lncRNA Pvt-1靶向ppm1b调控RIPK3磷酸化在视网膜缺血-再灌诱导节细胞程序性坏死中的作用机制研究

急性闭角型青光眼是重要的不可逆性致盲眼病，其特征为眼内压在短时间内急剧升高所引起的视网膜缺血-再灌（I/R）损伤，并最终导致视网膜神经节细胞（RGCs）程序性坏死。新近研究发现，lncRNA NRF能调控I/R所致心肌细胞程序性坏死，但哪些lncRNA在I/R损伤RGCs程序性坏死中起作用则未知。lncRNA Pvt-1在I/R损伤后RGCs中高表达，敲低Pvt-1可显著抑制RGCs坏死水平，提示其可能与程序性坏死有关。RIPK3磷酸化在程序性坏死中起关键作用，敲低Pvt-1则显著抑制I/R损伤所致RIPK3磷酸化水平上调，且Pvt-1能与ppm1b（RIPK3特异性去磷酸化酶）相互结合。由此我们提出假设：Pvt-1可能通过靶向ppm1b调控RIPK3磷酸化最终影响RGCs程序性坏死。本项目拟采用多种技术探索Pvt-1在程序性坏死中的调控机制，为青光眼视功能保护提供潜在分子靶点和理论依据。

Acute angle-closure glaucoma (AACG) is the leading cause of irreversible vision loss and blindness. Elevated intraocular pressure, which is one of the major characteristics of AACG, causes retinal ischemic reperfusion (I/R) injury. I/R contributes to cellular damage and causes RGCs necroptosis. Recent studies showed that lncRNA NRF regulate I/R induced necroptosis in cardiomyocytes, but the mechanism of lncRNAs on I/R induced RGCs necroptosis have not been completely cleared. According to RNA sequencing data, we have found lncRNA Pvt-1 up-regulate in RGCs after I/R, knockdown of Pvt-1 significantly inhibits the cellular numbers of RGCs necrosis, indicating that Pvt-1 may participate in necroptosis. Phosphorylation of RIPK3 play an important role in cellular necroptosis, knockdown of Pvt-1 significantly reduced the level of phosphorylated RIPK3. Moreover, we found Pvt-1 could bind with ppm1b (one of the specific dephosphorylase of RIPK3) after I/R. Therefore, we hypothesized that Pvt-1 may targeted with ppm1b to regulate phosphorylation level of RIPK3 to influence RGCs necroptosis. Our proposal aims to study the effects of lncRNA Pvt-1 on RGCs necroptosis following I/R injury, and to explore the underlying mechanisms for protective strategies of glaucoma in future by using single-molecule fluorescence in situ hybridization, elecrtoretinogram, western blot and RNA pull down techniques.