NUMB shRNA真核表达载体构建及对人U87胶质瘤细胞NUMB、p53表达的影响研究

目的：构建NUMB shRNA（短发夹RNA）真核表达载体，体外评价其对人U87胶质瘤细胞NUMB及p53 mRNA及P53蛋白表达的影响。方法：免疫荧光细胞化学染色证实NUMB、P53蛋白在U87胶质瘤细胞的表达。构建针对NUMB mRNA表达的一对反向shRNA互补序列，克隆入psilencer 3．1／hygro真核表达载体，在测序鉴定无误后将NUMB shRNA转染至U87胶质瘤细胞，用RT—PCR和Western blot检测U87细胞转染前后NUMB及p53基因和P53蛋白的表达水平变化。结果：基因测序证实质粒构建成功，RT—PCR和Western blot结果证实U87细胞转染NUMB shRNA后48hNUMB mRNA及蛋白表达水平均下降，P53蛋白表达水平也明显下调。结论：在U87胶质瘤细胞中，NUMB参与了P53蛋白表达水平的调控，提示NUMB是一个潜在的脑胶质瘤基因及药物治疗的靶点。

Construction of Human NUMB shRNA Eukaryotic Expression Vector and Its Effects on NUMB and p53 Expressions in U87 Glioma Cells

Aim： To construct a NUMB shRNA expression vector, and evaluate its effects on NUMB, p53 mRNA and protein expressions in U87 glioma cells in vitro. Methods： NUMB and P53 proteins in U87 glioma cells were confirmed by immunofluorescence cytochemical staining. A pair of reverse complementary sequence short hairpin RNA （shRNA） targetting NUMB mRNA was cloned into the eukaryotic expression vector psilencer 3.1/hygro, after sequencing, the vector was then transfected into U87 glioma cells. The changes of NUMB and p53 mRNA and protein expressions in transfected cells were demonstrated by RT-PCR and Western blot, respectively. Results： The vector had been constructed correctly by sequencing. RT-PCR and Western blot showed that the levels of NUMB mRNA and protein in U87 glioma cells were distinctly decreased after transfection with NUMB shRNA for 48 h, and the level of P53 protein was also downregulated obviously. Conclusion： In U87 glioma cells, NUMB was involved in the regulation of p53 protein, suggesting NUMB is a potential target for treating glioma with genes and drugs.