高级检索

微小RNA-15a-5p通过RUNX1增强胶质瘤细胞对替莫唑胺的敏感性机制研究

Mechanism of microRNA-15a-5p in enhancing the sensitivity of glioma cells to temozolomide by modulating RUNX1

    摘要
  • 摘要:
    目的 探讨微小RNA-15a-5p(miR-15a-5p)影响胶质瘤细胞增殖、侵袭、迁移及胶质瘤细胞对替莫唑胺(TMZ)敏感性的机制。
    方法 选取胶质瘤细胞H4、SHG-44、正常星形胶质细胞HA1800及TMZ耐药H4细胞, 将miR-NC、miR-15a-5p mimics质粒分别转染至H4细胞，记为miR-NC组、miR-15a-5p组; 将Vector、OE-RUNX1质粒分别转染至miR-15a-5p组细胞，并分别用400 μmoL/L TMZ处理，分别记为miR-15a-5p+Vector组、miR-15a-5p+RUNX1组、TMZ+Vector组及TMZ+RUNX1组。miR-NC组、miR-15a-5p组细胞用400 μmoL/L TMZ处理，记为TMZ+miR-NC组、TMZ+miR-15a-5p组。未进行任何处理的细胞设为Control组。采用四甲基偶氮唑蓝(MTT)检测细胞增殖能力。采用Transwell实验检测细胞侵袭、迁移能力。采用荧光定量聚合酶链反应(RT-PCR)检测细胞RUNX1 mRNA及miR-15a-5p表达水平。采用Western blot检测细胞RUNX1蛋白表达水平。采用TargetScan在线网站预测miR-15a-5p与RUNX1的结合位点。采用双荧光素酶报告基因实验验证miR-15a-5p与RUNX1的靶向关系。
    结果 H4、SHG-44细胞的RUNX1 mRNA及其蛋白表达水平高于HA1800细胞, H4、SHG-44细胞miR-15a-5p表达水平低于HA1800细胞，差异有统计学意义(P < 0.000 1)。与Control组比较, TMZ耐药H4细胞miR-15a-5p表达水平降低，差异有统计学意义(t=18.89, P < 0.000 1); 与Control组比较, TMZ耐药H4细胞RUNX1 mRNA及其蛋白表达水平升高，差异有统计学意义(t=34.11、18.07, P < 0.000 1)。上调miR-15a-5p、TMZ均可抑制细胞的增殖、侵袭及迁移，而上调miR-15a-5p联合TMZ可显著抑制细胞的增殖、侵袭及迁移。miR-15a-5p可负性调控RUNX1表达。RUNX1过表达可逆转上调miR-15a-5p、TMZ对胶质瘤细胞增殖、侵袭及迁移的抑制作用。
    结论 miR-15a-5p负性调控RUNX1抑制胶质瘤细胞的增殖、侵袭、迁移，以及提高肿瘤细胞对TMZ的敏感性。

     

    Abstract:
    Objective To investigate the effects of microRNA-15a-5p (miR-15a-5p) on the proliferation, invasion and migration of and its mechanism of sensitivity of glioma cells to temozolomide (TMZ).
    Methods Glioma cell lines H4 and SHG-44, normal astrocytes HA1800 and TMZ-resistant H4 cells were selected. H4 cells were transfected with miR-NC or miR-15a-5p mimics and recorded as the miR-NC group and the miR-15a-5p group, respectively. The cells of miR-15a-5p group was further transfected with Vector or OE-RUNX1 plasmids and treated with 400 μmoL/L TMZ, and recorded as miR-15a-5p+Vector group, miR-15a-5p+RUNX1 group, TMZ+Vector group and TMZ+RUNX1 group. Additionally, the miR-NC and miR-15a-5p groups were treated with 400 μmoL/L TMZ, and recorded as TMZ+miR-NC group and TMZ+miR-15a-5p group, respectively. Untreated cells served as the Control group. Cell proliferation was assessed using the Methyl Thiazolyl Tetrazolium (MTT) assay. Invasion and migration were evaluated by Transwell assays. RUNX1 mRNA and miR-15a-5p expression levels were determined by quantitative real-time polymerase chain reaction (RT-PCR). RUNX1 protein expression was analyzed by Western blot. The binding sites between miR-15a-5p and RUNX1 were predicted using the TargetScan online database. The targeting relationship between miR-15a-5p and RUNX1 was validated by dual-luciferase reporter assays.
    Results RUNX1 mRNA and its protein expression levels were significantly higher in H4 and SHG-44 cells compared to HA1800 cells, while miR-15a-5p expression was significantly lower in H4 and SHG-44 cells (P < 0.000 1). Compared with Control group, the expression level of miR-15a-5p in TMZ-resistant H4 cells was significantly lower (t=18.89, P < 0.000 1); compared with Control group, the expression level of RUNX1 mRNA and protein in TMZ-resistant H4 cells was significantly higher (t=34.11, 18.07, P < 0.000 1). Upregulation of miR-15a-5p and TMZ treatment both inhibited cell proliferation, invasion and migration, and the combination of miR-15a-5p upregulation and TMZ significantly enhanced these inhibitory effects. MiR-15a-5p negatively regulated RUNX1 expression. Overexpression of RUNX1 reversed the inhibitory effects of miR-15a-5p upregulation and TMZ on glioma cell proliferation, invasion, and migration.
    Conclusion MiR-15a-5p negatively regulates RUNX1, thereby inhibiting the proliferation, invasion and migration of glioma cells and enhancing their sensitivity to TMZ.

     

    • HTML全文
    • 参考文献(22)
    • 相关文章
    • 施引文献
  • 资源附件(0)

/

返回文章
返回