miR-181b过表达对胶质瘤U87细胞中MT1-MMP和TIMP3蛋白表达和细胞侵袭能力的影响  

Influence of overexpression of miR-181b in expressions of MT1-MMP and TIMP3 protein and invasive ability in glioma U87 cells

作　　者：李蕴潜[1] 赵丽艳[2] 石艳[3] 刘辉[4] 苗春生[3] 许万振[1] 杨志青[1] 

机构地区：[1]吉林大学第一医院神经外科,吉林长春130021 [2]吉林大学第二医院检验科,吉林长春130041 [3]吉林大学药学院实验药理与毒理学教研室,吉林长春130021 [4]吉林大学基础医学院解剖学系,吉林长春130021

出　　处：《吉林大学学报（医学版）》2015年第5期925-931,共7页Journal of Jilin University：Medicine Edition

基　　金：吉林省科技厅自然科学基金资助课题(201115054)

摘　　要：目的:探讨微小RNA-181b(miR-181b)过表达对人胶质瘤细胞中膜型基质金属蛋白酶1(MT1-MMP)和金属蛋白酶组织抑制剂3(TIMP3)表达的调控作用及对细胞侵袭能力的影响。方法:将培养于DMEM培养基的人胶质瘤U87细胞分为阴性对照组(不做任何处理)、空载组(转染Lipofetamine 2000)、乱序组(转染乱序has-miR-181b寡核苷酸)和miR-181b组(转染has-miR-181b寡核苷酸)。qRT-PCR法检测各组细胞中miR-181b基因表达水平,Western blotting法检测各组细胞中MT1-MMP和TIMP3蛋白的相对表达水平,基质胶侵袭试验检测各组细胞的侵袭能力,测定各组细胞的趋化运动抑制率、迁移率和细胞黏附率;生物信息学数据库预测miR-181b的侯选靶基因。结果:miR-181b组U87细胞中miR-181b基因表达水平明显高于其他各组(P<0.01),呈过表达。miR-181b组U87细胞中MT1-MMP蛋白表达水平低于其他各组(P<0.01),miR-181b与MT1-MMP蛋白表达水平呈负相关关系(r=-0.787,P<0.05);miR-181b组U87细胞中TIMP3蛋白表达水平高于其他各组(P<0.01),miR-181b与TIMP3蛋白表达水平呈正相关关系(r=0.801,P<0.05)。基质胶侵袭试验中miR-181b组穿膜细胞数明显低于其他各组(P<0.05)。与其他3组比较,miR-181b组U87细胞的趋化运动抑制率升高、迁移率和细胞黏附率降低(P<0.05)。生物信息学数据库预测,在MT1-MMP的3′非翻译区(3′UTR)有1个与miR-181b的结合位点,在TIMP3的3′UTR有2个与miR-181b的结合位点。结论:miR-181b过表达可下调MT1-MMP和上调TIMP3蛋白的表达,从而抑制胶质瘤细胞的侵袭能力;miR-181b作为关键性调节miR,可能成为胶质瘤治疗的重要靶标。Objective To explore the modulating effect of miR-181 b overexpression on the expressions of membrane-type 1 metalloprotease（MT1-MMP）and tissue inhibitor of metalloprotease-3（TIMP3）and invasive ability in glioma cells.Methods The human glioma U87 cells cultured in DMEM medium were divided into negative control（without treatment）,empty vector（transfected with Lipofetamine 2000）,scramble（transfected with scrambled has-miR-181 b oligonucleotide）and miR-181b（transfected with has-miR-181 b oligonucleotide）groups.The expression levels of miR-181 bgene in the U87 cells in various groups were detected by qRT-PCR method;the expression levels of MT1-MMP and TIMP3 proteins in the U87 cells in various groups were examined by Western blotting method;the invasive ability of the U87 cells in various groups was determined by matrigel invasion assay;the inhibitory rate of chemotactic movement,migrated rate and cell adhesion rate in various groups were detected;the candidate target genes of miR-181 bwere predicted by Bioinformatics Database.Results The expression level of miR-181 bmRNA in the U87 cells in miR-181 bgroup was significantly higher than those in other three groups（P 0.01）,resulting in miR-181 b overexpression.Compared with other three groups,the expression level of MT1-MMP protein in the U87 cells in miR-181 bgroup was decreased（P〈0.01）,and the miR-181 blevel was negatively correlated with the expression level of MT1-MMP protein（r=-0.787,P〈0.05）;while compared with other three groups,the expression level of TIMP3 protein in the U87 cells in miR-181 bgroup was increased（P〈0.01）,and the miR-181 blevel was positively correlated with the expression level of TIMP3protein（r=0.801,P〈0.05）.The matrigel invasion assay results showed that the number of transmembrane cells in miR-181 bgroup was significantly lower than those in other three groups（P〈0.05）.The inhibitory rate of chemotactic movement was increased,the migrated rate and cell adhesion rate of U87 cells in miR-

关 键 词：微小RNA-181b 膜型基质金属蛋白酶1 金属蛋白酶组织抑制剂3 胶质瘤细胞 侵袭 

分 类 号：R739.4[医药卫生—肿瘤]