DCLRE1A Contributes to DNA Damage Repair and Apoptosis in Age-Related Cataracts by Regulating the lncRNA/miRNA/mRNA Axis

Age-related cataract (ARC) is associated with the deregulation of transcription and defects in DNA repair in lens epithelial cells (LECs). DCLRE1A acted in DNA interstrand cross-links pathway to improve DNA replication and transcription. The aim of this study was to examined the further regulatory effect on DCLRE1A in the lncRNA-miRNA-mRNA network using a cell model of DCLRE1A overexpression (OE-DCLRE1A) in LECs. The expression level of DCLRE1A in ARC tissues and SRA01/04 cells after H₂O₂ treatment was measured as protein and mRNA by qRT-PCR and Western Blot(WB). CCK8, and TUNEL assays detected the change in cell viability and apoptosis, respectively. Furthermore, Immunofluorescence assays detect the expression of DNA damaged and repair marker proteins after OE-DCLRE1A. The global expression profiles of lncRNAs, miRNAs, and mRNAs were determined using high-throughput sequencing. KEGG and GO enrichment analysis disclose the possible function of differentially expressed (DE) lncRNA, miRNA, and mRNA. The protein and mRNA of DCLRE1A were decreased in the anterior capsule of ARC and SRA01/04 cells treated by H₂O₂. OE-DCLRE1A improved damaged-DNA repair and enhanced cell viability against apoptosis after H₂O₂ treatment. Furthermore, we demonstrated the DE-molecules between the OE-DCLRE1A and control groups including 595 DE-lncRNAs, 221 DE-miRNAs, and 4718 DE-mRNAs. Next, bioinformatics analysis not only found that the DE-mRNAs are mainly involved in DNA repair-related signaling pathways after OE-DCLRE1A, but also screened two lncRNA-miRNA-mRNA networks focusing on DNA damage activated by OE-DCLRE1A, which involved 2 lncRNAs, 2 miRNAs, and 53 mRNAs. We revealed that DCLRE1A activated the lncRNA/miRNA/DNA-repair network to take part in DNA repair processes, which not only represents a new regulatory mechanism employed by DCLRE1A but also uncovers the screening lncRNA may hold potential therapeutic values in ARC formation. However, these conclusions will need to be confirmed by future studies <i>in vitro</i> and <i>in vivo</i> models.

年龄相关性白内障（Age-related cataract, ARC）与晶状体上皮细胞（lens epithelial cells, LECs）中转录失调及DNA修复缺陷密切相关。DCLRE1A参与DNA链间交联通路，以促进DNA复制与转录。本研究旨在通过晶状体上皮细胞过表达DCLRE1A（OE-DCLRE1A）的细胞模型，进一步探究其在长链非编码RNA（long non-coding RNA, lncRNA）-微小RNA（microRNA, miRNA）-信使RNA（messenger RNA, mRNA）调控网络中的调控作用。采用实时荧光定量聚合酶链反应（quantitative real-time polymerase chain reaction, qRT-PCR）与蛋白质印迹（Western Blot, WB）技术，检测年龄相关性白内障患者前囊膜组织及经H₂O₂处理的SRA01/04细胞中DCLRE1A的蛋白与mRNA表达水平。通过CCK-8实验与TUNEL实验分别检测细胞活力与细胞凋亡情况。此外，采用免疫荧光实验检测过表达DCLRE1A后DNA损伤及修复标记蛋白的表达变化。采用高通量测序技术测定lncRNA、miRNA及mRNA的全局表达谱。通过KEGG通路富集分析与GO功能富集分析，揭示差异表达（differentially expressed, DE）的lncRNA、miRNA及mRNA的潜在生物学功能。研究发现，年龄相关性白内障患者前囊膜组织及经H₂O₂处理的SRA01/04细胞中，DCLRE1A的蛋白与mRNA表达水平均显著下调。过表达DCLRE1A可改善DNA损伤修复能力，并增强H₂O₂处理后细胞的抗凋亡活性与细胞活力。此外，本研究鉴定出过表达DCLRE1A组与对照组间的差异表达分子，包括595个差异表达lncRNA、221个差异表达miRNA及4718个差异表达mRNA。后续生物信息学分析不仅发现差异表达mRNA主要富集于DNA修复相关信号通路，还筛选出2个靶向由DCLRE1A激活的DNA损伤的lncRNA-miRNA-mRNA调控网络，该网络涉及2个lncRNA、2个miRNA及53个mRNA。本研究揭示，DCLRE1A可通过激活lncRNA/miRNA/DNA修复通路参与DNA修复过程，这不仅阐明了DCLRE1A的全新调控机制，同时也提示筛选得到的lncRNA在年龄相关性白内障的发生发展中具有潜在的治疗价值。不过，上述结论尚需后续体外（in vitro）与体内（in vivo）实验模型进一步验证。