靶向MR-1基因shRNA表达载体的构建及其对白血病K562细胞增殖的抑制作用  

Construction shRNA expression vector against MR-1 and its inhibitory effect on the proliferation of K562 leukemic cells

作　　者：赵午莉[1] 任开环[1] 李保卫[1] 邵荣光[1] 

机构地区：[1]中国医学科学院北京协和医学院医药生物技术研究所肿瘤室,北京100050

出　　处：《中国医药生物技术》2013年第1期6-12,共7页Chinese Medicinal Biotechnology

基　　金：国家自然科学基金(30772583;30701011);国家重点基础研究发展计划(973计划)(2009CB521807);"重大新药创制"国家科技重大专项(2012ZX09301-002)

摘　　要：目的构建一种靶向MR-1基因的shRNA稳定表达载体,并检验其对白血病K562细胞增殖能力的影响。方法以带有H1启动子可稳定表达靶基因shRNA的pCD-shRNA为载体,采用BamHI和HindIII酶切pCD-shRNA并回收线性空载体,采用T4DNA连接酶将MR-1shRNA模板DNA双链与酶切后线性化的载体连接,从而建立稳定表达MR-1-shRNA的载体。将构建的pCD-shRNA-MR-1载体转染人白血病K562细胞,建立稳定沉默MR-1的细胞系,采用细胞计数法和westernblot对MR-1沉默后细胞的增殖能力和信号通路进行检测分析。结果成功构建了可稳定表达MR-1-shRNA的载体pCD-shRNA-MR-1,经酶切鉴定及基因测序测定,结果表明载体中的MR-1-shRNA与设计序列完全相符,目的基因序列准确无误。将pCD-shRNA-MR-1转染白血病K562细胞,建立了两株稳定沉默MR-1的K562/MR-1细胞株。细胞增殖实验表明,MR-1稳定沉默细胞的增殖能力显著降低;细胞信号通路结果表明,MR-1稳定沉默细胞Jak2和Bcr-Abl的磷酸化水平降低。结论利用pCD-shRNA质粒构建了一个可表达MR-1-shRNA的表达载体,该载体转染K562细胞后引起细胞增殖能力降低。研究表明MR-1基因在肿瘤细胞增殖过程具有促增殖作用,其机制是降低Jak2和Bcr-Abl的磷酸化水平。Objective To construct a vector of targeting MR-1 and study its effect on proliferation of chronic myelocytic leukemia K562 cells. Methods The recombinant expressive vector pCD-shRNA-MR-1 was constructed by inserting DNA sequence into the pCD-shRNA vector with H1 promoter via enzyme BamH I, Hind III and T4 DNA ligase. The vector pCD-shRNA-MR-1 was transfected into the K562 cells and MR-1 stably silenced cells were established. Cell proliferation and signal pathway were investigated by cell counts and western blot, respectively. Results The recombinant expressive vector pCD-shRNA-MR-1 was constructed and identified by enzyme digestion and DNA sequencing. MR-1 stably silenced cells were established with vector pCD-shRNA-MR-1 in K562 cells, and the proliferation and p-Jak2 and p-Bcr-Abl of MR-1 stably silenced cells were decreased markedly as compared to mock transfected cells. Conclusions The recombinant expressive vector pCD-shRNA-MR-1 is constructed and identified. The proliferation is decreased after pCD-shRNA-MR-1 is transfected into K562 cells. MR-1 participates into the control of tumor proliferation bymodulation of p-Jak2 and p-Bcr-Abl.

关 键 词：基因表达调控 白血病 RNA干扰 MR-1 

分 类 号：R733.7[医药卫生—肿瘤]