Circular RNA HIPK3 regulates human lens epithelial cell dysfunction by targeting the miR-221-3p/PI3K/AKT pathway in age-related cataract
Circular RNA HIPK3 regulates human lens epithelial cell dysfunction by targeting the miR-221-3p/PI3K/AKT pathway in age-related cataract
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DOI：
10.1016/j.exer.2020.108128
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发表时间：
2020-09-01
影响因子：
3.4
通讯作者：
Huang, Wenjuan
中科院分区：
文献类型：
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作者：
Cui, Gangfeng;Wang, Ledan;Huang, Wenjuan
环状RNA同源结构域相互作用蛋白激酶3（circHIPK 3）参与了年龄相关性白内障（ARC）的发病机制。本文进一步揭示了circHIPK 3在ARC进展中的相关靶基因及其分子机制。实时荧光定量PCR检测circHIPK 3、microRNA（miR）-221-3p的表达。采用MTT法和流式细胞仪分别检测人透镜上皮细胞（HLEC）增殖和凋亡。采用Western blot法检测细胞凋亡相关蛋白、磷脂酰肌醇3-激酶（PI 3 K）/β-蛋白激酶B（AKT）通路相关蛋白的表达。用试剂盒测定丙二醛（MDA）和谷胱甘肽过氧化物酶（GSH-PX）水平。通过双荧光素酶报告基因检测和RNA免疫沉淀检测证实了miR-221- 3 p与circHIPK 3之间的相互作用。在ARC患者的人透镜上皮样品中，CircHIPK 3下调，而miR-221- 3 p上调。CircHIPK 3上调或miR-221- 3 p下调可促进HLEC增殖，抑制HLEC凋亡，降低MDA水平，升高GSH-PX水平。miR-221- 3 p是circHIPK 3的靶点，miR-221- 3 p过表达逆转了circHIPK对HLEC功能的保护作用。此外，circHIPK 3通过调控miR-221- 3 p激活PI 3 K/AKT通路，而沉默miR-221- 3 p则通过激活PI 3 K/AKT通路保护HLEC功能。我们证明circHIPK 3通过调节miR-221- 3 p/PI 3 K/AKT通路保护HLECs免受功能障碍，这为ARC的发病机制提供了新的见解，并为ARC提供了潜在的治疗靶点。
Circular RNA Homeodomain Interacting Protein Kinase 3 (circHIPK3) was found to involve in the pathogenesis of age-related cataract (ARC). Here, we further disclosed the related target genes and molecular mechanism of circHIPK3 in the ARC progression. The expression of circHIPK3, microRNA (miR)-221-3p was detected using the quantitative real-time polymerase chain reaction. Human lens epithelial cell (HLEC) proliferation and apoptosis were measured by 3-(4, 5)-dimethylthiahiazo (-z-y1)-3, 5-di-phenytetrazoliumromide (MTT) assay and flow cytometry, respectively. Western blot was used to detect the levels of apoptosis-related proteins, and phosphoinositide 3-kinase (PI3K)/p-protein kinase B (AKT) pathway-related proteins. Levels of malondialdehyde (MDA) and glutathione peroxidase (GSH-PX) were measured by kits. The interaction between miR-221-3p and circHIPK3 was confirmed by dual-luciferase reporter assay and RNA immunoprecipitation assay. CircHIPK3 was down-regulated while miR-221-3p was up-regulated in human lens epithelium samples of ARC patients. CircHIPK3 up-regulation or miR-221-3p down-regulation mediated the promotion of proliferation, inhibition of apoptosis, decrease of MDA level as well as increase of GSH-PX level in HLECs. MiR-221-3p was a target of circHIPK3, and miR-221-3p overexpression reversed the protective action of circHIPK in HLEC functions. In addition, circHIPK3 activated PI3K/AKT pathway via regulating miR-221-3p, and silencing miR-221-3p protected HLECs from dysfunction by activating PI3K/AKT pathway. We demonstrated that circHIPK3 protected HLECs from dysfunction by regulating miR-221-3p/PI3K/AKT pathway, indicating a new insight into the pathogenesis of ARC and providing a potential therapeutic target for ARC.