CAJ | 학술논문

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.
목적 관찰응용RNA간우침묵U251인효질류세포중부함포안산적락안산격매2(Pyk2)적표체대세포침습천이능력적영향.방법 장Pyk2단발협RNA(shRNA)화대조shRNA분별전염U251세포,병장세포분위Pyk2 shRNA조、대조shRNA조급미처리조.채용Western blot검측전염전후U251세포중Pyk2적단백표체급린산화수평.채용Transwell천이급침습실험검측전염전후U251세포적천이급침습능력.응용면역형광염색관찰전염전후세포점착반적형태변화,응용Western blot검측침습상관기질금속단백매(MMPs)적표체.결과 Pyk2 shRNA조U251세포중Pyk2적단백표체급린산화수평명현하조.Pyk2침묵능현저억제U251세포적천이급침습능력분별체72％급75％.미처리조세포점착반면적교소,정세사상,이Pyk2 shRNA조세포점착반면적증대,정점상혹반편상.Pyk2 shRNA조세포MMP-2화MMP-9적표체수평분별위0.24±0.07화0.18±0.06,현저저우미처리조(설정위1,P＜0.05).결론 Pyk2침묵능구억제U251효질류세포적천이화침습능력,가능여기삼여조공점착반적주전급MMPs적표체유관.
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.