MicroRNA Let-7c-5p-Mediated Regulation of ERCC6 Disrupts Autophagic Flux in Age-Related Cataract via the Binding to VCP
MicroRNA Let-7c-5p-Mediated Regulation of ERCC6 Disrupts Autophagic Flux in Age-Related Cataract via the Binding to VCP
复制标题
MicroRNA Let-7c-5p 介导的 ERCC6 调节通过与 VCP 结合扰乱年龄相关性白内障的自噬通量
DOI：
10.1080/02713683.2021.1900273
复制
发表时间：
2021-03-22
影响因子：
2
通讯作者：
Guan, Huaijin
中科院分区：
文献类型：
--
作者：
Cao, Yu;Li, Pengfei;Guan, Huaijin
摘要目的：DNA损伤有助于年龄相关性白内障（ARC）的发病机制，并通过核苷酸切除修复（NER）途径进行修复，其中包括ERCC 6。有证据表明，有缺陷的自噬导致透镜细胞器降解和白内障。本研究旨在探讨ERCC 6在ARC中对自噬的影响及其机制。方法：对30例ARC患者和30例年龄相匹配的接受透明透镜摘除术的对照者进行临床病例对照研究。应用透射电镜观察透镜前囊组织和透镜上皮细胞系（SRA 01/04）自噬囊泡的超微结构。进行实时聚合酶链反应和蛋白质印迹分析以测量相对基因表达水平。通过免疫荧光法评估基因表达水平和定位。采用免疫共沉淀法研究ERCC 6编码的CSB与VCP的关系。使用ERCC 6-siRNA和let-7 c-5 p模拟物来改变ERCC 6和let-7 c-5 p的表达。结果如下：ARC患者的透镜前囊组织和UVB诱导的SRA 01/04细胞中发生自噬诱导，其中LC 3B斑点的数量增加。与该结果一致，除了p62的表达外，beclin 1（BECN 1）和LC 3B的表达也增加。此外，ERCC 6表达降低，ERCC 6沉默诱导BECN 1、LC 3B和p62表达增加。此外，CSB与VCP相互作用，let-7 c-5 p通过靶向ERCC 6诱导自噬失调。结论：在ARC中，Let-7 c-5 p介导的ERCC 6下调可能阻止自噬泡的降解。CSB与VCP结合，诱导自噬体与溶酶体联合收割机结合并降解。
ABSTRACT Purpose: DNA damage contributes to the pathogenesis of age-related cataract (ARC) and is repaired through the nucleotide excision repair (NER) pathway, which includes ERCC6. Evidence has demonstrated that defective autophagy leads to lens organelle degradation and cataract. This study aimed to investigate the effects of ERCC6 on autophagy and determine its mechanisms in ARC. Methods: The clinical case-control study comprised 30 patients with ARC and 30 age-matched controls who received transparent lens extraction. Transmission electron microscopy was used to assess the ultrastructure of autophagic vesicles in lens anterior capsule tissues and lens epithelial cell line (SRA01/04). Real-time polymerase chain reaction and western blot analyses were performed to measure relative gene expression levels. Gene expression levels and localization were assessed by immunofluorescence. A coimmunoprecipitation assay was used to investigate the relationship between CSB which encoded by ERCC6 and VCP. ERCC6-siRNA and let-7 c-5p mimic were used to alter the expression of ERCC6 and let-7 c-5p. Results: Autophagy induction occurred in lens anterior capsule tissues of patients with ARC and in UVB-induced SRA01/04 cells, where the number of LC3B puncta was increased. Consistent with this result, the expression of beclin1 (BECN1) and LC3B, in addition to that of p62, was increased. Additionally, ERCC6 expression decreased, and silencing ERCC6 induced increases in the expression of BECN1, LC3B and p62. Moreover, CSB interacted with VCP, and let-7 c-5p induced dysregulation of autophagy by targeting ERCC6. Conclusion: In ARC, Let-7 c-5p-mediated downregulation of ERCC6 might prevent the degradation of autophagic vacuoles. CSB binds to VCP, inducing autophagosomes to combine with lysosomes and be degraded.