lncRNA-PANC1在小鼠视网膜神经节细胞分化过程中的功能和作用机制研究

Glaucoma, characterized by progressive loss of retinal ganglion cells (RGCs), is the second leading cause of blindness. Understanding of the molecular mechanism underlying RGCs development is of great importance for early diagnosis and treatment of glaucoma. MATH5-ISL1/POU4F2 network plays an essential role in RGCs fate determination and differentiation. However, so far many regulatory factors involved in this network remains unclear, especially, the role of noncoding RNA in this network is rarely known. Based on our initially work on long noncoding RNAs (lncRNAs) expressed in mouse retina, as well as the subsequent investigation of their expression in MATH5-,ISL1- and POU4F2-null mouse, we found a total of twenty lncRNAs with the potential to be additional regulatory factors in MATH5-ISL1/POU4F2 network, including the POU4F2 Associated Non-Coding RNA 1 (PANC1). Our previous work gives rise to the followed hypothesis about the role and mechanism of PANC1, i.e., (1) PANC1 is involved in the regulatory network of RGC fate determination and differentiation; (2) Functions of PANC1 is largely coupled with POU4F2, but PANC1 regulates a set of downstream genes independently on POU4F2 or MATH5. In the next work, we plan to construct a PANC1 premature termination mouse model (PANC1-GFP) by CRISPR/Cas9 mediated homologous recombination, which will also insert the coding region of IRES-eGFP upstream the BGH polyadenylation terminator. Development of retinal neurons in PANC1-GFP will be investigated to check the hypothesis (1). The hierarchical relationship between POU4F2 and PANC1 will be investigated by checking their expression in PANC1-GFP or Pou4f2-null mouse. Additionally, some parallel work will be performed for further checking of hypothesis (2), such as identification of PANC1 interacted DNA/RNA /proteins by CHIRP-seq and RNA pull down-mass spectrometry, as well as identification of PANC1 downstream genes by identification of the varied expressed lncRNAs in embryonic PANC1-GFP mouse. In summary, our work aims to reveal the function and mechanism of PANC1 in RGC development, which would largely extend the knowledge about the MATH5-ISL1/POU4F2 network and also the regulatory mechanism of retinal neuron development.

MATH5-ISL1/POU4F2网络是视网膜神经节细胞(retinal ganglion cell, RGC)分化调控的必要途径，但目前对其在RGC分化过程中功能的认识尚不统一，主要原因之一即该网络中仍有相当数量的未知调控因子。申请人前期工作首次发现lncRNA基因PANC1是该网络的重要组成，其特异表达于RGC中并极可能受POU4F2直接调控。但PANC1在RGC分化过程中的功能是否与POU4F2直接相关尚不明确。本项目将构建PANC1单敲小鼠和PANC1/POU4F2双敲小鼠，并分析其胚胎期视网膜的表型，以明确PANC1是否影响RGC的命运决定和凋亡；并将通过鉴定PANC1的互作基因，明确PANC1与POU4F2的协同作用机制。本项目的完成将能加深对MATH5-ISL1/POU4F2网络组成基因及其互作关系的理解，并为RGC损伤导致相关疾病的诊断和治疗提供新的思路。

视觉是人类获取外界信息的主要方式。视网膜中的神经节细胞(RGCs)负责将视觉信号从视网膜传递到大脑相应部位的视觉中心神经元，其损伤或发育不良会引起青光眼等多种眼科疾病，但现有技术尚不能实现RGCs的再生。因此对RGCs分化调控基因及其互作机制进行深入研究，将对RGCs再生技术的发展有积极的促进作用。.此前关于RGCs分化和发育调控的研究主要集中在转录因子上，但对长链非编码RNA (lncRNA) 等非编码RNA在该过程中的调控作用尚不明确。本项目通过表达研究发现PANC1(linc-3b)和linc-3a等lncRNA基因在视网膜中特异表达于RGCs中，且其表达水平受MATH5-POU4F2/ISL1途径的影响，提示其可能参与RGCs的分化和发育调控。我们通过基因编辑技术构建了PANC1基因的突变体小鼠，实现了PANC1转录本的提前终止。表型分析表明PANC1与RGCs等多种视网膜神经细胞的分化和缪勒细胞等的形态发育有关，从而明确了PANC1具有参与视网膜神经细胞分化发育调控的生物学功能。同时，我们通过原位杂交技术明确了PANC1的转录提前终止并不影响POU4F2(Brn3b)及POU4F1(Brn3a)基因的表达。另外，我们通过敲除启动子的策略，在转录水平沉默了linc-3a的表达，但表型观察表明linc-3a的转录沉默对小鼠视网膜神经细胞的分化和发育没有显著影响，但可能造成了部分小鼠虹膜的发育滞后。.此外，我们结合全长转录本测序及lncRNA-seq，系统鉴定了发育过程中小鼠视网膜中表达的lncRNA基因，共获得4500个左右多外显子的视网膜lncRNA基因，其中约37% 为本项目首次注释。通过共表达分析，我们对其中2097个lncRNA的生物学功能进行了初步推测，发现其中462个lncRNA功能富集于视觉形成和神经细胞发育相关生物学过程。我们同时结合本次获得的lncRNA注释，对CRX等多个转录因子突变体小鼠视网膜的转录组数据进行了分析，发现这些转录因子共引起233个lincRNA的表达水平发生了显著变化。.本项目的实施补充了对RGCs分化调控机制中lncRNA功能的认识，促进了对小鼠视网膜各种类型神经元形成的调控机制的系统理解，也对更准确地控制视神经元细胞的再生过程具有重要的参考价值。