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微小RNA-498通过直接靶向CCPG1促进视网膜母细胞瘤细胞增殖并抑制其凋亡。

miR-498 promotes cell proliferation and inhibits cell apoptosis in retinoblastoma by directly targeting CCPG1.

作者信息

Yang Ling, Wei Ning, Wang Li, Wang Xiang, Liu Qing-Huai

机构信息

Department of Ophthalmology, The First Affiliated Hospital of Nanjing Medical University, 300 Guangzhou Road, Nanjing, Jiangsu, 210029, China.

Department of Child Healthcare, Obstetrics and Gynecology Hospital Affiliated to Nanjing Medical University, Nanjing, China.

出版信息

Childs Nerv Syst. 2018 Mar;34(3):417-422. doi: 10.1007/s00381-017-3622-8. Epub 2017 Dec 15.

Abstract

PURPOSE

Retinoblastoma (Rb) is the most common intraocular tumor in children. MicroRNAs (miRNAs) play a crucial role in gene regulation and cell growth/apoptosis/differentiation. The current study aimed to investigate the role of miR-498 in Rb.

METHODS

Quantitative real-time polymerase chain reaction (QRT-PCR) was used to test mRNA level of miR-498. http://www.targetscan.org and http://www.microrna.org were applied to predict target of miR-498. Dual-luciferase reporter assay was applied to investigate if miR-498 targeted cell cycle progression 1 (CCPG1). Western blot (WB) was carried out to assess CCPG1 protein levels. 3-(4, 5-dimethyl-2-thiazolyl)-2, 5-diphenyl-2-H-tetrazolium bromide (MTT) assay was used to evaluate cell proliferation. Annexin-V Fluorescein (FITC) was adopted to explore cell apoptosis.

RESULTS

In Y79 cells, miR-498 was higher than in normal ARPE-19 cells. MiR-498 could recognize CCPG1-3' untranslated region (UTR). CCPG1 protein level was remarkably decreased when overexpressed miR-498, nevertheless, significantly increased when inhibiting miR-498. Y79 cells that were transfected with miR-498 mimics manifested notable cell apoptosis down-regulation and cell proliferation promotion; whereas, those transfected with miR-498 inhibitor displayed significant cell apoptosis up-regulation and cell proliferation inhibition compared with control group.

CONCLUSION

Taken together, miR-498 promotes cell proliferation and inhibits cell apoptosis in Rb by directly targeting CCPG1.

摘要

目的

视网膜母细胞瘤(Rb)是儿童最常见的眼内肿瘤。微小RNA(miRNA)在基因调控和细胞生长/凋亡/分化中起关键作用。本研究旨在探讨miR-498在Rb中的作用。

方法

采用定量实时聚合酶链反应(QRT-PCR)检测miR-498的mRNA水平。应用http://www.targetscan.org和http://www.microrna.org预测miR-498的靶标。采用双荧光素酶报告基因检测法研究miR-498是否靶向细胞周期进程1(CCPG1)。进行蛋白质免疫印迹法(WB)评估CCPG1蛋白水平。采用3-(4,5-二甲基-2-噻唑基)-2,5-二苯基-2-H-四氮唑溴盐(MTT)法评估细胞增殖。采用膜联蛋白-V异硫氰酸荧光素(FITC)法探究细胞凋亡。

结果

在Y79细胞中,miR-498高于正常ARPE-19细胞。miR-498可识别CCPG1的3'非翻译区(UTR)。过表达miR-498时,CCPG1蛋白水平显著降低,而抑制miR-498时则显著升高。转染miR-498模拟物的Y79细胞表现出明显的细胞凋亡下调和细胞增殖促进;而与对照组相比,转染miR-498抑制剂的细胞则表现出明显的细胞凋亡上调和细胞增殖抑制。

结论

综上所述,miR-498通过直接靶向CCPG1促进Rb细胞增殖并抑制细胞凋亡。

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