多发性骨髓瘤患者骨髓基质细胞调节miRNA-15a／-16表达在骨髓瘤细胞耐药中的作用及机制

Regulation of miRNA-15a/-16 expression on the drug resistance of myeloma cells

目的探讨骨髓基质细胞在骨髓瘤耐药中的作用及其分子机制。方法收集2011年5—7月中国医学科学院血液病医院淋巴瘤骨髓瘤诊疗中心初诊的多发性骨髓瘤（MM）患者骨髓标本5份，健康供者骨髓标本5份，分离培养其中骨髓基质细胞（BMSC），收集细胞培养上清液，酶联免疫吸附试验（ELISA）法检测细胞因子分泌水平。将MM患者BMSC与人骨髓瘤细胞系共同培养，分别加入美法仑、硼替佐米；采用四甲基偶氮唑盐（MTY）法观察骨髓瘤细胞增殖情况；采用实时定量PCR（qRT—PCR）方法检测骨髓瘤细胞微小RNA（miRNA）15a／-16的表达。人骨髓瘤细胞系中加入细胞因子，qRT．PCR检测miRNA-15a／-16的表达。转染上调骨髓瘤细胞miRNA-15a表达，采用膜联蛋白（Annexin）V／碘化丙啶（PI）染色及流式细胞术观察miRNA-15a上调表达对细胞周期、细胞凋亡的影响。结果成功分离得到BMSC细胞，MM患者BMSC中白细胞介素6（IL-6）和血管内皮生长因子（VEGF）的表达水平均明显高于健康对照[（189±9）比（115±15）pg／ml，（1497±40）比（1239±21）pg／ml，均P〈0．05]。美法仑、硼替佐米均上调骨髓瘤细胞miRNA-15a和miRNA-16的表达（均P〈0．05）；而MM患者BMSC与骨髓瘤细胞共培养后，可抑制美法仑和硼替佐米对miRNA-15a和miRNA-16的上调作用[美法仑：4．690±0．050比34．440±4．100，0．760±0．070比12．030±1．020；硼替佐米：1．440±230比11．480±1．488，0．880±0．040比3．680±0．420；均P〈0．05l。IL-6可抑制骨髓瘤细胞miRNA-15a／-16表达，并呈时间和剂量依赖性（P〈0．05）。转染上调骨髓瘤细胞miRNA-15a表达，导致细胞周期G1／S阻滞，抑制细胞增殖（P〈0．05）。结论骨髓瘤微环境中基质细胞可能通过分泌高水平IL-6抑制骨髓瘤细胞miRNA-15a／-16表达，抑制细胞增殖，降低骨髓瘤细胞对化疗药物的敏感性，参与MM耐药的发生。

Objective To explore the effects and mechanism of bone marrow stromal cells （BMSCs） on the drug resistance of multiple myeloma （MM）. Methods Fresh untreated MM patient （5 samples） and health donor （5 samples） bone marrow samples were collected from May 2011 to July 2011 at our hospital. Their BMSCs were separated respectively. The supernatant expression of cytokines in BMSCs was detected by enzyme-linked immunosorbent assay （ELISA）. Myeloma cells were co-cuhured with BMSCs and treated with melphalan or bortezomib. Cell proliferation and miRNA-15a/-16 expression of myeloma ceils were measured in different culture conditions with thiazoyl blue tetrazolium bromide （MT＇F） assay and quantitative real-time PCR （qRT-PCR） . miRNA-15a was transfected into myeloma cells. And the miRNA-15a functions on cell cycle and cell apoptosis were detected by flow cytometry. Results ELISA assay showed that cytokine IL-6 and VEGF were at higher levels in MM-BMSCs than healthy BMSCs （ （ 189 ± 9） vs （ 115 ± 15 ） pg/ml, （ 1497 ± 40） vs （ 1239 ± 21 ） pg/ml, both P 〈 0. 05 ）. The miRNA-15a/-16 expressions of myeloma cells were up-regulated after the treatment of melphalan and bortezomib （ all P 〈 0. 05 ）. However, the miRNA-15a/-16 up-regulation by melphalan or bortezomib became inhibited when MM cells were co-cultured with MM-BMSCs （melphalan：4. 690 ± 0. 050 vs 34. 440 ± 4. 100,0. 760 ± 0. 070 vs 12. 030 ± 1. 020, bortezomib： 1. 440 ± O. 230 vs 11. 480 ± 1. 488,0. 880 ±0. 040 vs 3. 680 ± 0. 420, all P 〈 0. 05）. Furthermore, IL-6 suppressed the expression of miRNA-15a/-16 in a dose and time-dependent pattern. The transfeetion of miRNA-15a led to the arrest of MM cell cycle in GI/S phase. Conclusions BMSCs suppress the proliferation of myeloma cells and regulate the drug sensitivity of myeloma ceils through the inhibited expression of miRNA-15a/-16. IL-6 plays a pivotal role in the occurrence of drug resistance. [ Key words ] Multiple myeloma; Drug resistance, neoplasm ; MicroRNAs ; Bone marrow stromal ceils