富含脯氨酸的酪氨酸激酶2在U251胶质瘤细胞侵袭迁移中的作用

摘要

目的 观察应用RNA干扰沉默U251人胶质瘤细胞中富含脯氨酸的酪氨酸激酶2(Pyk2)的表达对细胞侵袭迁移能力的影响.方法 将Pyk2短发夹RNA(shRNA)和对照shRNA分别转染U251细胞,并将细胞分为Pyk2 shRNA组、对照shRNA组及未处理组.采用Western blot检测转染前后U251细胞中Pyk2的蛋白表达及磷酸化水平.采用Transwell迁移及侵袭实验检测转染前后U251细胞的迁移及侵袭能力.应用免疫荧光染色观察转染前后细胞黏着斑的形态变化,应用Western blot检测侵袭相关基质金属蛋白酶(MMPs)的表达.结果 Pyk2 shRNA组U251细胞中Pyk2的蛋白表达及磷酸化水平明显下调.Pyk2沉默能显著抑制U251细胞的迁移及侵袭能力分别达72％及75％.未处理组细胞黏着斑面积较小,呈细丝状,而Pyk2 shRNA组细胞黏着斑面积增大,呈点状或斑片状.Pyk2 shRNA组细胞MMP-2和MMP-9的表达水平分别为0.24±0.07和0.18±0.06,显著低于未处理组(设定为1,P＜0.05).结论 Pyk2沉默能够抑制U251胶质瘤细胞的迁移和侵袭能力,可能与其参与调控黏着斑的周转及MMPs的表达有关.%Objective To investigate the impact of proline-rich tyrosine kinase 2 (Pyk2) silencing by RNA interference on invasion and migration of human U251 glioma cells.Methods U251 cells were transfected with Proline-rich tyrosine kinase 2 (Pyk2) short hairpin RNA (shRNA) and control short hairpin RNA (shRNA) and divided into three groups:the Pyk2 shRNA group,the control shRNA group,and the untreated group.The protein expression and phosphorylation levels of Pyk2 after transfection were detected by Western blotting.The migration and invasion ability of U251 cells was determined by Transwell migration and invasion assay.The morphology of focal adhesions was observed by immunofluorescence.The expression of invasion related matrix metallproteinases (MMPs) was detected by Western blotting.Results Compared with the untreated group,the expression and activity of Pyk2 was significantly downregulated in the Pyk2 shRNA group.Pyk2 downregulation significantly suppressed the migration and invasion ability of U251 cells by 72％ and 75％,respectively.Cells showed a threadlike pattern of small focal adhesions in the peripheral area of cells in the untreated group.However,cells showed a plaques pattern of large focal adhesions in the Pyk2 shRNA group.The relative expression of MMP-2 and MMP-9 in the Pyk2 shRNA group was 0.24 ±0.07 and 0.18 ±0.06,which was significantly decreased compared with the untreated group (set to 1) (P ＜ 0.05).Conclusion Pyk2 silencing can inhibit migration and invasion of U251 cells,which may be closely associated with the regulation of focal adhesion turnover and MMPs expression.The study indicates the potential value of Pyk2 as a molecular target for anti-invasion therapy.