长链非编码RNA p22857/TSP1调控自噬在iPSCs诱导的人晶状体混浊中的作用及机制

In recent studies, we found that the autophagy process in human lens epithelial cells (HLECs) was likely regulated by the newly discovered long non-coding RNA p22857 (lncRNA p22857) which could activate CD47 signaling through its target gene TSP1.In order to further prove and investigate the molecular mechanism of lncRNA p22857/TSP1 pathway in human lens development and cataractogenesis through autophagy, we’d like to establish a human lens opacity (cataract) model based on our newly developed protocol for functional lentoid bodies (LBs) generation using human induced pluripotent stem cells (iPSCs) in vitro—‘fried egg” method by which LBs exhibiting both structural and functional properties similar to human lens could be generated. Molecular mechanisms were then explored using techniques such as mRNA+lncRNA co-expression microarray, CRISPER-Cas9, siRNA, RNA pull down, mass spectrometry assay, lncRNA FISH and et al. The human lens opacity (cataract) model that were established in this study not only could the in vivo process of cataract be mimicked, but also could the species differences which are faced by researchers be avoided. Moreover, it provides the possibility in search for the molecular mechanism of lncRNA p22857 in cataract in human. Exploration may lead to clinical opportunities for new therapy and medicine target in cataract.

申请人前期工作新发现长链非编码RNA（lncRNA）p22857很可能通过靶基因TSP1调节人晶状体上皮细胞自噬过程。为了证实并深入研究lncRNA p22857/TSP1通路调控自噬在人晶状体发育及白内障中的作用及机制，本项目拟在本研究团队近期成功创立的体外晶状体（LBs）诱导新方法—“荷包蛋”法的基础上构建人晶状体混浊模型，模拟自噬异常诱发白内障发病进程，并通过lncRNA芯片、CRISPER-Cas9、siRNA、RNA pull down、lncRNA FISH等实验手段，在细胞、LB整体及病人水平探索lncRNA p22857/TSP1调控自噬在白内障发生发展中的作用及机制。本课题构建的LB混浊模型即避免了动物疾病模型的种属差异、模拟白内障在体发展进程，又为人来源lncRNA p22857调控白内障病理机制研究提供了可能性。研究结果将为白内障治疗新药物的寻找提供新思路和理论基础。

申请人前期创立体外再生晶体新方法“荷包蛋”法，利用iPSCs构建在结构、分子生物、光学特性上与人晶体类似的再生晶体（lentoid bodies，LBs），对比iPSCs与LB表达谱结合生物统计学分析发现lncRNAp22857很可能作用TSP1调控自噬参与晶状体发育及白内障发生发展。以上述基础为依托，本研究以再生晶体模型为基础构建体外年龄相关性LB模型，结合细胞、体外晶体发育及白内障模型和白内障病人囊膜样本，探索lncRNAp22857作用TSP1调控自噬或其他因子在晶体发育及白内障发生发展中的分子机制。结果表明：首先，以再生晶体为基础构建年龄相关性LB模型，发现成熟透明LB继续培养会逐渐变混浊，且混浊程度与LB蛋白聚集水平一致，成功再现了白内障的发生发展；其次，利用上述模型，结合小鼠胚胎期晶体、LB发育及年龄相关性白内障病人囊膜证实自噬在晶状体发育及白内障发生发展中具有关键作用；随后，系统检测LB诱导过程中自噬相关mRNA及lncRNA的表达，发现多个自噬相关lncRNA表达发生明显差异，并在晶状体上皮细胞（human lens epithelial cells, HLECs）自噬模型、LB发育模型中证实lncRNAp22857的重要生物学功能；再次，明确lncRNAp22857全长，定位于细胞核，siRNA敲减后对HLECs增值、迁移等具有重要影响，在LB诱导模型、年龄相关性白内障病人囊膜、年龄相关性LB模型上发现lncRNAp22857表达水平与LB形成率和混浊程度一致，且在病人囊膜样本中进一步得到证实，同时发现与TSP1表达相一致；最后，CHIRP实验发现lncRNAp22857在TSP1的DNA区域具有较高的结合峰度，提示lncRNAp22857对TSP1表达具有直接调控作用。有趣的是，我们还发现lncRNAp22857与晶状体标志性蛋白CRYGC及CRYBA的DNA区也具有较高的结合峰度，相互作用也在不同模型上得到证实。本研究通过不同模型证实lncRNAp22857通过调节TSP1调控自噬在晶状体发育及白内障发生发生中的关键作用，首次发现lncRNA也很可能参与晶状体标志性蛋白如CRYGC及CRYBA等的表达参与晶状体正常生物学功能及白内障病理过程，为白内障防治新药研发提供了新的方向，也为lncRNA在晶状体中生物学功能的探索提供了新的思路及理论基础。