南方医科大学学报

南方医科大学学报 ›› 2020, Vol. 40 ›› Issue (09): 1246-1252.doi: 10.12122/j.issn.1673-4254.2020.09.04

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miR-200c-3p靶向CCNE2抑制肾母细胞瘤细胞增殖

曹 娟，孙丽萍，安建红，张 欢，何潇潇，申 洪   

  • 出版日期:2020-09-20 发布日期:2020-09-20

MicroRNA-200c-3p inhibits proliferation of nephroblastoma cells by targeting CCNE2

  • Online:2020-09-20 Published:2020-09-20

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摘要： 目的 预测和验证miR-200c-3p的靶基因并探讨其对肾母细胞瘤增殖的抑制作用。方法 应用生物信息学软件对miR-200c-3p在肾母细胞瘤中的靶基因进行预测，建立SK-NEP-1及G401的miR-200c-3p过表达及抑制表达的稳定细胞系。实验设未转染组（Blank）、模拟物阴性对照组（mimic NC）、miR-200c-3p模拟物组（miR-200c-3p mimic）、抑制剂阴性对照组（inhibitor NC）及miR-200c-3p抑制剂组（miR-200c-3p inhibitor）。采用 RT-PCR及Western blot方法检测CCNE2在SK-NEP-1及G401不同组中的表达水平，荧光素酶报告基因检测miR-200c-3p 和CCNE2的靶向关系，细胞计数盒（CCK-8）和软琼脂克隆形成实验检测miR-200c-3p对SK-NEP-1及G401细胞增殖的抑制作用。结果 生物信息学方法预测CCNE2为miR-200c-3p的靶基因之一。RT-PCR实验结果示肾母细胞瘤细胞中miR-200c-3p模拟物组的CCNE2的表达水平低于模拟物阴性对照组（P<0.05）；荧光素酶报告基因检测证实CCNE2是miR-200c-3p的靶基因（P<0.01）。Western blot示在肾母细胞瘤细胞中miR-200c-3p模拟物组的CCNE2蛋白的表达水平低于模拟物阴性对照组（P<0.05）；CCK-8和软琼脂克隆形成实验证实miR-200c-3p对肾母细胞瘤细胞的增殖能力有明显抑制作用（P<0.01）；而miR-200c-3p抑制剂组结果相反。结论 miR-200c-3p通过靶基因 CCNE2抑制肾母细胞瘤细胞的增殖。

关键词: miR-200c-3p, CCNE2, 肾母细胞瘤

Abstract: Objective To predict and verify the target gene of miR-200c-3p and evaluate the inhibitory effect of miR-200c-3p on the proliferation of nephroblastoma cells. Methods The putative target genes of miR-200c-3p were predicted by bioinformatics approach. Nephroblastoma cell models with miR-200c-3p overexpression or knockdown were established in SK-NEP-1 and G401 cells with corresponding control groups. The expressions of CCNE2 in SK-NEP-1 and G401 cells in different groups were detected by RT-PCR and Western blotting. A luciferase reporter assay was used to determine the targeting relationship between miR-200c-3p and CCNE2. The effects of miR-200c-3p overexpression or knockdown on cell proliferation was detected by cell counting kit-8 (CCK-8) assay and soft agarose assay. Results CCNE2 was one of the target genes of miR-200c-3p as predicted by bioinformatics methods. Transfection of the two nephroblastoma cell lines with miR-200c-3p mimic resulted in significantly lowered CCNE2 mRNA and protein expressions (P<0.05). The results of dual-luciferase assay confirmed that miR-200c-3p bound to the 3’UTR of CCNE2. CCK-8 assay and soft agarose assay demonstrated that overexpression of miR-200c-3p significantly inhibited the proliferation of the nephroblastoma cells (P<0.01), and knocking down miR-200c-3p in the cells produced the opposite effects. Conclusion miR-200c-3p overexpression inhibits the proliferation of nephroblastoma cells by down-regulating its target gene CCNE2.

Key words: microRNA-200c-3p, CCNE2, nephroblastoma