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长链非编码RNA ZEB1反义RNA1通过靶向微小RNA-224调控淋巴瘤细胞增殖、侵袭、迁移的分子机制研究

Molecular mechanisms of long non-coding RNA ZEB1-antisense RNA1 in regulating lymphoma cell proliferation, invasion, and migration by targeting microRNA-224

    摘要
  • 摘要:
    目的  探讨长链非编码RNA ZEB1反义RNA1(LncRNA ZEB1-AS1)通过靶向微小RNA(miR)-224调控淋巴瘤细胞增殖、侵袭、迁移的分子机制。
    方法  体外培养淋巴瘤细胞系Raji, 分别转染si-NC、si-ZEB1-AS1、miR-NC、miR-224 mimics、si-ZEB1-AS1+anti-miR-NC、si-ZEB1-AS1+anti-miR-224。采用实时荧光定量聚合酶链式反应(RT-qPCR)评估LncRNA ZEB1-AS1和miR-224在淋巴瘤细胞中的表达量; 采用细胞计数试剂盒(CCK-8)检测细胞活力; 采用Transwell实验检测细胞迁移和侵袭能力; 通过双荧光素酶报告实验确定LncRNA ZEB1-AS1对miR-224的调控作用; 采用Western blot检测细胞周期素D1(CyclinD1)、基质金属蛋白酶(MMP)-2、MMP-9蛋白的表达水平。
    结果  与对照组比较, 淋巴瘤组LncRNA ZEB1-AS1水平升高, miR-224表达水平降低，差异有统计学意义(P < 0.05)。与si-NC组比较, si-ZEB1-AS1组中LncRNA ZEB1-AS1的表达水平、光密度(OD)值、迁移细胞和侵袭细胞数量，以及CyclinD1、MMP-2和MMP-9表达水平均降低，差异有统计学意义(P < 0.05)。与miR-NC组比较, miR-224组miR-224表达水平提高，而OD值、细胞迁移和侵袭数量以及CyclinD1、MMP-2和MMP-9表达水平降低，差异有统计学意义(P < 0.05)。上调miR-224水平可降低WT-ZEB1-AS1的荧光素酶活性，但不影响MUT-ZEB1-AS1的荧光素酶活性。与pcDNA-NC组相比, pcDNA-ZEB1-AS1组miR-224表达水平降低，差异有统计学意义(P < 0.05); 与si-NC组比较, si-ZEB1-AS1组miR-224表达升高，差异有统计学意义(P < 0.05)。与si-ZEB1-AS1+anti-miR-NC组比较, si-ZEB1-AS1+anti-miR-224组的OD值、迁移和侵袭数量，以及CyclinD1、MMP-2和MMP-9蛋白水平提高，差异有统计学意义(P < 0.05)。
    结论  抑制LncRNA ZEB1-AS1从而靶向上调miR-224的表达，能够有效降低淋巴瘤细胞的增殖、侵袭及迁移能力。

     

    Abstract:
    Objective  To investigate the molecular mechanisms of long non-coding RNA ZEB1-antisense RNA1 (LncRNA ZEB1-AS1) in regulating lymphoma cell proliferation, invasion, and migration by targeting microRNA (miR)-224.
    Methods  The lymphoma cell line Raji was cultured in vitro and transfected with si-NC, si-ZEB1-AS1, miR-NC, miR-224 mimics, si-ZEB1-AS1+anti-miR-NC, and si-ZEB1-AS1+anti-miR-224, respectively. Real-time fluorescent quantitative polymerase chain reaction (RT-qPCR) was used to assess the expression levels of LncRNA ZEB1-AS1 and miR-224 in lymphoma cells. Cell viability was detected using the Cell Counting Kit-8 (CCK-8). Cell migration and invasion abilities were evaluated by Transwell experiments. Dual luciferase reporter experiments were conducted to confirm the regulatory effect of LncRNA ZEB1-AS1 on miR-224. Western blot was performed to detect the expression levels of cyclin D1 (CyclinD1), matrix metalloproteinase (MMP)-2, and MMP-9 proteins.
    Results  Compared with the control group, the lymphoma group showed increased LncRNA ZEB1-AS1 levels and decreased miR-224 expression levels (P < 0.05). Compared with the si-NC group, the si-ZEB1-AS1 group exhibited decreased expression levels of LncRNA ZEB1-AS1, optical density (OD) values, the number of migrated and invaded cells, and expression levels of CyclinD1, MMP-2, and MMP-9 protein (P < 0.05). Compared with the miR-NC group, the miR-224 group showed increased miR-224 expression levels but decreased OD values, the number of migrated and invaded cells, and expression levels of CyclinD1, MMP-2, and MMP-9 proteins (P < 0.05). Upregulation of miR-224 levels reduced the luciferase activity of WT-ZEB1-AS1 but had no effect on MUT-ZEB1-AS1 luciferase activity. Compared with the pcDNA-NC group, the pcDNA-ZEB1-AS1 group showed decreased miR-224 expression levels (P < 0.05); compared with the si-NC group, the si-ZEB1-AS1 group showed increased miR-224 expression levels (P < 0.05). Compared with the si-ZEB1-AS1+anti-miR-NC group, the si-ZEB1-AS1+anti-miR-224 group showed increased OD values, the number of migrated and invaded cells, and expression levels of CyclinD1, MMP-2, and MMP-9 proteins(P < 0.05).
    Conclusion  Inhibition of ZEB1-AS1 to targetedly upregulate of miR-224 expression can effectively reduce lymphoma cell proliferation, invasion, and migration abilities.

     

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