反义miRNA-221/222上调p27kip1抑制胶质瘤细胞生长的体外研究

目的 探讨敲低miRNA-221/222表达上调p27kip1抑制U251人脑胶质瘤细胞株生长的效果及机制.方法 脂质体共转染反义miRNA-221/222下调U251人脑胶质瘤细胞株miRNA-221、miRNA-222的表达.使用Northern blot方法 鉴定转染后U251细胞miRNA-221、miRNA-222表达水平下调;MTT法评价反义miRNA-221/222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;Western blot分析p27kip1蛋白的表达变化.结果 Northern blot显示反义miRNA-221/222共转染组使miRNA-221、miRNA-222的表达水平明显下降.反义miRNA-221转染组仅使miRNA-221的表达水平明显下降,反义miRNA-222转染组仅使miRNA-222的表达水平明显下降.转染无意序列组及对照组的miRNA-221、miRNA-222表达水平没有改变.MTT结果 显示反义miRNA-221/222共转染组肿瘤细胞生长速度小于对照组、转染无意序列组、转染反义miRNA-221组、转染反义miRNA-222组.流式细胞术检测可见反义miRNA-221/222共转染组细胞周期存在G0/G1期阻滞且明显高于其他各组.Western blot显示反义miRNA-221/222共转染组的p27kip1蛋白表达明显上调.结论 反义miRNA-221/222通过上调p27kip1蛋白表达来抑制胶质瘤细胞U251的生长.

Antisense miRNA-221/222 inhabits U251 glioblastoma cell growth through up-regulating p27kip1

Objective To study the suppressive effect of enhancing p27kip1 by knocking down of miRNA-221/222 on the U251 human glioma cell line growth and the possible mechanism. Methods Oligofectamine was used to transfect miRNA-221/222 antisense oligonucleotides to knock down the miRNA-221/221 expression level of U251 human glioma cell line in vitro. Northern blot was conducted to detect the miRNA expression of miRNA-221/222 among different treated groups. The proliferation ability of anti-miRNA-221/222 treated U251 glioma cell was determined by MTT. The cell cycle distribution was detected by cell flow cytometry. Western blot assay was used for p27kip1 expression. Results The expression level of miRNA-221/222 in the anti-miRNA-221/222 group was significantly knocked down than in the other group was determined by northern blot. During the MTT observation, the tumor cell growth was slower in anti-miRNA-221/222 treated group than that in the other treated group. Cell cycle was blocked in G0/G1 phase in the anti-miRNA-221/222 group than in the other group. The expression of p27kip1 was up-regulated in the anti-miRNA-221/222 group by western blot. Conclusion By up-regnlateing p27kip1, anti-miRNA-221/222 suppresses growth of U251 human glioma cell line.