反义microRNA-221与反义microRNA-222抑制人脑胶质瘤细胞的体外与体内生长  

Inhibitory effect of knocking down microRNA221 and microRNA222 on glioma cell growth in vitro and in vivo

作　　者：张春智[1] 康春生[1] 浦佩玉[1] 王广秀[1] 贾志凡[1] 张安玲[1] 韩磊[1] 许鹏[1] 

机构地区：[1]天津医科大学总医院神经外科天津市神经病学研究神经肿瘤研究室,300052

出　　处：《中华肿瘤杂志》2009年第10期721-726,共6页Chinese Journal of Oncology

基　　金：教育部新世纪优秀人才支持计划（NCET-07-0615）;国家自然科学基金（30772231）;天津市科技计划项目（07ZCGHHZ1000）

摘　　要：目的探讨敲低microRNA（miR）-221和miR-222表达以抑制人脑胶质瘤U251细胞生长的作用及其机制。方法脂质体介导转染反义寡聚核苷酸（AS—miR-221和AS．miR-222）于人脑胶质瘤细胞13251。采用Northern blot鉴定转染后U251细胞的miR-221和miR-222表达水平；四甲基偶氮唑蓝（Myr）法评价AS—miR-221和AS—miR-222抑制U251细胞生长的作用；Transwell实验检测细胞侵袭能力；流式细胞术检测细胞周期的分布和凋亡；Western blot检测转染后U251细胞相关蛋白表达的变化，并用AS—miR-221和AS—miR-222治疗裸鼠皮下移植瘤，观察其在活体内对肿瘤生长的抑制作用。结果Northern blot检测结果显示，AS-miR-221和AS—miR-222共转染后，肿瘤细胞miR-221和miR-222表达明显下降，细胞生长速度降低，细胞穿过率为14．5％，细胞周期出现G0／G1期阻滞，凋亡率（13．7％）增高，并可见connexin43、p27、PUMA、caspase-3、PTEN、TIMP3和Bax等相关蛋白表达增高，而bcl-2表达降低，p53无明显变化。经AS—miR-221和AS—miR-222治疗后，裸鼠皮下移植瘤生长明显受抑。结论AS-miR-221和AS—miR-222共转染可抑制U251细胞的增殖与侵袭，miR-221和miR-222可以作为人脑胶质瘤基因治疗的侯选靶点。Objective To study the inhibitory effect of knocking down microRNA （miR）-221 and miR-222 on human glioma cell growth and its possible mechanism. Methods miRNA-221/222 antisense oligonucleotides （antisense miR221/222）were transfected into human glioma U251 ceils by lipofectamine. Northern blot analysis was conducted to detect the mRNA expression of miR-221/222 in the control and transfected cell groups. The proliferation activity of cells was determined by MTT assay. Cell invasion ability was examined by transwell assay, and cell cycle kinetics and apoptosis were detected with flow cytometry. The expression of relevant proteins was analyzed by Western blotting. The therapeutic efficacy of antisense miR221/222 on the growth of xenograft tumors in nude mice were also observed. Results In the antisense miR-221/222-transfected cells, the expression of miR-221/222 was significantly reduced; the cell invasion ability was suppressed, cell cycle was blocked at G0/G1 phase, and apoptotic ceils were increased. The growth of xenograft tumors treated with antisense miR-221/222 was also inhibited. In antisense miR-221/222 treated tumor cells, the expression of bcl-2 was down-regulated while connexin43, p27, PUMA, caspase-3, PTEN, TIMP3 and Bax up-regulated, and p53 expression not changed. Conclusion There is a significant inhibitory effect of antisense miR-221/222 on the growth of human glioma U251 ceils, miR-221/222 may be considered as a candidate target for gene therapy of human gliomas.

关 键 词：人脑胶质瘤细胞系 microRNA-221 microRNA-222 基因疗法 

分 类 号：R686[医药卫生—骨科学]