腺病毒载体介导shRNAs共调控胶质母细胞瘤细胞中miR-221／222的表达及功能的体外研究  

Expressions and functions of miR-221 and miR-222 in glioblastoma co-modulated by adenovirus-mediated shRNAs

作　　者：王轩[1] 韩磊[2] 张安玲[2] 王广秀[2] 浦佩玉[2] 于春江[3] 申长虹[2] 佟小光[1] 康春生[2] 

机构地区：[1]天津市环湖医院神经外科,天津市神经外科研究所,天津市脑血管与神经变性重点实验室,300060 [2]天津医科大学总医院神经外科,天津市神经病学研究所神经肿瘤实验室,天津300052 [3]首都医科大学三博脑科医院神经外科,北京100093

出　　处：《中华神经医学杂志》2016年第3期224-230,共7页Chinese Journal of Neuromedicine

基　　金：国家自然科学基金（81402079、81172399）;天津市应用基础与前沿技术研究计划（15JCQNJC44700）

摘　　要：目的构建能够对胶质母细胞瘤小分子RNA（miR）-221和miR-222达到多层次、半定量、共调控效果的腺病毒载体系统。方法应用载体融合和短发夹RNA（shRNA）引导链3＇端突变的策略构建出突变型（mut）-shmiR-221/222和经典型（class）-shmiR-221/222 2种共表达腺病毒载体并转导入LN229和U251细胞中，分别为class-shmiR-221/222转导组、mut-shmiR-221/222转导组，同时设空白对照组，采用实时荧光定量PCR检测3组细胞miR-221/222的表达，Western blotting检测细胞周期蛋白依赖性激酶抑制因子（1Bp27kip1）蛋白的表达，流式细胞仪检测细胞周期G1期阻滞情况，倒置相差显微镜观察细胞形态的变化，Caspase-3/7萤光素酶法检测细胞凋亡，编写基于核苷酸循环基序（NCM）的软件，推断shRNA和靶miR的准确三维结构并计算相应的自由能状态。结果空白对照组、mut-shmiR-221/222转导组、class-shmiR-221/222转导组细胞miR-221/222的表达依次下调；p27kip1表达水平依次上调；G0/G1期比例依次上调，S期比例依次下调，G1期阻滞比例依次上调；细胞破坏和丧失原有形态的程度依次上调，细胞数量依次下调；细胞凋亡依次增加，差异均有统计学意义（P〈0.05）；自由能计算结果显示，mut-shmiR-221/222转导组（-32.7、-32.7）的引导链与靶miR结合时发生作用的热力学稳定性较class-shmiR-221/222转导组（-42.1、-41.5）减低。 结论成功构建shRNA共表达腺病毒载体，利用shRNA引导链3＇端突变的策略影响热力学稳定性可以对胶质母细胞瘤细胞miR-221/222多层次、半定量调控。Objective To establish a adenovirus vector system to co-encode two shRNAs targeting miR-221 and miR-222, respectively, which has multilevel, semiquantitative and co-modulated abilities. Methods The co-modulated shRNA-encoding adenoviruses were constructed by restriction enzyme digestion method （mutant-shmiR-221/222 and class-shmiR-221/222）. The two adenovirus vectors were transduced into LN229 and U251 cells （class-shmiR-221/222 transduction group and mut-shmiR-221/222 ransduction group）; and blank control group was established. The expression of miR-221/222 in the three groups was examined by quantitative real time-PCR, increase of targeted protein expression was determined by Western blotting, and flow cytometry was used to detect the cell cycle kinetecs. The cellular damage was observed under a phase contrast microscope, whereas the cell apoptosis was measured by caspase-3/7 activating assay. Furthermore, the flee energy states were analyzed after applying a NCM-based algorithm for predicting the accurate binding structures. Results The co-modulated shRNA-encoding adenoviruses were successfully constructed and transduced into glioblastoma cell lines. In the blank control group, mut-shmiR-221/222 ransduction group and class-shmiR-221/222 transduction group, the miR-221/222 expression level and rate of cells at stage S decreased accordingly; p27kipl expression level, rate of cells at G0/G1 stage and rate of cells arrested at G1 stage increased accordingly; degrees of cells being destroyed and loss their own morphology and cell apoptosis rate increased accordingly; number of cells decreased accordingly; the differences among them were significant （P〈0.05）. The free energy states revealed that the mutated type was less stable in binding with miRNA than the classic type. Conclusions ShRNA encoding adenovirus to co-modulate the expressions ofmiR-221 and miR-222 in glioblastoma cells is successfully established. Together with the strategy of mutation to influence the stability of binding, the modulation

关 键 词：胶质母细胞瘤 小分子RNA RNA干扰 腺病毒载体 基因治疗 

分 类 号：R739.41[医药卫生—肿瘤]