miRNA-663在不同的白血病细胞系的表达水平及其生物学功能的初步研究  

Expression Level of miRNA-663 in Different Leukemic Cell Lines and Its Biological Function

作　　者：杨洋[1] 王莉莉[1] 李永辉[1] 高晓宁[1] 于力[1] 

机构地区：[1]中国人民解放军总医院血液科,北京100853

出　　处：《中国实验血液学杂志》2011年第2期279-283,共5页Journal of Experimental Hematology

基　　金：国家自然科学基金资助(编号90919044;81000221);国家973项目资助(编号2005CB522400)

摘　　要：本研究通过5-氮杂胞苷(5-aza)处理K562细胞系,寻找去甲基化后表达上调的microRNA(miRNA),检测其在正常人、慢性髓系白血病(CML)病人和白血病细胞系的表达水平,探讨其对K562细胞系增殖的影响。采用miRNA芯片筛选5-aza处理后表达上调的miRNA,结合生物信息学在上调的芯片结果中选取miRNA前体上游带有CpG岛的miR-663、miR-638及miR-92b,通过荧光实时定量PCR的方法验证在芯片结果中这3个上调的miRNA,证实miR-663在去甲基化后表达上调。分别在K562细胞系、U937细胞系、Kasumi细胞系、初治CML患者外周血白细胞及正常人骨髓白细胞检测miR-663的表达水平。通过甲基化特异性PCR(MSP)的方法,在K562细胞系检测miR-663前体上游CpG岛是否存在甲基化。通过瞬时转染的方法在K562细胞系过表达合成的miR-663成熟体,观察其对K562细胞增殖的影响。结果表明:去甲基化后能在K562细胞系上调miR-663的表达水平,与正常人骨髓白细胞相比,初治CML病人的白细胞、白血病细胞系K562、U937及Kasumi的miR-663表达水平相对较低;MSP检测表明,在K562细胞系miR-663上游的CpG岛是甲基化的。体外K562细胞系过表达miR-663可以抑制K562细胞系的增殖。结论:在K562细胞系miR-663上游的CpG岛存在甲基化。与正常人相比,miRNA-663在CML病人、白血病细胞系K562、U937及Kasumi表达水平相对较低,5-aza可以上调miRNA-663在K562细胞系的表达,在K562细胞系过表达miRNA-663可抑制K562细胞的增殖,提示miRNA-663在白血病中可能具有抑癌作用。The aim of the research was to find the up-regulated microRNA（miRNA） in K562 cells treated by 5-aza, to detect the miRNA expression in healthy people,CML patients and leukemia cell lines and to investigate the influence of miRNA on K562 cell proliferation.Up-regulated miRNA in K562 cells after 5-aza treatment was screened by microarray.The up-regulated miR-638,miR-663 and miR-92b with CpG islands in upstream region were screened by microarray in combination with bioinformatics.The miRNA mentioned above was further ascertained by SYBR-green real-time PCR.Up-regulated miR-663 was confirmed by real-time PCR.Expression level of miR-663 was detected in K562,U937 and Kasumi cell lines,and white blood cells from bone marrow of normal donor and from peripheral blood of newly diagnosed CML patient.Methylation-specific PCR（MSP） was applied to analyze the methylated status of miR-663 CpG island in K562 cells.Proliferation of K562 cells was observed after miR-663 was over expressed by transient transfection.The results showed that the expression level of miR-663 in K562 cells was up-regulated after 5-aza treatment,and the expressions of miR-663 were lower in K562,U937,Kasumi cell lines and newly diagnosed patients,compared with healthy people.The CpG island of miR-663 was methylated in K562 cell line according to detection result of MSP.The proliferation of K562 cell could be suppressed by over-expression of miR-663 in vitro.It is concluded that miR-663 CpG island is methylated in K562 cell line.The miR-663 is down-regulated in K562,U937,Kasumi cell lines and CML patients,compared with healthy people.miRNA-663 in K562 cells is up-regulated after 5-aza treatment. Over-expression of miR-663 can suppress the proliferation of K562 cells,which suggestes that miR-663 may possesses suppressive effect for leukaemia.

关 键 词：miR-663 K562细胞系 去甲基化 白血病 

分 类 号：R733.7[医药卫生—肿瘤]