PAX6/GTL2-DIO3 miRNAs/WNT轴调控表皮干细胞向角膜上皮细胞转分化的机制研究

Epidermal stem cells can be transdifferentiated into corneal epithelial cells for tissue engineering corneal construction. The mechanism of the transdifferentiation has not been fully elucidated. PAX6 and WNT signaling pathway are main factors during the transdifferentiation. Through miRNA sequencing we found 75% of the differently expressed miRNAs in epidermal stem cells compared with corneal epithelial cells were miRNAs in Gtl2-Dio3 imprinted region. The preliminary results suggest that PAX6 bind to the promoter of Gtl2-Dio3 miRNAs and the target genes of Gtl2-Dio3 miRNAs are WNT suppressor genes. So in this project, we attempt to confirm the regulation network constructed by PAX6, Gtl2-Dio3 miRNAs and WNT signaling pathway, and then confirm the regulation effect of PAX6/Gtl2-Dio3 miRNAs/WNT axis on epidermal stem cells to corneal epithelial cells transdifferentiation by using gene overexpression, interference, miRNA transfection and transplantation. It is a new point of view that the PAX6/Gtl2-Dio3 miRNAs/WNT axis regulates the transdifferentiation of epidermal stem cells into corneal epithelial cells and the uncovered molecular mechanism will provide fundamental basis for promoting the tissue engineering corneal constructed by epidermal stem cells to clinical application.

表皮干细胞向角膜上皮细胞转分化是获得自体来源组织工程角膜的重要途径，PAX6和WNT通路是影响该转分化的重要因素。课题组通过miRNA测序发现表皮干细胞和角膜上皮细胞差异表达的miRNA中75%位于GTL2-DIO3印记区，本项目前期结果显示GTL2-DIO3印记区上游启动子能够被PAX6结合，WNT信号通路抑制基因是GTL2-DIO3 miRNA下游靶基因。为此本项目拟进一步揭示由PAX6、GTL2-DIO3 miRNA和WNT通路构建的调控网络，并通过过表达、干扰、miRNA转染、移植等方法确定PAX6/GTL2-DIO3 miRNA/WNT轴对表皮干细胞向角膜上皮细胞转分化的作用，从而围绕GTL2-DIO3印记区miRNA这一全新视点阐明PAX6/GTL2-DIO3 miRNA/WNT轴对表皮干细胞向角膜上皮细胞转分化的调控机制，为推进自体来源细胞构建组织工程角膜向临床应用奠定基础。

皮肤表皮干细胞具有多向分化潜能，同时其和角膜缘干细胞有着相同的起源，因而皮肤表皮干细胞向角膜上皮细胞转分化是获得自体来源组织工程角膜的重要途径，然而该转分化过程中所涉及到的分子机制尚不明确。本项目通过miRNA测序发现表皮干细胞和角膜上皮细胞差异表达的miRNA中76%位于GTL2-DIO3印记区，并且该印记区的差异miRNA在两种细胞中的有着相同的表达模式，即在表皮干细胞中的表达水平高于角膜上皮细胞且不同的miRNA表达水平趋于一致。通过对表皮干细胞中GTL2-DIO3印记区miRNA抑制后发现，表皮干细胞标志基因K5表达下调，而角膜上皮细胞标志基因K3和K12表达上调。将GTL2-DIO3印记区miRNA抑制后的细胞构建细胞植片进行眼表移植后发现，移植后的细胞能够在很大程度上重建眼表角膜。这表明GTL2-DIO3印记区miRNA能够调控表皮干细胞向角膜上皮细胞的转分化过程。进一步地，本项目探索了GTL2-DIO3印记区miRNA的上下游调控网络，结果表明GTL2上游存在角膜上皮细胞关键转录因子PAX6的结合位点，通过和该位点结合PAX6能够负调控GTL2-DIO3印记区miRNA的表达；同时，对GTL2-DIO3印记区miRNA的靶基因分析显示Wnt信号通路抑制基因sFRP4和DKK2是其靶基因，对GTL2-DIO3印记区miRNA的抑制能够抑制Wnt信号通路的活性。在此基础上，本项目进一步的研究表明在表皮干细胞向角膜上皮细胞转分化的过程中，PAX6通过负调控GTL2-DIO3印记区miRNA的表达从而解除sFRP4和DKK2的抑制，进而使得Wnt信号通路失活。过表达GTL2-DIO3印记区miRNA或利用Wnt信号激动剂都能够逆转此转分化过程。综合本项目的研究结果，我们以GTL2-DIO3区miRNAs为中心，揭示了一条全新的、影响皮肤表皮干细胞向角膜上皮细胞转分化的分子途径（PAX6-GTL2-DIO3 miRNAs-WNT 轴）。该分子机制的阐明将为实现皮肤表皮干细胞向角膜上皮细胞的有效转分化，推进表皮干细胞构建组织工程角膜向临床应用奠定基础。