低表达miR-27a对胶质瘤细胞侵袭和凋亡的影响

目的 探讨下调miR-27a表达后对U87胶质瘤细胞的影响。方法 常规培养U87细胞系，分别用慢病毒干扰质粒miR-27a-3p、miR-27a-5p转染U87细胞（Lt-I），同时设立无义序列转染组（Lt-NC组）和空白对照组。通过嘌呤霉素处理后筛选出稳定感染的U87胶质瘤细胞。采用实时定量聚合酶链反应（RT-PCR）检测转染后miR-27a-3p、miR-27a-5p在U87细胞系中的表达水平，划痕实验、Transwell侵袭试验检测胶质瘤细胞的侵袭、迁移能力，Western blot法分析转染后的侵袭和凋亡的变化，流式细胞术分析转染前后细胞凋亡的变化，磁共振成像（MRI）和裸鼠皮下胶质瘤模型分析裸鼠肿瘤的形态、直径等特征及随时间的变化规律。结果 相对于未感染慢病毒的空白U87胶质瘤细胞，稳定感染Lt-I的U87胶质瘤细胞miR-27a表达明显下调，同时迁移、侵袭能力降低，而感染Lt-NC组（阴性对照组）则未见此改变。Western blot法检测结果显示，低表达miR-27a细胞侵袭能力降低，同时也促进细胞凋亡。流式细胞术结果显示，敲减miR-27a后，细胞凋亡率增高。磁共振成像（MRI）和裸鼠皮下胶质瘤模型显示，实验组裸鼠肿瘤的生长明显受到抑制。结论 敲减miR-27a可抑制人脑胶质瘤细胞系U87细胞的迁移、侵袭，促进其凋亡，同时抑制其成瘤能力，这可能成为胶质瘤基因治疗的新靶点。

Effect of Low MicroRNA-27a Expression on the Invasiveness and Apoptotic Potential of Glioma Cells

Objective To investigate the effect of downregulating microRNA-27a(miR-27a) expression on invasion and apoptosis of U87 glioma cells. Methods The U87 glioma cell line was conventionally cultured, and the U87 cells were transfected with interfering lentiviral plasmids containing either miR-27a-3p or miR-27a-5p.In parallel,a scrambled-sequence transfection group (Lt-NC) and an empty-vector control group were set up. After puromycin treatment, stably transfected U87 glioma cells were selected.Quantificational real-time polymerase chain reaction (qRT-PCR) was used to detect miR-27a expression levels after transfection of U87 cells. Scratch assays and Transwell invasion assays were used to determine the invasion and migration of the glioma cells after miR-27a knockdown. Western blot techniques were used to analyze changes in invasion and apoptosis after miR-27a knockdown. Flow cytometry was used to determine the change in apoptotic potential due to miR-27a knockdown. Magnetic resonance imaging(MRI),In vivo bioluminescence imaging and Subcutaneous glioma model were used to analyze nude-mouse glioma models for morphology, size, and other characteristics, as well as how these properties change with time. Results Fluorescence microscope showed downregulated miR-27a expression of U87 glioma cells was stably established.Relative to control U87 glioma cells not transfected with lentivirus,U87 glioma cells stably transfected with lentiviral had a clearly downregulated miR-27a expression,a reduced invasion and migration, whereas no change was observed in the NC transfected group (negative control group). Western blot results showed invasion related protein MMP2 was low expression, whereas the expression of protein TIMP2, which inhibits glioma cells invasion was increased.Flow-cytometric results showed that after miR-27a knockdown, the rate of apoptosis increased. Western blot results showed the apoptosis related protein Bax was highly expressed, whereas the expression of protein Bcl-2, which inhibits apoptosis was decreased after miR-27a knockdown.MRI,In vivo bioluminescence imaging and Subcutaneous glioma model showed that tumor growth in the experimental group of nude-mouse was clearly inhibited. Conclusion miR-27a knockdown has an inhibitory effect on invasion and migration whereas promotes apoptosis in the U87 human glioma cells, and thus miR-27a can serve as a new target for gene therapy of glioma.