摘要
目的探讨敲低miR-21表达抑制U251人脑胶质瘤细胞株增殖能力的效果和机制。方法脂质体介导转染反义寡聚核苷酸（AS—miR-21）下调U251人脑胶质瘤细胞株miR-21的表达。使用实时定量PCR和原位杂交法鉴定转染后U251细胞miR-21表达水平下调；MTF法评价AS—miR-21抑制U251细胞生长效果；流式细胞术检测转染后U251细胞周期分布和凋亡；采用细胞免疫荧光技术（PCNA、CyclinDl、Bcl-2、PTEN和Septin-7）评价转染后U251细胞肿瘤生物学性状改变。结果MTY结果显示AS—miR-21转染组肿瘤细胞生长速度小于对照组与无义寡核苷酸（F=78．926，P=0．000）组；实时定量PCR法：AS—miR-21转染组miR-21表达下调为对照组0．0424-0．012；LNA—miR-21原位杂交显示AS—miR-21转染组miR-21表达水平较对照组与无义寡核苷酸组下调；流式细胞术检测可见AS—miR-21转染组细胞周期存在G0／G1期阻滞（Х^2=14．160，P=0．007）且凋亡比例高于对照组与无义寡核苷酸组（F=23341．25，P=0．000）；细胞免疫荧光法表明AS—miR-21治疗后U251细胞PCNA、CyclinDl、Bcl-2表达下调，PTEN、Septin-7表达上调。结论以miR-21作为靶点抑制U251人脑胶质瘤细胞株生长结果肯定，miR-21可以作为人脑胶质瘤基因治疗的侯选靶点。
Objective To study the inhibition of U251 human glioma cell line proliferation by knocking down of miR -21 expression in vitro and the possible mechanism. Methods Oligofectamine was used to transfect miRNA - 21 antisense oligonucleotides to knock down the miR - 21 expression level of U251 human glioma cell line in vitro. In - situ hybridization and real - time PCR were conducted to detect the miRNA expression of miR - 21 among different treated groups. The proliferation ability of AS - miR - 21, scramble and control treated U251 glioma cell were determined by MTT assay. The biological characteristics of U251 ceils were evaluated by immunofluoresence staining and cell flow cytometry. Results The expression level of miR -21 was significantly knocked down by AS- miR- 21 treatment. As MTT assay showed, the tumor cell survival rate was inhibited significantly （ F = 78. 926, P = 0. 000）. G0/G1 phase cell cycle arrest was founded （ Х^2 = 14. 160, P =0. 007） and tumor cell apoptosis was induced in As-miR-21 group （ F =23341.25, P =0.000）. The expression of PCNA, CyclinD1 and Bcl-2 were down - regulated and PTEN and Septin - 7 were up - regulated in the AS - miR - 21 treated tumor cells. Conclusions The suppressive effect of anti - miR -21 ODNs on the growth of U251 human glioma cell line is significant and miR -21 can be taken as a candidate for gene therapy of human glioma.
出处
《中华神经外科杂志》
CSCD
北大核心
2009年第8期746-749,共4页
Chinese Journal of Neurosurgery
基金
国家自然科学基金（30772231）
天津市科委应用基础计划重点项目（09JCZOJC17600）
教育部新世纪优秀人才支持计划资助（NCET-07-0615）
