ABL2基因表达沉默对胶质瘤细胞迁移和侵袭的影响

Effect of silencing abelson nonreceptor tyrosine kinases 2 expression on invasion and migration of glioma cells

目的 研究非受体酪氨酸激酶ABL2基因表达沉默对胶质瘤细胞迁移和侵袭能力的影响及机制. 方法 免疫组化染色检测正常脑组织和经病理证实的胶质母细胞瘤组织标本（分别来自天津医科大学总医院神经外科自2012年9月至2014年8月期间的癫痫、胶质瘤手术患者）中ABL2的表达.将携带ABL2抑制物核苷酸序列（shRNA-ABL2）、阴性对照核苷酸序列（shRNA-NC）的重组慢病毒转染体外培养的SNB19胶质瘤细胞,分别作为shRNA-ABL2组、shRNA-NC组,同时设不做任何处理的空白对照组,转染后48 h实时荧光定量PCR检测3组细胞ABL2 mRNA的表达;Western blotting检测3组细胞ABL2、皮层肌动蛋白、Y-421位点酪氨酸磷酸化的皮层肌动蛋白（pY-421 cortactin）的表达;划痕实验和Transwell实验评价各组细胞的迁移和侵袭能力;免疫荧光染色检测ABL2、皮层肌动蛋白在SNB19细胞中的定位情况. 结果 免疫组化染色显示ABL2在正常脑组织中表达较低,在胶质母细胞瘤中表达则较高.与shRNA-NC组、空白对照组比较,转染后shRNA-ABL2组细胞ABL2 mRNA和蛋白、pY421 cortactin蛋白的表达降低,差异均有统计学意义（P＜0.05）;划痕实验显示shRNA-ABL2组细胞迁移数（72.33 ±7.64）少于shRNA-NC组、空白对照组（187.67±5.03、190.33±7.23）,Transwell实验显示shRNA-ABL2组穿膜细胞数低于shRNA-NC组、空白对照组,差异均有统计学意义（P＜0.05）.免疫荧光染色结果显示ABL2与皮层肌动蛋白在细胞前端伪足处共定位. 结论 ABL2在胶质母细胞瘤组织中表达较正常脑组织高.沉默ABL2表达可能通过调节pY-421 cortactin的水平来抑制胶质瘤细胞的迁移和侵袭.

Objective To investigate the inhibitory effect of abelson nonreceptor tyrosine kinases 2 （ABL2） silenced by shRNA on invasion and migration in glioma cells as well as its potential mechanism.Methods The ABL2 expressions in normal brain tissues and glioblastoma tissues,collected from patients performed epilepsy and glioma surgeries,respectively,in our hospital from September 2012 to August 2014,were detected by immunohistochemistry.The recombinant lentivirus aimed at ABL2 silencing was prepared.Human glioma cells SNB19 were employed in this study and assigned into three groups：group of ABL2 silenced by shRNA （shRNA-ABL2）,shRNA-negative control group （shRNA-NC） and non-silencing shRNA group （shRNA-N）.Forty-eight h after the transfection,the following experiments were performed：Real time quantitative-PCR was employed to detect the mRNA expression of ABL2; and Western blotting was used to detect the protein expression ofABL2,cortactin and phosphorylated cortactin at tyrosine 421 （pY-421cortactin）,respectively; migration and invasion ability were evaluated by wound-healing assay and Transwell assay; immunofluorescence was used to disclose the expression relationship between ABL2 and cortactin at protein level.Results Immunohistochemistry indicated that the ABL2 expression level in glioblastoma tissues was higher as compared with that in normal brain tissues.The mRNA and protein expression levels of ABL2 in shRNA-ABL2 group were significantly lower than those in the shRNA-NC group and shRNA-N group （P〈0.05）.Wound-healing assay indicated that the number of cell migration in the shRNA-ABL2 group （72.33±7.64） was significantly smaller than that in the shRNA-NC group and shRNA-N group （187.67± 5.03 and 190.33±7.23,P〈0.05）; Transwell assay showed that the invasiveness capability of cells in the shRNA-ABL2 group was significantly decreased as compared with that in the shRNA-NC group and shRNA-N group （P〈0.05）.Double immunofluorescent staining revealed ABL2 enjoyed co-localization with cortactin at cell protruded membrane.Conclusions The ABL2 expressions in glioblastoma tissues are higher than those in normal brain tissues.Silencing ABL2 expression will inhibit glioma cell migration and invasion owing to down-regulating the level ofphosphorylated form of cortactin.