lncRNA XLOC-150632经Fgf2/PI3K调控新生小鼠视网膜新生血管形成的新机制

ROP is a place in premature and low birth weight infants induced retinopathy, the pathological features of retinal vascular dysplasia. Up to now, the pathogenesis of ROP has not been fully elucidated. At present, it is believed that the relative hypoxia mediated VEGF increase, which leads to the formation of neovascularization in the pathogenesis of the disease. LncRNA is a XLOC_150632 lncRNA expression was significantly up-regulated in the study group with the application of high-throughput sequencing technology found in retinal tissue of newborn mice OIR screening, previous studies showed that lncRNA overexpression of XLOC_150632 can lead to the proliferation of newborn mouse retinal vascular endothelial cells; bioinformatic analysis confirmed the potential target genes for FGF2, associated with PI3K pathway; expression of OIR PI3K in neonatal mice retinal vascular endothelial cells, increased expression of VEGF promote retinal neovascularization. Therefore, this project intends to study the role and mechanism of XLOC-150632 in the regulation of retinal neovascularization in neonatal mice by lncRNA Fgf2/PI3K in order to find the potential targets for ROP therapy and prevention.

ROP是一种发生于早产儿和低体重儿的致盲性视网膜病变，其病理特征为视网膜血管异常增生。迄今为止，其发病机制尚未完全阐明，目前认为视网膜相对缺氧介导的VEGF增加，进而导致新生血管形成在ROP发病机制中起主导作用。lncRNA XLOC_150632是本研究小组应用高通量测序技术从OIR新生小鼠视网膜组织筛查发现的一条表达显著上调的lncRNA，前期研究发现：lncRNA XLOC_150632过表达可导致新生小鼠视网膜血管内皮细胞增殖；生物信息学分析证实其潜在靶基因为FGF2，与PI3K通路相关；OIR新生小鼠视网膜血管内皮细胞PI3K表达下调，而VEGF表达上调促进视网膜新生血管形成。因此，本项目拟通过lncRNA XLOC-150632经Fgf2/PI3K调控新生小鼠视网膜新生血管形成的作用与新机制进行研究，以寻找ROP治疗和预防潜在的作用靶点。

本研究通过高通量测序技术筛选出OIR新生小鼠和正常新生小鼠视网膜组织lncRNAs的差异表达，分析获得1118个与OIR相关的lncRNAs。我们根据lncRNAs表达差异及表达量在差异表达在2倍以上的lncRNAs中选择了6条lncRNAs（XLOC_150632、XLOC_150636、XLOC_122045、XLOC_100454、XLOC_170009、XLOC_122042），其中lncRNAXLOC_150632变化倍数最大，且在前期研究中发现lncRNA XLOC_150632过表达可导致新生小鼠视网膜血管内皮细胞增殖，对其进行生物学分析预测其潜在靶基因为FGF2，与PI3K通路相关。研究组建立了lncRNA XLOC_150632过表达和沉默慢病毒、FGF2过表达慢病毒，并对OIR小鼠进行了玻璃体注射，通过石蜡切片、HE染色观察突出视网膜内界膜的血管内皮细胞核数；通过心脏灌注荧光染色剂FITC后进行视网膜铺片观察视网膜血管增殖情况；通过Real-time PCR检测正常小鼠（A组）、OIR小鼠（B组）、OIR+玻璃体注射lncRNA XLOC_150632过表达慢病毒小鼠（C组）、OIR+玻璃体注射lncRNA XLOC_150632沉默慢病毒小鼠（D组）、OIR+玻璃体注射FGF2过表达慢病毒小鼠（E组）、OIR+玻璃体注射lncRNA XLOC_150632过表达慢病毒+FGF2过表达慢病毒小鼠（F组）视网膜中lncRNA XLOC_150632、FGF2、PI3K/Akt、VEGF/VEGFR家族mRNA的表达；通过Western blot检测各组小鼠视网膜中FGF2、PI3K/Akt、VEGF/VEGFR家族蛋白表达情况。结果显示，OIR小鼠较正常新生小鼠HE染色可见突破视网膜内界膜的血管内皮细胞核，视网膜组织中的血管迂曲、扩张明显，RT-PCR示LncRNA XLOC_150632及其靶基因FGF2的表达上调，PI3K/Akt表达下调，而VEGFA显著上调；Western blot检测结果提示FGF2表达上调，PI3K/Akt表达下调。OIR小鼠注射lncRNA XLOC_150632过表达慢病毒后，突出视网膜内界膜的血管内皮细胞核数增多，视网膜血管扭曲、扩张情况更加明显，RT-PCR示LncRNA XLOC_150632及其靶基因FGF2