miRNA-181a对多发性骨髓瘤细胞生物学特性影响的初步研究

A preliminary study on the effect of miRNA-181a on biological features of multiple myeloma cells

目的 探讨miRNA-181a（miR-181a）在多发性骨髓瘤（MM）患者中的表达情况及其对MM细胞生物学特性的影响.方法 应用免疫磁珠筛选的方法分离并纯化25例初治、复发难治MM患者及10例非恶性血液病患者骨髓CD138＋细胞,应用实时定量聚合酶链反应（qRT-PCR）检测miR-181a表达情况.同时检测RPMI 8226、H929及U266三种MM细胞株中miR-181a的表达.应用miR-181a抑制剂及激动剂观察下调和上调miR-181a表达对MM细胞生物学特性的影响,并对miR-181a进行靶基因预测.结果 与非恶性血液病患者相比,MM患者CD138＋细胞中miR-181a表达上调.对MM细胞株的观察发现,与非恶性血液病患者骨髓CD138＋细胞相比,RPMI 8226及U266细胞株miR-181a高表达,而H929细胞株则低表达.相比对照组,应用100 nmol/L miR-181a抑制剂下调miR-181a后,U266细胞的增殖活力在24、48及72 h分别为（50.5±4.1）%、（52.3±2.2）%和（69.5±4.3）%,低于对照组的（67.1±3.3）%、（71.5±3.6）%和（78.1±5.4）%（均P〈0.05）,提示细胞增殖受抑.而应用100 nmol/L miR-181a激动剂上调miR-181a后,H929细胞在24、48 h时的增殖活力分别为（38.5±3.6）%和（82.2±6.9）%,高于对照组的（21.2±2.4）%和（61.3±5.4）%（均P〈0.01）,提示细胞增殖增强.细胞周期分析提示,miR-181a抑制剂使U266细胞周期阻滞在G0/G1期.细胞药敏实验结果显示,多柔比星、紫杉醇及5-氟尿嘧啶作用后,与药物单独处理的细胞相比,miR-181a抑制剂下调U266细胞miR-181a表达的细胞增殖活力下降.在48 h和72 h的吸光度（A）值显著低于对照组.细胞迁移实验显示,miR-181a抑制剂下调miR-181a能抑制U266细胞的迁移能力,实验组细胞的迁移比例为（62±10）%,低于对照组的（89±12）%（P〈0.05）,而miR-181a激动剂则能提高H929细胞的迁移能力,实验组细胞的迁移比例[（242±9）%]高于对照组的（98±8）%（P〈0.01）.结论 MM细胞高表达miR-181a.miR-181a的高表达可能会促进MM细胞的增殖、转移及耐药,可能与MM患者预后有关,是MM预后评估一个重要的候选生物标志物.对miR-181a靶基因进行进一步预测,同时应用基因缄默的策略可能会改善MM患者的预后.

Objective To explore the expression of miRNA-181a （miR-181a） in patients with multiple myeloma （MM） and its effect on biological features of MM cells. Methods CD138＋cells of bone marrow from 25 MM patients and 10 patients with hematological non-malignancies were purified by using immunomagnetic separation, and the expression of miR-181a in CD138＋cells and MM cell lines including RPMI 8226, H929 and U266 were detected by real-time quantitative PCR. The effects of down-regulation and up-regulation of miR-181a expression on the biological characteristics of MM cells were studied with miR-181a antagomir and agomir. Results Compared with patients with hematological non-malignant diseases, the expression of＆amp;nbsp;miR-181a in CD138＋ cells was upregulated in MM patients. Compared with CD138＋ cells in hematological non-malignancies, high expressions of miR-181a were observed in RPMI 8226 and U266 myeloma cell line, while low expressions of miR-181a were observed in H929 cells. Down-regulation of miR-181a with 100 nmol/L miR-181a antagomir could inhibit the proliferation of U266 cells at 24,48 and 72 h [（67.1 ± 3.3） %vs. （50.5 ± 4.1） %, （71.5 ± 3.6） % vs. （52.3 ± 2.2） %, （78.1 ± 5.4） % vs. （69.5 ± 4.3） %, P 〈 0.05 respectively], whereas up-regulation of miR-181a with 100 nmol/L miR-181a agomir could significantly promote the proliferation of H929 cells at 24 h and 48 h [（21.2 ± 2.4） %vs. （38.5 ± 3.6） %, （ 61.3 ± 5.4） %vs. （82.2 ±6.9）%, P〈0.01 respectively]. Cell cycle analysis showed that miR-181a antagomir made U266 cell cycle arrest in the G0/G1 phase. Meanwhile, susceptibility test results indicated that the apoptosis of U266 cells induced by doxorubicin, paclitaxel and 5-fluorouracil was increased when the proliferation of miR-181a expression was down-regulated with miR-181a antagomir. In migration assay, the data showed that down-regulation of miR-181a with miR-181a antagomir could inhibit the migration of U266 cells, and the proportion of migrated cells in the experimental group （62 ± 10） %was lower than that in the control group （89 ± 12） %（P〈 0.05）, whereas up-regulation of miR-181a with miR-181a agomir could improve the migration of H929 cells, and the proportion of migrated cells in the experimental group （242 ± 9） % was higher than that in the control group （98 ± 8）%（P〈0.01）. Conclusions The high expression of miR-181a expressed highly by MM cells may promote the proliferation, migration and drug resistance of myeloma cells, indicating that miR-181a could be an important prognostic biomarker candidate, and the application of gene silencing may improve the prognosis of MM.