Chen Jun, Rao Xueyi, Wang Xiaoqian, Li Yang, Shen Yajun, Gan Jing
Department of Pediatrics, West China Second University Hospital, Sichuan University, Chengdu, Sichuan, China.
Ministry of Education, Key Laboratory of Birth Defects and Related Diseases of Women and Children (Sichuan University), Chengdu, China.
J Cell Mol Med. 2025 May;29(9):e70593. doi: 10.1111/jcmm.70593.
Long non-coding RNAs (lncRNAs) play significant roles in neurological diseases, including epilepsy. Our previous study identified lncRNA-GPHN as specifically downregulated in a rat model of status epilepticus (SE). Investigate the role and regulatory mechanism of lncRNA-GPHN in the pathogenesis of epilepsy. SE rat and in vitro cell models were used to analyse expression dynamics, cellular localisation, and effects of lncRNA-GPHN on epileptic seizures, followed by HE staining, Nissl staining, and TUNEL staining. Luciferase Reporter assay, ChIRP assay, real-time quantitative PCR, and Western blotting accompanied with TUNEL assay and whole-cell patch-clamp techniques were employed to determine the molecular mechanism in lncRNA-GPHN regulating epilepsy in neurons. Post-seizure, lncRNA-GPHN in SE rats' hippocampus was markedly downregulated, hitting a nadir at 24 h. FISH and qPCR confirmed its cytoplasmic localization in neurons. EEG showed that lncRNA-GPHN overexpression significantly curtailed seizure frequency and intensity, elevating the threshold, while MWM results pointed to enhanced cognition in SE rats. Histological staining revealed less neuronal damage and better cellular integrity in overexpressing rats, accompanied with a reduction in neuronal apoptosis. In vitro, lncRNA-GPHN reduced neuronal excitability and epileptic potentials dose-dependently. q-PCR and ChIRP showed lncRNA-GPHN upregulates YWHAH by sequestering miR-320. Dual-luciferase and Western blot validated miR-320's direct suppression of YWHAH and lncRNA-GPHN's counteracting effect. TUNEL staining confirmed that miR-320 overexpression increased apoptosis, mitigated by lncRNA-GPHN overexpression and further reduced with combined overexpression. lncRNA-GPHN ameliorates epilepsy by inhibiting apoptosis via the miR-320/YWHAH axis, providing insights into epilepsy pathogenesis and potential targeted therapeutic strategies.
长链非编码RNA（lncRNAs）在包括癫痫在内的神经疾病中发挥着重要作用。我们之前的研究发现lncRNA-GPHN在癫痫持续状态（SE）大鼠模型中特异性下调。研究lncRNA-GPHN在癫痫发病机制中的作用及调控机制。利用SE大鼠和体外细胞模型分析lncRNA-GPHN的表达动态、细胞定位及其对癫痫发作的影响，随后进行苏木精-伊红（HE）染色、尼氏染色和TUNEL染色。采用荧光素酶报告基因检测、染色体RNA免疫沉淀（ChIRP）检测、实时定量聚合酶链反应（qPCR）、蛋白质免疫印迹法以及TUNEL检测和全细胞膜片钳技术来确定lncRNA-GPHN调控神经元癫痫的分子机制。癫痫发作后，SE大鼠海马中的lncRNA-GPHN明显下调，在24小时时达到最低点。荧光原位杂交（FISH）和qPCR证实其在神经元中的细胞质定位。脑电图显示lncRNA-GPHN过表达显著降低癫痫发作频率和强度，提高阈值，而 Morris 水迷宫（MWM）结果表明SE大鼠的认知能力增强。组织学染色显示过表达大鼠的神经元损伤较少，细胞完整性较好，同时神经元凋亡减少。在体外，lncRNA-GPHN剂量依赖性地降低神经元兴奋性和癫痫电位。q-PCR和ChIRP显示lncRNA-GPHN通过隔离miR-320上调14-3-3蛋白ζ（YWHAH）。双荧光素酶和蛋白质免疫印迹验证了miR-320对YWHAH的直接抑制作用以及lncRNA-GPHN的拮抗作用。TUNEL染色证实miR-320过表达增加细胞凋亡，lncRNA-GPHN过表达可减轻这种凋亡，联合过表达则进一步降低凋亡。lncRNA-GPHN通过miR-320/YWHAH轴抑制细胞凋亡来改善癫痫，为癫痫发病机制和潜在靶向治疗策略提供了见解。
