沉默LIMK1的表达抑制体外胶质瘤细胞的侵袭与迁移  

Silencing expression of LIM kinase 1 inhibits invasion and migration of human glioma cells in vitro

作　　者：周华[1] 杨学军[1] 赵鹏飞[1] 张辰[1] 陈磊[1] 朱蒙[1] 于圣平[1] 周星辰[1] 海龙[1] 刘波[1] 李帅[1] 林雨[1] 

机构地区：[1]天津医科大学总医院神经外科,天津300052

出　　处：《中华神经医学杂志》2015年第6期541-546,共6页Chinese Journal of Neuromedicine

基　　金：国家自然科学基金（81272782、81472352）;高等学校博士学科点专项科研基金（20131202110006）

摘　　要：目的 研究LIM激酶1（LIMK1）小干扰RNA （siRNA）敲低后对人胶质瘤细胞系SNB19侵袭迁移能力的影响及机制. 方法 （1）免疫组化染色检测对照脑组织及胶质母细胞瘤组织中LIMK1蛋白的表达.（2）将SNB19分为3组：siRNA-LIMK1组（转染siRNA-LIMK1干扰片段）、siRNA-NC组（转染对照siRNA片段）、空白对照组（未转染）.采用实时定量（RT-PCR）法和Western blotting法检测3组细胞中mRNA和蛋白的表达情况.划痕实验和Transwell侵袭实验评价3组细胞的迁移和侵袭能力.免疫荧光法观察3组细胞的定位以及细胞片状伪足形态变化.Western blotting法检测3组细胞基质金属蛋白酶（MMP）-2、MMP-9以及丝切蛋白（cofilin）、磷酸化cofilin （p-cofilin）的表达. 结果 （1）免疫组化结果显示蛋白在对照脑组织呈弱阳性表达,而在胶质母细胞瘤中呈强阳性表达.（2）siRNA-LIMK1组细胞mRNA和蛋白较其他2组细胞均明显下降,差异有统计学意义（P＜0.05）.划痕实验和Transwell侵袭实验显示3组细胞创面愈合率、侵袭细胞数分别为34.33％±1.53％、75.67％±2.08％、78.33％±1.53％及40.33±5.03、136.67±5.13、143.33±2.52,差异均有统计学意义（F=608.59,P=0.000;F=515.52,P=0.000）;其中siRNA-LIMK1组创面愈合率、侵袭细胞数较其他2组明显下降,差异有统计学意义（P＜0.05）.免疫荧光结果显示LIMK1表达于细胞质特别富集于细胞前缘的片状伪足处,siRNA-LIMK1组细胞片状伪足较siRNA-NC组和空白对照组明显变小甚至不伸展.Western blotting结果显示,与siRNA-NC组和空白对照组比较,siRNA-LIMK1组MMP-2、MMP-9以及p-cofilin的表达量均明显下降,差异有统计学意义（P＜0.05）;而cofilin的表达量无明显变化,差异无统计学意义（P＞0.05）. 结论 沉默LIMK1的表达可有效抑制体外胶质瘤细胞系的侵袭和迁移,LIMK1可能成为脑胶质瘤的新的治疗靶点.Objective To investigate the effect of silencing LIM kinase 1 （LIMK1） expresssion on invasion and migration of glioma cells,and investigate its possible mechanism.Methods Immunohistochemistry was used to detect the LIMK1 protein expression in normal brain tissues and glioblastoma tissues.Human glioma cells SNB19 were employed and assigned into three groups：siRNA-LIMK1 treated group （siRNA-LIMK1）,siRNA-negative control group （siRNA-NC） and blank control group;siRNA-LIMK1 and non-sipencing siRNA were transfected into SNB19 cells by lipofectamine TM3000.The mRNA and protein expressions of LIMK1 were detected by real time-PCR and Western blotting,respectively.The migration and invasion of SNB 19 glioma cells were evaluated by wound-healing assay and Transwell invasion assay.Immunofluorescence was used to localise the expression of LIMK1 in glioma cells and observe the lamellipodia morphology of glioma cells.The expressions of matrix metalloproteinase-2 （MMP-2）,MMP-9,cofilin and phosphorylated-cofilin （p-cofilin） in SNB19 cells were determined by Westem blotting.Results Immunohistochemistry showed that LIMK1 expressed in both normal brain tissues and glioblastoma tissues,but the LIMK1 expression was elevated in glioblastoma tissues as compared with that in normal brain tissues.The mRNA and protein expressions of LIMK1 in SNB19 cells of the siRNA-LIMK1 treated group were remarkably down-regulated as compared with those in the other two groups （P〈0.05）.The wound healing rate in the siRNA-LIMK1 treated group,siRNA-NC group and blank control group were 34.33%±1.53%,75.67%±2.08% and 78.33%±1.53%,and invaded cell population were 40.33±5.03,136.67±5.13 and 143.33±2.52,with significant differences （F=608.59,P=0.000;F=515.52,P=0.000）;as compared with those in the siRNA-NC group and blank control group,the migration and invasion capacities of glioma cells in siRNA-LIMK1 treated group were markedly inhibited （P〈0.05）.LIMK1 expressed in the cytoplasm,especially enriched at the lamellipo

关 键 词：神经胶质瘤 侵袭 迁移 LIM激酶1 RNA干扰 

分 类 号：R739.41[医药卫生—肿瘤]