Fan Fan, Zhuang Jianhui, Zhou Peng, Liu Xin, Luo Yi
Department of Ophthalmology, Eye & ENT Hospital, Fudan University, Shanghai, China.
Myopia Key Laboratory of The Health Ministry and Visual Impairment and Reconstruction Key Laboratory of Shanghai, Shanghai, China.
Oncotarget. 2017 Nov 21;8(66):110209-110220. doi: 10.18632/oncotarget.22597. eCollection 2017 Dec 15.
Human lens epithelial cell (HLEC) apoptosis is a common pathogenic mechanism in age-related cataracts (ARC). While the function of microRNAs (miRNAs) in the eye is beginning to be explored using miRNA expression array, the role of miR-34a in regulating HLEC apoptosis remains unknown and requires further investigation.
Quantitative reverse-transcript polymerase chain reaction (RT-PCR) was used to determine the expression level of miR-34a in cataractous and control samples. MiR-34a mimics and small interfering RNAs were transfected into SRA01/04. Cell apoptosis and oxidative stress were assessed by flow cytometry. The Dual-Luciferase Reporter Assay System was used to confirm whether miR-34a bound to the 3'-UTR of the target gene and blocked its activity. The potential roles of the identified target genes in apoptosis and mitochondria dysfunction were also evaluated.
The expression of miR-34a increased in lens epithelial samples of ARC compared with the transparent group (cataract 2.41±0.81 vs. control 1.20±0.44, P=0.005). In cultured SRA01/04, miR-34a increased reactive oxygen species production and induced apoptosis (early apoptosis: 45.55%±5.96% vs. 15.85%±4.93%, P<0.01; late apoptosis: 6.10%±2.67% vs. 0.95%±0.42%, P<0.01). Overexpression of miR-34a promoted mitochondria-mediated apoptosis through activation of caspase-9, disruption of the mitochondrial membrane potential, blocking of mitochondrial energy metabolism and enhancement of cytochrome C release. Furthermore, Notch1 and Notch2 were confirmed as putative targets of miR-34a, but only Notch2 was verified as the effector that triggered mitochondria-mediated apoptosis.
MicroRNA-34a is increased in the cataractous lens and triggers mitochondria-mediated apoptosis and oxidative stress by suppressing Notch2.
人晶状体上皮细胞（HLEC）凋亡是年龄相关性白内障（ARC）常见的致病机制。虽然利用微小RNA（miRNA）表达阵列对miRNA在眼中的功能进行了初步探索，但miR-34a在调节HLEC凋亡中的作用仍不清楚，需要进一步研究。
采用定量逆转录聚合酶链反应（RT-PCR）测定白内障样本和对照样本中miR-34a的表达水平。将miR-34a模拟物和小干扰RNA转染至SRA01/04细胞。通过流式细胞术评估细胞凋亡和氧化应激。采用双荧光素酶报告基因检测系统确认miR-34a是否与靶基因的3'-非翻译区（3'-UTR）结合并阻断其活性。还评估了已鉴定的靶基因在凋亡和线粒体功能障碍中的潜在作用。
与透明组相比，ARC晶状体上皮样本中miR-34a的表达增加（白内障组2.41±0.81 vs.对照组1.20±0.44，P=0.005）。在培养的SRA01/04细胞中，miR-34a增加活性氧生成并诱导凋亡（早期凋亡：45.55%±5.96% vs. 15.85%±4.93%，P<0.01；晚期凋亡：6.10%±2.67% vs. 0.95%±0.42%，P<0.01）。miR-34a的过表达通过激活caspase-9、破坏线粒体膜电位、阻断线粒体能量代谢和增强细胞色素C释放促进线粒体介导的凋亡。此外，Notch1和Notch2被确认为miR-34a的假定靶标，但只有Notch2被证实是触发线粒体介导凋亡的效应因子。
在白内障晶状体中，微小RNA-34a增加，并通过抑制Notch2触发线粒体介导的凋亡和氧化应激。
