首页|circHIPK3/miR-330-5p对人晶状体上皮细胞损伤的影响及其调控机制研究

circHIPK3/miR-330-5p对人晶状体上皮细胞损伤的影响及其调控机制研究

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目的 探究环状RNA同源域相互作用蛋白激酶3(circHIPK3)、微小RNA-330-5p(miR-330-5p)表达对人晶状体上皮细胞损伤的影响及其调控机制.方法 研究对象为人晶状体上皮细胞(lens epithelial cells,LECs)SRA01/04,采用过氧化氢(H2O2)诱导构建SRA01/04细胞损伤模型,随机分为正常对照组(NC)组、模型组(A)组、过表达质粒(OE-Vector)+H2O2组(B组)、circHIPK3过表达质粒(OE-circHIPK3)+H2O2组(C 组)、OE-circHIPK3+miR-330-5p 阴性对照 mimic NC 序列(miR-NC)+H2O2组(D 组)、OE-circHIPK3+miR-330-5p 寡核苷酸模拟物(miR-330-5p mimics)+H2O2组(E 组),共 5 组.qRT-PCR 法检测各组细胞中 circHIPK3、miR-330-5p水平.双荧光素酶报告法验证circHIPK3与miR-330-5p的靶向关系.分别采用CCK-8法和流式细胞术检测细胞存活率、凋亡率.分析细胞中氧化应激[超氧化物歧化酶(superoxide dismutase,SOD)、丙二醛(malondialdehyde,MDA)、谷胱甘肽过氧化物酶(glutathione peroxidase,GSH-Px)]水平.蛋白免疫印迹法(Western blot)检测凋亡相关蛋白[B淋巴细胞瘤-2相关蛋白(BCL2-associated X,Bax)、B淋巴细胞瘤-2(B-cell lymphoma-2,Bcl-2)].结果 A组细胞中circHIPK3水平低于NC组,miR-330-5p水平高于NC组(P<0.05);与NC组比较,A组细胞存活率、Bcl-2蛋白水平及SOD、GSH-Px水平降低,凋亡率、Bax蛋白水平及MDA水平升高(P<0.05);与A组比较,D组细胞存活率升高,凋亡率、Bax蛋白水平降低,Bcl-2蛋白水平升高,SOD、GSH-Px水平升高,MDA水平降低(P<0.05);circHIPK3可靶向调控miR-330-5p表达.与D组比较,E组miR-330-5p水平升高,细胞存活率、Bcl-2蛋白水平及SOD、GSH-Px水平降低,凋亡率、Bax蛋白水平及MDA水平升高(P<0.05).结论 circHIPK3过表达可靶向抑制miR-330-5p表达,促进LECs存活,抑制LECs凋亡,增强其抗氧化应激能力,减轻LECs损伤.
Effect and Regulatory Mechanism of CircHIPK3/miR-330-5p on Injury of Human Lens Epithelial Cells
Objective To explore the effect and regulatory mechanisms of circHIPK3 and miR-330-5p expression on human lens epithelial cells injury.Methods Human lens epithelial cells(LECs)SRA01/04 were used as the cell experimental material,and a cell injury model of SRA01/04 was induced with hydrogen peroxide(H2O2).The SRA01/04 cells were randomly divided into 6 groups:NC group,model group,overexpression plasmid(OE-Vector)+H2O2 group,circHIPK3 overexpression plasmid(OE-circHIPK3)+H2O2 group,OE-circHIPK3+miR-330-5p negative control mimic NC sequence(miR-NC)+H2O2 group and OE-circHIPK3+miR-330-5p oligonucleotide mimics(miR-330-5p mimics)+H2O2 group.The qRT-PCR method was used to detect the levels of circHIPK3 and miR-330-5p in each group.The dual luciferase reporter assay was used to verify the targeting relationship between circHIPK3 and miR-330-5p.CCK-8 method and flow cytometry were used to detect cell survival rate and apoptosis rate respectively.The levels of superoxide dismutase(SOD),malondialdehyde(MDA)and glutathione peroxidase(GSH-Px)were detected.Western blot was used to detect the expression of B lymphomatoma-2 related protein(Bax)and B lymphomatoma-2(Bcl-2).Results The level of circHIPK3 was lower and the level of miR-330-5p was higher in the model group cells than in the NC group(P<0.05).Compared with those in the NC group,the cell survival rate,Bcl-2 protein level,SOD and GSH-Px level decreased in the model group,but apoptosis rate,Bax protein level and MDA level increased(P<0.05).Compared with those in the model group and OE-Vector+H2O2 group,the cell survival rate in the OE-circHIPK3+H2O2 group increased,the apoptosis rate and Bax protein level decreased,the Bcl-2 protein level climbed,and the SOD,GSH-Px and MDA levels dropped(P<0.05).CircHIPK3 was found with the ability to target the expression of miR-330-5p.Compared with those in the OE-circHIPK3+miR-NC+H2O2 group,the miR-330-5p level of the OE-circHIPK3+miR-330-5p mimics+H2O2 group climbed,but the cell survival rate,Bcl-2 protein level,SOD and GSH-Px levels dropped.Moreover,the apoptosis rate and levels of Bax protein and MDA increased(P<0.05).Conclusions Overexpression of circHIPK3 will target the inhibition of miR-330-5p expression,promote LECs survival,inhibit LECs apoptosis,enhance their antioxidant stress resistance and alleviate LECs damage.

Human lens epithelial cellsCircular RNACircular RNA homologous domain interacting protein kinase 3Mi-cro RNA-330-5p

傅亦恬、郝燕生、李海霞

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西安爱尔眼科医院青光眼白内障科(西安市,710001)

西安爱尔眼科医院角膜眼表科(西安市,710001)

人晶状体上皮细胞 环状RNA 环状RNA同源域相互作用蛋白激酶3 微小RNA-330-5p

2024

中国激光医学杂志
中国光学学会

中国激光医学杂志

CSTPCD
影响因子:0.677
ISSN:1003-9430
年,卷(期):2024.33(5)