中文摘要：

目的 探讨反义miR-221/222上调PUMA表达促进人脑胶质母细胞瘤U251细胞凋亡及其机制.方法 脂质体共转染反义miR-221/222下调U251人脑胶质瘤细胞株miR-221、miR-222的表达.流式细胞术检测转染后U251细胞凋亡变化;Caspase 3/7活性检测转染后U251细胞中Caspase 3的活性;JC-1检测转染后U251细胞线粒体膜电位变化;免疫荧光检测Bax蛋白定位;Western blot分析凋亡相关蛋白的表达变化.结果 流式细胞术显示反义miR-221/222可增加U251细胞凋亡;Caspase 3/7活性检测显示反义miR-221/222可增加U251细胞Caspase 3活性;JC-1检测显示反义miR-221/222可增加U251细胞线粒体膜电位衰减;免疫荧光检测反义miR-221/222可促进Bax蛋白从胞质中向线粒体膜上转移;Western blot显示反义miR-221/222共转染组的PUMA、Caspase 3、Bax蛋白表达明显上调,bc1-2蛋白表达明显下调.结论 反义miR-221/222通过上调PUMA蛋白表达来增加胶质瘤细胞U251的凋亡.

英文摘要：

Objective To study the effect of increasing apoptosis by antisence miR - 221/222upregulating PUMA expression on U251 human glioma cell and its mechanism. Methods Oligofectamine was used to transfect miRNA- 221/222 antisense oligonucleotides to knock down the miR- 221/221expression level of U251 human glioma cell line in vitro. Flow cytometry was used to measure early apoptosis by Annexin V staining. Caspase 3/7 activity assay accessed Caspase 3 activity. Changes in mitochondrial membrane potential were determinated by JC - 1 Staining. Translocation of Bax was detected by immunofluorescence. Western blot assay validated expression of apoptotic protein. Results After miR -221/222 was knocked - down, the apoptosis was increased, Caspase 3 activity was enhanced, mitochondrial membrane potential was significantly decreased and Bax translocated from the cytosol to the mitochondria.Western blot assay showed that the expression of PUMA, Caspase 3 and Bax were up - regulated and the expression of bcl - 2 was down - regulated in the anti - miR - 221/222 group. Conclusions By upregulating PUMA, anti- miR-221/222 chould increase the apoptosis of U251 human glioma cell line.