Long non-coding RNAs Gabarapl2 and Chrnb2 positively regulate inflammatory signaling in a mouse model of dry eye

Purpose: To elucidate the expression profile and the potential role of long non-coding ribonucleic acids (RNAs; lncRNAs) in a dry eye disease (DED) model. Methods: A DED model was established in C57BL/6J mice with 0.2% benzalkonium chloride (BAC) twice a day for 14 days. The differentially expressed lncRNAs were detected by RNA-seq technology and the aberrantly expressed lncRNAs were further verified by RT-qPCR. Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analyses were conducted to predicate the related candidate genes and potential pathological pathways. Cells from a human corneal epithelial cell line (HCECs) were cultured under hyperosmolarity. The regulation of inflammatory factors by silencing potential targeted lncRNAs was verified in vitro in HCECs. Results: In our study, a significant increase in corneal fluorescence staining and a reduction in tear production were observed in DED mice at all follow-ups compared with the controls, and the differences were increasing over time. In total, 2649 upregulated and 704 downregulated lncRNAs were identified in DED mice. We selected six aberrantly expressed and most abundant lncRNAs and performed RT-qPCR using the samples for RNA-seq. Chrnb2, Gabarapl2, and Usp31 were thereby confirmed as the most significantly altered lncRNAs. Pathway analysis revealed that the neuroactive ligand–receptor interaction signaling pathway was the most enriched, followed by the calcium signaling pathway and cytokine–cytokine receptor interaction. Following treatment of Gabarapl2 siRNA and Chrnb2 siRNA, tumor necrosis factor-a (TNF-a), interleukin (IL)-1ß, and interleukin (IL)-6 were significantly downregulated in the HCECs. Conclusion: Our study suggests that Chrnb2 and Gabarapl2 may be involved in the inflammation response by regulating TNF-a, IL-1ß, and IL-6 in DED. These candidate lncRNAs may be both potential biomarkers and therapeutic targets for DED. Overall design: CK and TREAT are the names of samples, where CK stands for blank control group and TREAT stands for dry eye mice model group. RNA-seq was performed to identify lncRNAs involved in the cornea with the dry eye model.

研究目的：阐明长链非编码RNA（long non-coding ribonucleic acids，lncRNAs）在干眼症（dry eye disease，DED）模型中的表达谱及其潜在作用。
研究方法：采用0.2%苯扎氯铵（benzalkonium chloride，BAC）每日两次给药，持续14天，构建C57BL/6J小鼠干眼症模型。通过RNA测序（RNA-seq）技术检测差异表达的lncRNAs，并采用实时定量聚合酶链式反应（RT-qPCR）对异常表达的lncRNAs进行验证。通过基因本体（Gene Ontology，GO）富集分析与京都基因与基因组百科全书（Kyoto Encyclopedia of Genes and Genomes，KEGG）富集分析，预测相关候选基因及潜在病理通路。将人角膜上皮细胞系（human corneal epithelial cell line，HCECs）置于高渗条件下培养，在体外HCECs中验证靶向沉默潜在lncRNAs对炎症因子的调控作用。
研究结果：与对照组相比，干眼症模型小鼠在各随访时间点均出现角膜荧光染色评分显著升高、泪液分泌量降低，且上述差异随时间推移逐渐增大。本研究共鉴定出2649个上调表达、704个下调表达的lncRNAs。选取6个异常表达且丰度最高的lncRNAs，采用RNA-seq样本进行RT-qPCR验证，最终确认Chrnb2、Gabarapl2及Usp31为表达变化最显著的lncRNAs。通路富集分析显示，神经活性配体-受体相互作用信号通路富集程度最高，其次为钙信号通路及细胞因子-细胞因子受体相互作用通路。分别用Gabarapl2 siRNA与Chrnb2 siRNA处理HCECs后，肿瘤坏死因子-α（tumor necrosis factor-α，TNF-α）、白细胞介素-1β（interleukin-1β，IL-1β）及白细胞介素-6（interleukin-6，IL-6）的表达量均显著下调。
研究结论：本研究表明，Chrnb2与Gabarapl2可能通过调控TNF-α、IL-1β及IL-6的表达参与干眼症的炎症反应。上述候选lncRNAs有望成为干眼症潜在的生物标志物及治疗靶点。
整体实验设计：CK与TREAT为两组样本名称，其中CK代表空白对照组，TREAT代表干眼症模型组。通过RNA-seq技术鉴定干眼症模型小鼠角膜组织中差异表达的lncRNAs。