摘要
目的 探讨miR-30-5p对视网膜母细胞瘤细胞增殖的影响及作用机制。方法 采用实时荧光定量聚合酶链式反应(qRT-PCR)检测miR-30-5p在视网膜母细胞瘤细胞系和人视网膜内皮细胞(HRECs)中的表达。利用TargetScan数据库进行生物信息学分析并预测miR-30-5p靶基因,双荧光素酶报告基因实验验证miR-30-5p与FOXG1的3?UTR结合能力及靶向关系,qRT-PCR和Western blotting分别检测miR-30-5p对FOXG1的mRNA与蛋白表达影响。瞬时转染Y79细胞后,通过CCK8法检测各组Y79细胞的增殖。结果 与HRECs比较,miR-30-5p在视网膜母细胞瘤细胞中表达降低,FOXG1在视网膜母细胞瘤细胞中表达升高。生物信息学预测结果显示miR-30-5p与FOXG1存在结合位点,qRT-PCR和Western blotting显示miR-30-5p可负调控FOXG1的mRNA和蛋白的表达,双荧光素酶实验结果证实miR-30-5p与FOXG1 3?UTR存在靶向关系。瞬时转染miR-30-5p可升高Y79细胞中miR-30-5p的表达水平,抑制Y79细胞的增殖活力。过表达FOXG1可逆转miR-30-5p上调对Y79细胞增殖的影响。结论 miR-30-5p通过下调FOXG1表达抑制视网膜母细胞瘤Y79细胞的增殖。
Objective To investigate the effect of microRNA-30-5 p(miR-30-5 p) on the proliferation of retinoblastoma cells.Methods The expression of miR-30-5 p in retinoblastoma cell lines and normal human retinal epithelial cells(HRECs) was determined using quantitative Real-time polymerase chain reaction(qRT-PCR).Bioinformatic analysis and prediction of miR-30-5 p target genes were performed using the TargetScan database.Dual-luciferase reporter experiments verified the 3?-UTR binding ability and targeting relationship of miR-30-5 p and FOXG1.The effect of miR-30-5 p on the mRNA and protein expression of FOXG1 was determined using qRT-PCR and Western blotting,respectively.The effects of the miR-30-5 p-FOXG1 pathway on cell proliferation were examined in Y79 cells by transient transfection.Results miR-30-5 p expression was decreased and FOXG1 expression was increased in retinoblastoma cells compared with that in HRECs.Bioinformatics prediction results revealed the binding sites of miR-30-5 p and FOXG1.qRT-PCR and western blot results showed that miR-30-5 p negatively regulated FOXG1 mRNA and protein expression.Dual-luciferase assay confirmed a targeting relationship between miR-30-5 p and FOXG1 3?-UTR.Transient transfection of miR-30-5 p increased the expression level of miR-30-5 p in Y79 cells.Overexpression of FOXG1 reversed the inhibition of Y79 cell proliferation.Conclusion miR-30-5 p inhibits the proliferation of Y79 retinoblastoma cells by downregulating FOXG1 expression.
作者
颜繁诚
蒋现
柴一杰
王昊森
孟照洋
汪晓磊
王艳玲
冯雪
YAN Fancheng;JIANG Xian;CHAI Yijie;WANG Haosen;MENG Zhaoyang;WANG Xiaolei;WANG Yanling;FENG Xue(Department of Ophthalmology,Beijing Friendship Hospital,Capital Medical University,Beijing 100050,China;Department of Ophthalmology,Anhui No.2 Provincial People's Hospital,Hefei 230041,Anhui,China;Department of Pathology,School of Basic Medical Sciences,Peking University Health Science Center,Beijing 100191,China;State Key Laboratory of Tribology,Tsinghua University,Beijing 100084,China)
出处
《山东大学耳鼻喉眼学报》
CAS
2022年第5期63-69,共7页
Journal of Otolaryngology and Ophthalmology of Shandong University
基金
国家自然科学基金(81870686)
首都卫生发展科研专项项目(2018-1-2021)。
