关键词: CRISPR/Cas9, pyk2基因, 人脑胶质瘤细胞, 增殖、迁移及侵袭能力

Abstract: Two pairs of CRISPR/Cas9 recombinant plasmids which targets pyk2 genes were constructed to investigate the effects of Pyk2 (proline rich tyrosine kinase 2) on the proliferation and invasion of human glioma cell line U251. The plasmids were transfected into human glioma U251 cells after they were identificatied, and the wild type U251 cells were set as the control group. Positive monoclonal cells were selected by enzyme digestion and Western blot analysis, then the mutation sites were revealed by sequencing. Compared with the control group, the proliferation ability of the experimental group (the positive monoclonal cells) was decreased by the RTCA method (P<0.05). The invasion and migration ability of the cells in experimental group were significantly inhibited by Transwell chamber method and Scratch test, and the differences were statistically significant (P<005). The matrix metalloproteinase 9 (MMP9), which was used to verify the invasion ability, was significantly decreased in the experimental group than in the control group by Western blot method. Through the above methods, we successfully obtained the stable cell lines with target gene knockout, and confirmed that knockout of pyk2 gene could significantly inhibit the proliferation, migration and invasion of human glioma cells.

Key words: CRISPR/Cas9, pyk2 genes, human glioma cells, proliferation, migration and invasion