摘要
目的探讨miR-195过表达对人脑胶质瘤细胞系U251增殖和凋亡的影响及其机制。方法实验分为Blank组、NC组和miR-195组，Blank组处理时仅加等体积PBS，后两组通过脂质体将无义序列或miR-195模拟物转染至U251，RT—PCR检测转染前后miR-195表达水平；用CCK-8法计算细胞生长抑制率和平板克隆形成实验评价细胞的增殖能力；流式细胞仪分析细胞凋亡和周期；Hoechst33342和PI双染法检测原位细胞凋亡坏死；利用生物信息学技术预测miR-195的靶基因，Westernblot检测Bcl-2的蛋白水平。结果RT—PCR测得转染后miR-195表达水平提高6倍左右；细胞生长抑制率和平板克隆形成结果分别显示过表达miR-195可以抑制U251生长和克隆形成能力（与NC组比较P〈0．001）；流式细胞仪检测细胞凋亡和Hoeehst33342和PI双染法结果示miR-195组细胞凋亡和坏死比例明显增高（与Blank组及NC组相比P〈0．001）；流式细胞仪检测细胞周期发现miR-195能够阻滞U251在G0／G1期，与Blank组及NC组相比P〈0．05；TargetSean预测发现Bcl-2是miR-195的靶基因；Westernblot检测示过表达miR-195可以下调Bcl-2的表达，与Blank组及NC组相比p〈0．05。结论过表达miR-195可以抑制胶质瘤细胞U251的增殖，促进胶质瘤细胞凋亡和坏死并产生G0／G1期阻滞，可能成为胶质瘤基因治疗的新靶点。
Objective To explore the function and mechanism of miR - 195 over - expression in glioblastoma U251 cell proliferation and apoptosis. Methods The experiment is divided into Blank , NC and miR - 195. Blank is treated with equal volume PBS. Negative control or miR - 195 mimics were transfected into U251 cells by Lipofectamine RNAiMAX. miR - 195 expression levels were detected by Real -time PCR. Cell growth inhibition rates and proliferation were analyzed by CCK -8 assay and colony formation. Flow cytometry was used to monitor changes in cell apoptosis and cycle. Apoptotic cells and necrotic cells were also qualified by Hoechst 33342 and propidium iodide staining. The target of miR - 195 was forecasted by hioinformatics. The expression level of Bcl - 2 protein was detected through Western blotting. Results After the over - expression of miR - 195, Real - time PCR showed that miR - 195 expression level was up - regulated about six times. Cell growth inhibition rate and colony formation revealed that the growth and proliferation ability of the miR- 195 group were significantly suppressed（ compared with NC, P 〈0. 001）. Over- expression of miR- 195 could promote cell apoptosis and death（ compared with Blank and NC, P 〈 0. 001 ）. Predicting outcomes of TargetScan indicated that Bcl - 2 was a target of miR - 195. Furthermore, the over- expression of miR- 195 could block G0/G1 transition and down-regulate the Bcl - 2 expression （ compared with Blank and NC, P 〈 0. 05 ）. Conclusion Over - expression of miR - 195 could inhibit proliferation, induce apoptosis and death, and block G0/S1 transition, miR- 195 may serve as an attractive target of gene therapy for glioblastoma.
出处
《中华神经外科杂志》
CSCD
北大核心
2013年第5期473-477,共5页
Chinese Journal of Neurosurgery
基金
山西省基础研究计划（青年科技研究基金）项目（2012021035-4）
山西省科技攻关项目（20110313011-3）
