PTEN及相关miRNA介导NGF抗角膜上皮细胞凋亡的机制研究

Neurotrophic keratitis is a degenerative corneal condition induced by corneal nerve damage that results from a variety of ocular and systemic diseases. Several clinical studies confirmed that NGF plays a pivotal role in modulating wound-healing processes in the cornea. However, the molecular mechanisms by which NGF functions in corneal epithelium are still unclear. Our preliminary study showed that the apoptosis of human corneal epithelial cells(HCECs) was suppressed by NGF treatment, and that the expressions of FoxO3a and its target Bim were abnormal. The HCECs treated by NGF was also subjected to microarray analysisto find out that PTEN was the vital factor for HCECs apoptosis. Therefore, we hypothesize that certain miRNAs targeting PTEN might be involved in NGF regulated cell apoptosis of HCECs. In the current study, we will explore the expression differences of miRNAs in the HCECs upon NGF treatment by Agilent's Human miRNA Microarray Kit and the Sanger MicroCosm search engine. SYBR? Premix Ex Taq TM II kit will be used to identify miRNAs that regulate the expression of PTEN. We will then investigate the effect of the candidated miRNA(s)on the expressions of PTEN, FoxO3a, Bim and the activity of PI3K/Akt signaling pathway, and finally discover the mechanism by which NGF inhibite the apoptosis of HCECs. Regulation of the apoptosis factorsby NGF-induced miRNAs on human corneal epithelium may lead to a new vision into the healing of neurotrophic keratitis.

神经源性角膜炎是由多种原因引起角膜神经损伤造成角膜上皮退行性变的疾病，它的预后差、治疗棘手。临床研究表明NGF可以促进上皮溃疡的愈合，但其确切的作用机制仍未明确。我们在前期工作中发现，NGF处理角膜上皮细胞后，细胞凋亡得到了抑制；进一步的检测发现FoxO3a及其靶基因Bim的表达发生了异常；借助miRNA芯片技术，分析得到潜在的关键作用位点PTEN基因。因此我们设想，PTEN及相关miRNA可能是介导NGF抗角膜上皮细胞凋亡的关键因素。本项目拟在此基础上研究NGF处理角膜上皮细胞后PTEN及相关miRNA的表达规律；明确作用于PTEN的miRNA；探讨关键miRNA对PTEN、PI3K/Akt信号通路、以及FoxO3a和Bim的调控，明确角膜上皮细胞凋亡受调控的机制。通过上述实验，可望为NGF治疗神经源性角膜炎提供更为充分的实验依据，并为明确NGF在角膜上皮自我修复中的作用提供新的视角。

神经源性角膜炎是由多种原因引起角膜神经损伤造成角膜上皮退行性变的疾病，它的预后差、治疗棘手。临床研究表明NGF可以促进上皮溃疡的愈合，但其确切的作用机制仍未明确。NGF处理角膜上皮细胞后，细胞凋亡得到了抑制；NGF与受体TrkA结合，并导致TrkA磷酸化，激活TrkA通路。激活的TrkA能是Akt磷酸化，从而磷酸化FoxO3a，并使其发生细胞质转位，阻止FoxO3a结合到Bim的3‘启动子区域，造成细胞凋亡抑制因子Bim的高表达，从而抑制角膜上皮细胞凋亡。运用miRNA芯片技术，初步挑选出与PTEN基因相关、且表达上调的miRNA，分析miRNA和PTEN 3’ UTR序列，并运用qPCR法检测验证这些候选miRNA表达的变化趋势是否与芯片技术结果一致，找出实验目标miRNA。然而，我们在检测原代角膜上皮细胞及永生化角膜上皮细胞中PTEN表达时发现，在上述两种细胞内均没有PTEN的表达。将NGF处理后的样本通过miRNA芯片筛选，得到与角膜上皮细胞增殖有关的差异miRNA: miRNA-494, miRNA-1305, miRNA-15, miRNA-18, and miRNA-320c。qRT-PCR验证发现NGF能显著抑制miRNA-494的表达，将miRNA-494过表达至角膜上皮细胞发现，miRNA-494显著增加细胞阻滞于G1期，然而NGF处理后，却能促进角膜上皮细胞的细胞周期进展以及增殖能力。通过数据库比对发现，miRNA-494直接靶向cyclin D1的3'-UTR，luciferase re porter实验证实了这一发现。我们的研究结果，为NGF治疗神经源性角膜炎提供更为充分的实验依据，并为明确NGF在角膜上皮自我修复中的作用提供新的视角。