下调Pygo2抑制脑胶质瘤U251细胞增殖、侵袭及其cyclin D1的表达

Down-regulated Pygo2 expression suppresses proliferation,invasion,and cyclin D1 expression of glioblastoma U251 cells

目的构建RNA干扰(RNAi)重组体抑制Pygo2的表达,并探讨其对脑胶质瘤U251细胞增殖、侵袭的影响及其机制。方法针对Pygo2cDNA序列设计并合成一对特异性的含有短发卡的寡核苷酸序列及其阴性对照序列,经退火后插入pSuper中构建重组体。经EcoRⅠ和HindⅢ双酶切鉴定和DNA测序后,用脂质体2000将其转染脑胶质瘤U251细胞,采用实时定量PCR和蛋白质印迹方法检测Pygo2shRNA对U251细胞Pygo2mRNA和蛋白表达的干扰效果,MTT法检测细胞增殖,流式细胞术检测细胞周期分布,BrdU掺入法检测DNA合成,Transwell检测细胞侵袭。采用蛋白质印迹和免疫荧光法检测Pygo2shRNA对U251细胞cyclin D1、β-catenin蛋白水平和亚细胞定位的影响。结果双酶切和测序鉴定证实插入序列完全正确;Pygo2shRNA显著抑制了U251细胞Pygo2mRNA和蛋白的表达;Pygo2shRNA抑制U251细胞Pygo2表达后,细胞增殖显著降低,细胞更多地阻滞在G1期,且BrdU掺入显著减少,侵袭细胞数显著减少。此外,抑制Pygo2表达可显著下调U251细胞cyclin D1的表达但不改变其亚细胞定位。U251细胞β-catenin表达及其亚细胞定位无明显改变。结论成功构建了抑制Pygo2表达的重组载体。抑制Pygo2表达能有效地抑制脑胶质瘤U251细胞DNA合成,可能通过下调Wnt信号靶基因cyclin D1的表达,使细胞阻滞于G1期而抑制细胞增殖和侵袭。

Objective To construct recombinant vectors for RNA interference（RNAi）targeting Pygo2, and to assess its influence on the proliferation, invasion of glioblastoma U251 cells and the related mechanism. Methods A pair of oligonucleotides containing short hairpin structure targeting Pygo2 cDNA sequences were designed and synthesized, and their negative control sequences were also synthesized. After anmealed, they were inserted into pSuper vector to generate the recombinant plasmids. Then the recombinant plasmids were digested with EcoR I and Hind Ⅲ for identification, and the sequence was assayed by DNA sequencing. The recombinant plasmids were transfected into cultured glioblastoma U251 cells using Lipofectamine^TM 2000. The effect of Pygo2 shRNA on Pygo2 mRNA and protein in U251 ceils was detected by real time PCR and Western blotting analysis, respectively. MTT assay was used to detect the cell proliferation; cell cycle was analyzed by flow eytometry; Bromodeoxyuridine （BrdU） incorporation analysis was used to examine DNA synthesis; and cell invasion assay was performed using Transwell chambers. The effect of Pygo2 shRNA on the protein level and subcellular location of cyclin D1 and β catenin was detected by Western blotting analysis and immunofluorescent staining. Results The recombinant plasmids were completely coincided with the design by the restriction map and the sequence analysis. Pygo2 mRNA and protein expression was significantly suppressed by Pygo2 shRNA. Furthermore, the proliferation of cells in Pygo2 shRNA group was notably inhibited, cell cycle was arrested at the G1 phase, and BrdU incorporation and migrating cells were significantlyinhibited. In addition, Pygo2 knockdown significantly down-regulated cyclin D1 expression without altering the subcellular location, and the expression level and subcellular location of β-catenin had no noticeable changes. Conclusion The recombinant vectors for specific suppression of Pygo2 expression have been constructed successfully. Inhibition of Pygo2 expression can suppress cell proliferation and invasion of glioma U251 cells, decrease DNA synthesis, arrest cell cycle at the G~ phase, and decrease expression of the Wnt target gene evclin D1.