靶向抑制miR155表达对耐阿霉素多发性骨髓瘤细胞株RPM18226／DOX耐药性的影响  

Targeting miR155 restores chemotherapy sensitivity in drug-resistant myeloma cell-line RPMI8226/ DOX cells

作　　者：肖翠容[1] 洪秀理[2] 胡嘉升[2] 陈亚玫[2] 鹿全意[2] 

机构地区：[1]福建省漳州市医院,363000 [2]厦门大学附属中山医院血液科,361004

出　　处：《中华血液学杂志》2017年第1期55-59,共5页Chinese Journal of Hematology

基　　金：福建省医学创新课题（2014-CXB-42）

摘　　要：目的分析miR155异常表达在多发性骨髓瘤（MM）化疗耐药机制中的作用，探讨靶向抑制miR155表达对耐阿霉素MM细胞株RPMI8226/DOX耐药性的影响，进一步分析其作用机制。 方法通过浓度梯度递增法建立耐阿霉素MM细胞系RPMI8226/DOX；采用实时荧光定量PCR法检测RPMI8226/DOX细胞和MM敏感细胞株RPMI8226/S miR155基因表达，Western blot检测FOXO3a、BCL-2蛋白表达。在RPMI8226/DOX细胞中分别转染miR155抑制物和模拟物，通过实时荧光定量PCR法检测miR155抑制物和模拟物的转染效率，应用CCK-8法检测转染后细胞对阿霉素的敏感性。在靶向抑制物干预MM细胞后，Western blot分析FOXO3a、BCL-2通路蛋白表达的变化。 结果①RPMI8226/DOX细胞miR155相对表达量为RPMI8226/S细胞的（26.860±2.340）倍，BCL-2蛋白表达上调，FOXO3a蛋白表达下调。②靶向抑制miR155表达72 h后，转染抑制率为64.57%，miR155基因表达下调，FOXO3a蛋白表达上调，BCL-2蛋白表达下调；RPMI8226/DOX细胞部分恢复了对阿霉素的敏感性，逆转耐药倍数为2.518。 结论miR155异常表达与MM的化疗耐药形成相关，靶向抑制miR155表达可以通过影响FOXO3a蛋白表达恢复MM耐药细胞对化疗药物的敏感性。目的分析miRl55异常表达在多发性骨髓瘤（MM）化疗耐药机制中的作用，探讨靶向抑制miRl55表达对耐阿霉素MM细胞株RPMl8226／DOX耐药性的影响，进一步分析其作用机制。方法通过浓度梯度递增法建立耐阿霉素MM细胞系RPMl8226／DOX；采用实时荧光定量PCR法检测RPMl8226／DOX细胞和MM敏感细胞株RPMl8226／SmiRl55基因表达，Westernblot检测FOX03a、BCL．2蛋白表达。在RPMl8226／DOX细胞中分别转染miRl55抑制物和模拟物，通过实时荧光定量PCR法检测miRl55抑制物和模拟物的转染效率，应用CCK一8法检测转染后细胞对阿霉素的敏感性。在靶向抑制物干预MM细胞后，Westernblot分析FOX03a、BCL一2通Objective To explore the mechanism of abnormal expression of microRNA155 （miR155） in myeloma drug-resistance to probe the possibility of inhibiting miR155 expression to restore chemotherapy sensitivity and its molecular mechanism in drug-resistant myeloma cells. Methods Drug- resistant myeloma cell-line RPMI8226/DOX was established by culturing RPMI8226 cells with continuous low concentration and intermittent gradually increasing concentration of doxorubicin in vitro; The levels of miR155 mRNA were measured by qRT-PCR, and both proteins FOXO3a and BCL-2 expressions were detected by Western blot in cell-lines RPMI8226/S and RPMI8226/Dox. RPMI8226/DOX cells were transfected by miR155 inhibitor and mimic using gene transfer method, and then CCK-8 was used to measure proliferation and inhibition ratio, the changes of miR155 expression were detected by RT-PCR. Proteins FOXO3a and BCL-2 were detected by Western blot. Results Comparing with RPMI8226 cells, the level of miR155 mRNA was obviously up-regulated with the relative expression of 26.860 ± 2.340, together with increased expression of Bcl-2 protein but decreased expression of FOXO3a in RPMI8226/ DOX cells. After 72 h treatment with miR155 inhibitor, the inhibition rate of transfection was 64.57%, miR155 expression decreased sharply, the level of FOXO3a expression was upregulated while BCL-2 expression decreased, chemotherapy sensitivity was restored on cell-line RPMI8226/DOX with reversed drug-resistance ratio of 2.518. Conclusions The abnormal expression of miR155 was closely associated with myeloma drug-resistance, targeting inhibition of miR155 expression could restore chemotherapy sensitivity by increasing FOXO3a expression in drug-resistant myeloma cells.

关 键 词：多发性骨髓瘤 抗药性 肿瘤 微RNAS FOXO3A 

分 类 号：R733.3[医药卫生—肿瘤]