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

doi:10.13481/j.1671-587x.20150508

吉林大学学报(医学版) ›› 2015, Vol. 41 ›› Issue (05): 925-931.doi: 10.13481/j.1671-587x.20150508

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

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

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

收稿日期:2015-05-20 出版日期:2015-09-28 发布日期:2015-09-29
通讯作者: 赵丽艳,副教授,硕士研究生导师(Tel:0431-88796607,E-mail:zhaoliyan406@gmail.com) E-mail:zhaoliyan406@gmail.com
作者简介:李蕴潜(1971-),男,吉林省长春市人,副教授,医学博士,主要从事胶质瘤基础与临床方面的研究。
基金资助:
吉林省科技厅自然科学基金资助课题(201115054)

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

LI Yunqian¹, ZHAO Liyan², SHI Yan³, LIU Hui⁴, MIAO Chunsheng³, XU Wanzhen¹, YANG Zhiqing¹

1. Department of Neurosurgery, First Hospital, Jilin University, Changchun 130021, China;
2. Clinical Laboratory, Second Hospital, Jilin University, Changchun 130041, China;
3. Department of Experimental Pharmacology and Toxicology, School of Pharmacy, Jilin University, Changchun 130021, China;
4. Department of Anatomy, School of Basic Medical Sciences, Jilin University, Changchun 130021, China

Received:2015-05-20 Online:2015-09-28 Published:2015-09-29

摘要/Abstract

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的候选靶基因。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。

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

Abstract:

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.

Key words: miR-181b, membrane-type 1 metalloprotease, tissue inhibitor of metalloprotease-3, glioma cell, invasion

中图分类号:

R739.4

李蕴潜, 赵丽艳, 石艳, 刘辉, 苗春生, 许万振, 杨志青. 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[J]. 吉林大学学报(医学版), 2015, 41(05): 925-931.

LI Yunqian, ZHAO Liyan, SHI Yan, LIU Hui, MIAO Chunsheng, XU Wanzhen, YANG Zhiqing. Influence of overexpression of miR-181b in expressions of MT1-MMP and TIMP3 protein and invasive ability in glioma U87 cells[J]. Journal of Jilin University(Medicine Edition), 2015, 41(05): 925-931.

参考文献

[1] Fillmore HL,Van Meter TE,Broaddus WC.Membrane-type matrix metalloproteinases (MT-MMPs):expression and function during glioma invasion[J].J Neurooncol,2001,53(2):187-202.

[2] Baker AH,George SJ,Zaltsman AB,et al.Inhibition of invasion and induction of apoptotic cell death of cancer cell lines by overexpression of TIMP-3[J].Br J Cancer,1999,79(9/10):1347-1355.

[3] Aigner A.MicroRNAs (miRNAs) in cancer invasion and metastasis:therapeutic approaches based on metastasis-related miRNAs[J].J Mol Med,2011,89(5):445-457.

[4] Li L,Li H.Role of microRNA-mediated MMP regulation in the treatment and diagnosis of malignant tumors[J].Cancer Biol Ther,2013,14(9):796-805.

[5] Liu J,Shi W,Wu C,et al.miR-181b as a key regulator of the oncogenic process and its clinical implications in cancer[J].Biomed Rep,2014,2(1):7-11.

[6] Shi L,Cheng Z,Zhang J,et al.has-mir-181a and hsa-mir-181b function as tumor suppressors in human glioma cells[J].Brain Res,2008,1236:185-193.

[7] Slaby O,Lakomy R,Fadrus P,et al.MicroRNA-181 family predicts response to concomitant chemoradiotherapy with temozolomide in glioblastoma patients[J].Neoplasma,2010,57(3):264-269.

[8] Abba M,Patil N,Allgayer H.MicroRNAs in the regulation of MMPs and metastasis[J].Cancers,2014,6(2):625-645.

[9] Zarrabi K,Dufour A,Li J,et al.Inhibition of matrix metalloproteinase 14 (MMP-14)-mediated cancer cell migration[J].J Biol Chem,2011,286(38):33167-33177.

[10] Ulasov I,Yi R,Guo D,et al.The emerging role of MMP14 in brain tumorigenesis and future therapeutics[J].Biochim Biophys Acta,2014,1846(1):113-120.

[11] Lin H,Zhang Y,Wang H,et al.Tissue inhibitor of metalloproteinases-3 transfer suppresses malignant behaviors of colorectal cancer cells[J].Cancer Gene Ther,2012,19(12):845-851.

[12] Cruz-Munoz W,Khokha R.The role of tissue inhibitors of metalloproteinases in tumorigenesis and metastasis[J].Crit Rev Clin Lab Sci,2008,45(3):291-338.

[13] Anania MC,Sensi M,Radaelli E,et al.TIMP3 regulates migration,invasion and in vivo tumorigenicity of thyroid tumor cells[J].Oncogene,2011,30(27):3011-3023.

[14] Shi ZM,Wang XF,Qian X,et al.MiRNA-181b suppresses IGF-1R and functions as a tumor suppressor gene in gliomas[J].RNA,2013,19(4):552-560.

[15] Wang J,Sai K,Chen FR,et al.miR-181b modulates glioma cell sensitivity to temozolomide by targeting MEK1[J].Cancer Chemother Pharmacol,2013,72(1):147-158.

[16] Sun YC,Wang J,Guo CC,et al.MiR-181b sensitizes glioma cells to teniposide by targeting MDM2[J].BMC Cancer,2014,14:611-617.

[17] She X,Yu Z,Cui Y,et al.miR-181 subunits enhance the chemosensitivity of temozolomide by Rap1B-mediated cytoskeleton remodeling in glioblastoma cells[J].Med Oncol,2014,31(4):892-901.

[18] Wang B,Hsu SH,Majumder S,et al.TGFbeta-mediated upregulation of hepatic miR-181b promotes hepatocarcinogenesis by targeting TIMP3[J].Oncogene,2010,29(12):1787-1797.

[19] Lu Y,Roy S,Nuovo G,et al.Anti-microRNA-222 (anti-miR-222) and-181B suppress growth of tamoxifen-resistant xenografts in mouse by targeting TIMP-3 protein and modulating mitogenic signal[J].J Biol Chem,2011,286(49):42292-42302.

[20] 温娜,唐泽波,金宏,等.五味子多糖对脑胶质瘤SHG-44细胞血管内皮生长因子表达水平的影响[J].中国老年学杂志,2014,34(24):7048-7049.

[21] 郭成永,梁朝辉,魏可欣,等.解旋酶RECQ1和细胞核相关抗原Ki-67在脑胶质瘤组织中的表达及相关性[J].中国老年学杂志,2015,35(12):3259-3260.

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

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