反义miR-21抑制U251人脑胶质瘤细胞株增殖的体外研究

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miB-21表达水平下调;MTY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、CyelinD1、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 MTT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0.042±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(X2=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、CyclinD1、Bcl-2表达下调,PrEN、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.

Inhibition of U251 human glioma cell line proliferation by knocking down of miR-21 expression in vitro

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 dioma ceill line in vitro.In-situ hybridization and real-time PCR were conducted to detect the miRNA expression of miR-21 anlong different treated groups.The proliferation ability of AS-miR21.scramble and control treated U251 glioma cell were determined by MTr assay.The biological characteristics of U251 cells were evaluated by immunofluoresenee staining and cell flow cytometry.Results The expression level of miR-21 was significantly knocked down bv AS-miR-21 treatment.As MTT assay showed,the tumor cell survival rate was inhihired significantly(F=78.926,P=0.000).G0/G1 phase cell cycle arrest was founded(X2=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.CyelinDi and Bcl-2were down-regulated and mN and Septin-7 were up-regulated iu 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.