摘要
目的 检测microRNA-181a（miR-181a）在急性淋巴细胞白血病（ALL）患儿中的表达水平,并研究其在ALL细胞株CCRF-CEM细胞及耐药株CEM-C1细胞中的功能.方法 采用实时荧光定量PCR方法检测ALL患儿骨髓样本、ALL细胞株CCRF-CEM细胞及其耐药株CEM-C1细胞中miR-181a的表达水平.采用电穿孔转染的方法抑制耐药株CEM-C1细胞并上调非耐药株CCRF-CEM细胞中miR-181a的表达,予不同浓度梯度（终浓度分别为0.01、0.1、1、10、100、1 000 ng/ml）喜树碱处理后,采用CCK-8法观察各浓度梯度喜树碱处理后细胞的存活情况,绘制细胞增殖抑制曲线并计算半数抑制浓度（IC50）.结果 初诊-复发组患儿初诊及复发骨髓样本miR-181a相对表达水平（4.84±2.71及6.53±2.20）均高于对照组（1.41±0.53）（P=0.017、0.001）,初诊-完全缓解组患儿初诊骨髓样本miR-181a水平（7.5±2.50）较对照组明显升高（P=0.000）,而完全缓解后miR-181a水平下降至1.35±0.35,与对照组比较差异无统计学意义（P=0.863）.CEM-C1细胞miR-181a相对表达水平（-4.39±0.08）较CCRF-CEM细胞（-2.32±0.03）明显升高（P=0.000）.转染miR-181a抑制剂CEM-C1细胞较转染阴性对照组增殖抑制率明显升高（P＜0.05）,IC50分别为30.61、2 255.00 ng/ml,耐药指数（RI） =73.67.miR-181a过表达CCRF-CEM细胞较阴性对照组增殖抑制率明显降低（P＜0.05）,IC50分别为126.60、1.34 ng/ml,RI=94.26.结论 ALL患儿骨髓及CEM-C1细胞中miR-181a异常高表达,抑制CEM-C1细胞中miR-181a的表达可明显增加CEM-C1细胞的药物敏感性,在CCRF-CEM细胞中上凋miR-181a的表达能明显增加CCRF-CEM细胞的耐药性.
Objective To investigate the expression of miR-181 a in bone marrow （BM） samples of pediatric acute lymphoblastic leukemia （ALL） and explore the mechanism of miR-181a on ALL cell line CCRF-CEM and drug resistance cell line CEM-C1.Methods BM samples were obtained from 18 patients where matched samples at initial diagnosis and first BM relapse or complete remission were available.BM samples and cord blood samples （normal controls） were used to confirm the differential expression of miRNA-181a by quantitative real-time polymerase chain reaction （qRT-PCR）.The expressions of miR-181a in both CCRF-CEM and its mutidrug-resistant counterpart CEM-C1 cells were also detected.Then,CCK-8 assay was performed to quantify the effects of miR-18 l a on CEM-C1 and CCRF-CEM cells growth and viability.Results Up-regulated miR-181a with higher fold changes in both initial diagnosis （4.84±2.71,7.58±2.50） and relapsed samples （6.53±2.20） compared to normal controls （1.41±0.53） （P=0.017,0.000,0.001,respectively） were observed,whereas the miR-181a expression in the samples of CR （1.35±0.35） compared to normal control showed no significant difference （P=0.863）.The miR-181a expression level was higher in CEM-C1 cells （-4.39±0.08） than of in CCRF-CEM cells （-2.32±0.03） （P=0.000）.CCK-8 assay revealed that suppression of miR-181a in CEM-C1 cells by transfecting the specific inhibitor of miR-18 1 a led to significantly higher cellular proliferation inhibition rate than negative control cells （P〈0.05）,IC50 were 30.61 ng/ml and 2 255.00 ng/ml with RI as 73.67.While increased miR-181a in CCRF-CEM cells led to significantly lower CPIR than negative control cells （P〈0.01）,IC50 were 126.60 ng/ml and 1.34 ng/ml with RI as 94.26.Conclusion Upregulation of miR-181a might play an important role in the development of drug resistance in CEM-C1 cells,and knockdown of miR-18 1 a could sensitize CEM-C1 cells to camptothecin; Meanwhile increased expression of miR-181a could promote CCRF-CE
出处
《中华血液学杂志》
CAS
CSCD
北大核心
2015年第1期53-57,共5页
Chinese Journal of Hematology
基金
国家自然科学基金（81272310）
