Down-regulated Pygo2 expression suppresses proliferation, invasion, and cyclin D1 expression of glioblastoma U251 cells / 第二军医大学学报

ABSTRACT

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 annealed, 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™ 2000. The effect of Pygo2 shRNA on Pygo2 mRNA and protein in U251 cells 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 cytometry; 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 significantly inhibited. 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 G1 phase, and decrease expression of the Wnt target gene cyclin Dl.