白血病细胞YB-1基因功能的初步研究  

Effect of YB-1 gene knockdown on human leukemia cell line K562/A02

作　　者：许文林[1] 周磊磊[1] 陈巧云[1] 陈琛[1] 方丽丽[1] 房新建[1] 沈慧玲[1] 

机构地区：[1]江苏大学附属人民医院血液科,镇江212002

出　　处：《中华医学遗传学杂志》2009年第4期400-405,共6页Chinese Journal of Medical Genetics

基　　金：江苏省卫生重大项目（K2005017）;江苏省医学重点人才资助项目（RC2007034）

摘　　要：目的探讨YB-1基因对白血病K562／A02细胞株基因表达谱及细胞生物学行为的影响。方法采用脂质体介导的方法将已构建的人YB—1基因特异性短发卡RNA（short hairpin RNA，shRNA）及随机片段HK的真核表达载体转染入K562／A02细胞中。采用基因芯片技术比较阳性转染组与对照组细胞的基因表达的差异，逆转录一PCR验证部分基凶表达的改变。应用甲基氮唑蓝比色法和细胞周期测定分析对白血病细胞增殖的影响，AnnexinV检测白血病细胞的凋亡程度。结果阳性转染的3组细胞YB-1基因和蛋白的表达量明显低于随机片段组和未转染细胞；芯片中共有表达差异的基因252条，其中143条表达下调，109条表达上调，包括转录调节、蛋白翻译合成、细胞增殖、细胞凋亡、细胞的免疫调节、细胞信号和传递蛋白表达等基因。逆转录PCR证实CARD8基因表达上调，RHOC基因表达下调，和芯片检测结果一致。生长曲线提示阳性转染组细胞生长缓慢，细胞周期动力学分析示阳性转染组G1期细胞增多，G2期与S期细胞显著减少；在小剂量三氧化二砷作用下，阳性转染组细胞的凋亡数量明显高于对照组细胞。结论YB-1基因下调可引起白血病K562／A02细胞株基因表达谱的改变，这些基因的改变可能对白血病细胞增殖、细胞周期和凋亡等生物学行为产生一定的影响。Objective To investigate the potential effects of YB-1 gene knockdown on gene expression profile, cell growth and apoptosis in leukemia cell line K562/A02. Methods The recombinant eukaryotic expression plasmid containing YB-1 short hairpin RNA （shRNA） or random sequence （HK） were transfected into K562/A02 cells by lipofectamine mediation, cDNA microarray was performed to explore the alteration of gene expression profile when YB I gene expression was decreased. Expression of CARD8 and RHOC genes were verified by semi quantitative reverse transcription-PCR （RT-PCR）. The proliferative ability of the cells was determined by methyl thiazolyhetrazolium（MTT） assay and cell cycle analysis. Cell apoptosis was assayed by AnnexinV FITC/PI double labeled flow cytometry. Results The levels of YB-1 mRNA and protein decreased dramatically in three positively transfected cells when compared with untransfected K562/A02 cells or K562/A02-HK thansfected cells. Gene expression profile was altered by transfection of YB-1 shRNA into K562/A02 cells. Among 47 000 genes on the microarray, 252 genes were detected to have changes, with 143 down-regulated and 109 up-regulated. They were functionally related to cell cycle progression, gene replication, metabolism, cell apoptosis, cell signal transduction, etc. An increase in CARD8 gene expression and a decrease in RHOC gene expression have been confirmed by RT- PCR in K562/A02-YBX13 cells. The introduction of exogenous YB 1 shRNA gene into K562/A02 cells resulted in decreased proliferation, higher G1 , lower G2 and S ratio in cell cycle distribution in comparison with the control groups. Annexin V/PI detection indicated higher Annex/n V^ - ratio in the three positively transfected cells 24 hours after cells were treated with 0.5 μmol/L of As2O3. Conclusion Down-regulation of YB-1 gene by shRNA-YB 1 can alter the gene expression profile in K562/A02 cells, leading to change of cell proliferation and apoptosis.

关 键 词：YB-1基因 RNA干扰 基因芯片 增殖 凋亡 

分 类 号：R686[医药卫生—骨科学]