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

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。

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

Objective To explore the modulating effect of miR-181b 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-181b oligonucleotide) and miR-181b (transfected with has-miR-181b oligonucleotide) groups.The expression levels of miR-181b gene 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-181b were predicted by Bioinformatics Database. Results The expression level of miR-181b mRNA in the U87 cells in miR-181b group was significantly higher than those in other three groups (P<0.01),resulting in miR-181b overexpression.Compared with other three groups,the expression level of MT1-MMP protein in the U87 cells in miR-181b group was decreased (P<0.01),and the miR-181b level 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-181b group was increased(P<0.01),and the miR-181b level was positively correlated with the expression level of TIMP3 protein (r=0.801,P<0.05).The matrigel invasion assay results showed that the number of transmembrane cells in miR-181b group 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-181b group were decreased compared with other three groups (P<0.05).The Bioinformatics Database prediction results revealed that one miR-181b-binding site within MT1-MMP 3' UTR and two binding sites of miR-181b within TIMP3 3' UTR were documented. Conclusion The miR-181b overexpression inhibits the invasive ability of glioma cells through downregulating the MT1-MMP expression and upregulating the TIMP3 expression;miR-181b serves as a critical regulator and may be an important therapeutic target for gliomas.