环状RNA HIPK3对晶状体上皮细胞间充质转化的调控及其机制研究

Epithelial-mesenchymal transition (EMT) of lens epithelial cells (LECs) is recognized as the major pathological feature of fibrotic posterior capsule opacification (PCO) and fibrotic cataract, including anterior/posterior polar cataracts. Recently, accumulating evidence indicates that circular RNAs, a class of stable and conserved non-coding RNA molecules, play an important role in the regulation of EMT. Our previous study has revealed that the expression level of circHIPK3 detected by circRNA sequencing and qRT-PCR is significantly decreased in cataract patients compared to age matched controls. Silencing of circHIPK3 regulates the proliferation and apoptosis of LECs. CircHIPK3 acts as a sponge with miR-193a. ERBB4, WT1 and CRYAA are the direct targets of miR-193a. These target genes participate in the process of EMT in an direct or indirect way through different pathways. This study will determine circHIPK3 expression pattern in EMT model of LECs and PCO model of mice at different time points, clarify the role of circHIPK3 in EMT of LECs and cell dysfunction through in vitro or in vivo intervention of circHIPK3 expression, and reveal the molecular mechanism of circHIPK3-mediated LECs dysfunction.This study will provide novel insights into the prevention and treatment of lens fibrosis.

晶状体上皮细胞（LECs）间充质转化（EMT）是晶状体纤维化继而发生极性白内障或后发性白内障（PCO）的重要病理特征。最新研究发现，一类稳定和保守表达的非编码RNA分子，环状RNA，对EMT有关键的调控作用。本项目预实验中，环状RNA测序筛选发现在白内障发病中环状RNA HIPK3（circHIPK3）的表达显著下调，作为miRNA海绵与miR-193a相互作用，miR-193a靶向调节ERBB4、WT1和CRYAA基因，直接或间接参与EMT过程。本研究拟建立LECs的EMT模型和大鼠PCO模型，分析多个刺激强度/时间点的circHIPK3表达规律；基于circHIPK3表达干预，从细胞、组织和动物水平明确circHIPK3在LECs的EMT中的调控作用；基于生物信息学和分子生物学技术，阐明circHIPK3调控EMT的分子机制；并进行临床验证。研究成果有望为防治白内障提供新的思路。

白内障是世界首位的致盲性眼病，晶状体上皮细胞（HLECs）间充质转化（EMT）是晶状体纤维化继而发生年龄相关性白内障（ARC）和极性白内障的重要病理特征。本研究临床验证结果显示，circHIPK3在皮质型、核型和后囊下型三种白内障亚型患者的晶状体囊膜中较眼库对照均显著下调。首先，对circHIPK3表达干预，下调后HLECs的活力下降、凋亡增加、线粒体膜电位下降、增殖减少。研究circHIPK3对HLECs EMT调控，Western blot结果发现，circHIPK3下调后间质细胞标志物（α-SMA、Vimentin）表达减少，上皮细胞标志物（ZO-1，E-Cadherin）表达增加。在晶状体上皮中下调circHIPK3可以上调miR-193a 的表达，miR-193a靶向下游靶基因CRYAA，miR-193a mimic 转染后人晶状体上皮细胞的活力下降，增殖减少，凋亡增加。第二、探索白内障发生发展中房水中微环境的改变及调控。糖尿病性白内障患者房水中TGF-β1及TGF-β2表达显著上调，房水中可分离得到大量外泌体，外泌体miRNA测序结果显示了糖尿病白内障和ARC差异表达的miRNA，包括295个microRNA上调，138个microRNA下调。外泌体miR-551b上调时CRYAA下调，细胞活力下降，凋亡增加；miR-29b通过上调CACNA1C介导HLECs钙离子相关信号转导。进一步研究了丝氨酸蛋白酶HTRA1/TGF-β/Smad信号通路调控EMT的分子机制。后囊下白内障患者晶状体前囊膜中HTRA1基因表达较对照组明显下调，随着年龄增大有逐渐下降的趋势。在原代培养晶状体上皮细胞中，HTRA1下调后激活了TGF-β/Smad信号通路，促进了EMT相关通路的关键靶标，包括α-SMA、PAI-1和CTGF的mRNA水平上调，FN1和α-SMA蛋白表达也上调，HTRA1调控TGF-β/Smad通路可能在调节HLEC的异常增殖和迁移中发挥作用。在小鼠前房注射AAV敲减HTRA1后4周，在敲减组中82.76%的晶状体出现了前囊膜下白内障，显著高于对照组，在原代小鼠晶状体上皮细胞中，HTRA1敲除上调p-Smad2/3、FN1和α-SMA的表达。该项目的施行，揭示了人晶状体上皮细胞的EMT网络调控的分子机制，研究成果有望为防治白内障提供新的思路。