microRNA⁃449a通过调控MDM4抑制神经母细胞瘤生长
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陕西省重点研发计划项目(2018KW?062)


MicroRNA⁃449a inhibits neuroblastoma by regulating MDM4
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    摘要:

    目的:研究microRNA-449a(miR-449a)对神经母细胞瘤细胞增殖和凋亡的影响及其作用机制。方法:在BE(2)-C细胞中过表达miR-449a后通过实时荧光定量PCR(quantitative real-time PCR,qPCR)分析基因表达水平的变化,MTT分析细胞活力,克隆形成试验观察肿瘤细胞增殖能力,同时,采用microRNA靶标预测和功能注释数据库miRDB预测miR-449a潜在靶向基因,对靶基因MDM4进行过表达和干扰表达后,Western blot分析相关蛋白表达水平,流式细胞术分析细胞凋亡。结果:miR-449a过表达抑制细胞增殖及克隆形成。经过软件预测和实验验证,证实MDM4为miR-449a的1个靶基因。另外干扰MDM4表达后亦可以抑制细胞增殖和克隆形成。过表达MDM4可以抵消由转染miR-449a模拟物所引起的细胞凋亡和p53蛋白量的增加。miR-449a可以促进BE(2)-C细胞凋亡,但是在稳定干扰p53表达的细胞株没有观察到这种效应。结论:miR-449a通过靶向调控MDM4基因进而稳定p53蛋白从而促进肿瘤细胞凋亡,提示miR-449a可能作为神经母细胞瘤潜在治疗靶标之一。

    Abstract:

    Objective:To investigate the mechanisms underlying the effects of microRNA-449a(miR-449a)on proliferation and suppression of neuroblastoma. Methods:After overexpressed miR-449a in BE(2)-C cells,the change of gene expression levels were analyzed by quantitative real-time PCR(qPCR),cell viability was tested by MTT,and the proliferation efficiency of tumor cell was observed by clone formation assay. Meanwhile,we also predicted potential target genes of miR-449a using miRDB,and manipulated the level of MDM4 by overexpression or knockdown the expression of MDM4. Then,the expression of related proteins was analyzed by Western blot and the apoptosis was detected by flow cytometry. Results:Overexpression of miR-449a inhibited cell proliferation and colony formation,and promoted cell apoptosis. MDM4 was confirmed to be a target gene of miR-449a by using software prediction and the experimental verification. In addition,knockdown of MDM4 expression inhibited cell proliferation and colony formation. Overexpression of MDM4 neutralized the increases of apoptosis and p53 protein level induced by miR-449a. Furthermore,miR-449a promoted apoptosis of BE(2)-C cells,but this effect was not observed in p53 knockdown cell line. Conclusion:miR-449a increases p53 protein level by suppressing MDM4 to promote neuroblastoma cell apoptosis. This study implies miR-449a could be a potential therapeutic target of neuroblastoma.

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程 涛,张 生,姚 远,张向宁,张爱辉,侯崇智. microRNA⁃449a通过调控MDM4抑制神经母细胞瘤生长[J].南京医科大学学报(自然科学版),2021,(1):22-28

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  • 收稿日期:2020-01-22
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  • 在线发布日期: 2021-02-04
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