miR-21调控人脑胶质瘤细胞侵袭能力的体外研究

目的 探讨miR-21调控人脑胶质瘤细胞侵袭能力的体外机制.方法 脂质体介导反义miR-21(AS-miR-21)转染体外常规培养的U251细胞,同时设无义寡脱氧核苷酸(ODN)组和对照组.测定荧光素酶活性验证3组细胞中miR-21的表达,Matrigel基质生长实验检测U251细胞的生长情况,Transwell细胞体外迁移实验检测U251细胞的迁移能力,Western blotting检测细胞侵袭相关蛋白局部粘着斑激酶(FAK)、基质金属蛋白酶9/2(MMP-9/2)、人基质金属蛋白酶抑制因子1(TIMP-1)、微管蛋白α(Tubulin-α)的表达,免疫荧光检测细胞Tubulin-α蛋白的形态. 结果 与对照组和无义ODN组比较,转染AS-miR-21组U251细胞miR-21表达水平降低,Matrigel基质生长实验显示转染AS-miR-21组U251细胞体外培养克隆平均直径较小,Transwell细胞体外迁移实验显示转染AS-miR-21组穿膜细胞数较少,Western blotting结果 显示转染AS-miR-21组U251细胞FAK、MMP-9/2表达较低,TIMP-1表达较高,差异均有统计学意义(P＜0.05).Tubulin-α的表达无明显变化,免疫荧光染色显示转染AS-miR-21组Tubulin-α蛋白形态扭曲. 结论 miR-21高表达可以促进U251胶质瘤细胞侵袭生长,提示miR-21可以作为基因治疗脑胶质瘤的候选靶点.

MiR-21 regulates the invasion of U251 human glioblastoma cells in vitro

Objective To study the mechanism of miR-21 in regulating the invasion of human glioblastoma (GBM) cells in vitro. Methods The transfection reagent oligofectamine was mixed with antisense miRNA-21 (AS-miR-21) and nonsense oligodeoxyribonucleotides (ODN), respectively, and then, they were added into the medium of U251 GBM cell line as AS-miR-21 treatment group and nonsense ODN treatment group, respectively; control group (treated with PBS) was also established.MiR-21 luciferase reporter assay was used to detect the miR-21 knocking down effect. Matrigel cell growth assay and Transwell assay were used to determine the invasion and migration abilities of U251 cells. Western blotting was employed to test the expressions of invasion-related proteins (FAK,MMP-9/2, TIMP-1 and Tubulin-α); immunofluorescence was also employed to observe the morphology of Tubulin-α protein in GBM cells. Results Luciferase intensity in as-miR-21 treated U251 cells was significantly suppressed as compared with that in the control group and nonsense ODN treatment group (P＜0.05). The diameter of cultured clone in as-miR-21 treated U251 cells was smaller than that in the controls and nonsense ODN treatment group (F=102.819, P=0.000). Decreased cells via the transwell member in thc AS-miR-21 treatment group were detected as compared with those in the controls and cnonsensc ODN treatment group (F=243.465, P=0.000). The expressions of FAK, MMP-2/9 weredown-regulated and that of TIMP-1 was up-regulated in the AS-miR-21 treated tumor cells as compared with the other 2 groups (P＜0.05). No obvious changes were noted on the expression of Tubulin α,however, the morphology of Tubulin α protein in the AS-miR-21 treatment group changed. Conclusion High expression of miR-21 induce the ability of U251 GBM cell invasion and miR-21 can be taken as a candidate for gene therapy of human glioma.