于KRT14附近的LncRNA KRT-AS，在糖尿病皮肤组织中异常低表达。本项目拟在前期研究基础上，通过体内外实验，证实KRT-AS调控KRT14的机制:通过防止KRT14受到核酸酶降解的方式增加KRT14 mRNA稳定性从而提高皮肤局部KRT14的表达，达到促进慢性创面愈合的目的。本项目有望揭示AGEs通过LncRNA调控KRT14低表达与糖尿病皮肤病变形成之间的机制，为将LncRNA作为糖尿病皮肤病变治疗的新靶点提供更充分的科学依据。

我们在芯片中筛查出一条在糖尿病大鼠皮肤中下调达68.4倍的lncRNA，命名为Lnc KRT AS，全长为2681nt，特异性表达于在大鼠角质形成细胞胞质，其5’端与Krt14有一段长631nt的序列互相配对，覆盖Krt14的4个外显子。敲低Lnc KRT AS后大鼠原代角质形成细胞角蛋白14表达量降低，qPCR显示Krt14下调2.35倍（P<0.0001,n=3）。过表达Lnc KRT AS后，角蛋白14上调，qPCR显示Krt14上调3倍（P=0.026, n=3）。用siRNA敲低Lnc KRT AS后进入G1期的细胞数增多，过表达Lnc KRT AS组的细胞进入G1期减少。敲低Lnc KRT AS后细胞增殖率降低（P=0.0002, n=6），而过表达Lnc KRT AS细胞增殖率增高（P=0.026, n=6）。si NC转染组细胞迁移率增高（P=0.019, n=3），过表达Lnc KRT AS组细胞细胞迁移率减低（P=0.017, n=3）。敲低Lnc KRT AS后的细胞凋亡率增高（P=0.036, n=3），过表达Lnc KRT AS后的细胞凋亡率减低（P=0.008, n=3）。空载体组第7天大鼠伤口愈合率低于Lnc KRT AS过表达组（P=0.0005，n=4）。H&E染色结果显示过表达Lnc KRT AS组表皮厚度大于空载体组（P=0.007，n=4）。过表达Lnc KRT AS组keratin14染色面积比率大于空载体组（P=0.002, n=4）。过表达Lnc KRT AS糖尿病大鼠伤口角蛋白14的表达量明显高于空载体组。核酸酶保护实验显示过表达Lnc KRT AS组中与Lnc KRT AS相配对的Krt14的5’端起第3个外显子表达量高于空载体组3.73倍（P=0.0078，n=3）。α-鹅膏蕈碱结果显示在第2小时过表达Lnc KRT AS组的细胞的Krt14表达量高于空载体组2.67倍（P=0.0066，n=3），第4小时过表达Lnc KRT AS组细胞的Krt14表达量高于空载体组17.4倍（P=0.0072，n=3）。我们发现一条新的的反义Lnc KRT AS，这条Lnc KRT AS和Krt14形成RNA-RNA双链结构增加其mRNA稳定性以上调角蛋白14的表达量，促进角质形成细胞的增殖和迁移，以促进糖尿病伤口愈合。