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Breast Cancer Research
Published in: Breast Cancer Research 1/2011

Open Access 01-02-2011 | Research article

Knockdown of miR-21 in human breast cancer cell lines inhibits proliferation, in vitro migration and in vivotumor growth

Authors: Li Xu Yan, Qi Nian Wu, Yan Zhang, Yang Yang Li, Ding Zhun Liao, Jing Hui Hou, Jia Fu, Mu Sheng Zeng, Jing Ping Yun, Qiu Liang Wu, Yi Xin Zeng, Jian Yong Shao

Published in: Breast Cancer Research | Issue 1/2011

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Abstract

Introduction

MicroRNAs (miRNAs) are a class of small non-coding RNAs (20 to 24 nucleotides) that post-transcriptionally modulate gene expression. A key oncomir in carcinogenesis is miR-21, which is consistently up-regulated in a wide range of cancers. However, few functional studies are available for miR-21, and few targets have been identified. In this study, we explored the role of miR-21 in human breast cancer cells and tissues, and searched for miR-21 targets.

Methods

We used in vitro and in vivo assays to explore the role of miR-21 in the malignant progression of human breast cancer, using miR-21 knockdown. Using LNA silencing combined to microarray technology and target prediction, we screened for potential targets of miR-21 and validated direct targets by using luciferase reporter assay and Western blot. Two candidate target genes (EIF4A2 and ANKRD46) were selected for analysis of correlation with clinicopathological characteristics and prognosis using immunohistochemistry on cancer tissue microrrays.

Results

Anti-miR-21 inhibited growth and migration of MCF-7 and MDA-MB-231 cells in vitro, and tumor growth in nude mice. Knockdown of miR-21 significantly increased the expression of ANKRD46 at both mRNA and protein levels. Luciferase assays using a reporter carrying a putative target site in the 3' untranslated region of ANKRD46 revealed that miR-21 directly targeted ANKRD46. miR-21 and EIF4A2 protein were inversely expressed in breast cancers (rs = -0.283, P = 0.005, Spearman's correlation analysis).

Conclusions

Knockdown of miR-21 in MCF-7 and MDA-MB-231 cells inhibits in vitro and in vivo growth as well as in vitro migration. ANKRD46 is newly identified as a direct target of miR-21 in BC. These results suggest that inhibitory strategies against miR-21 using peptide nucleic acids (PNAs)-antimiR-21 may provide potential therapeutic applications in breast cancer treatment.
Appendix
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Literature
1.
Parkin DM, Bray F, Ferlay J, Pisani P: Global cancer statistics, 2002. CA Cancer J Clin. 2005, 55: 74-108. 10.3322/canjclin.55.2.74.CrossRefPubMed
2.
Ambros V: MicroRNA pathways in flies and worms: growth, death, fat, stress, and timing. Cell. 2003, 113: 673-676. 10.1016/S0092-8674(03)00428-8.CrossRefPubMed
3.
Esquela-Kerscher A, Slack FJ: Oncomirs - microRNAs with a role in cancer. Nat Rev Cancer. 2006, 6: 259-269. 10.1038/nrc1840.CrossRefPubMed
4.
Chan JA, Krichevsky AM, Kosik KS: MicroRNA-21 is an antiapoptotic factor in human glioblastoma cells. Cancer Res. 2005, 65: 6029-6033. 10.1158/0008-5472.CAN-05-0137.CrossRefPubMed
5.
Fulci V, Chiaretti S, Goldoni M, Azzalin G, Carucci N, Tavolaro S, Castellano L, Magrelli A, Citarella F, Messina M, Maggio R, Peragine N, Santangelo S, Mauro FR, Landgraf P, Tuschl T, Weir DB, Chien M, Russo JJ, Ju J, Sheridan R, Sander C, Zavolan M, Guarini A, Foa R, Macino G: Quantitative technologies establish a novel microRNA profile of chronic lymphocytic leukemia. Blood. 2007, 109: 4944-4951. 10.1182/blood-2006-12-062398.CrossRefPubMed
6.
Yanaihara N, Caplen N, Bowman E, Seike M, Kumamoto K, Yi M, Stephens RM, Okamoto A, Yokota J, Tanaka T, Calin GA, Liu CG, Croce CM, Harris CC: Unique microRNA molecular profiles in lung cancer diagnosis and prognosis. Cancer Cell. 2006, 9: 189-198. 10.1016/j.ccr.2006.01.025.CrossRefPubMed
7.
Iorio MV, Ferracin M, Liu CG, Veronese A, Spizzo R, Sabbioni S, Magri E, Pedriali M, Fabbri M, Campiglio M, Menard S, Palazzo JP, Rosenberg A, Musiani P, Volinia S, Nenci I, Calin GA, Querzoli P, Negrini M, Croce CM: MicroRNA gene expression deregulation in human breast cancer. Cancer Res. 2005, 65: 7065-7070. 10.1158/0008-5472.CAN-05-1783.CrossRefPubMed
8.
Frankel LB, Christoffersen NR, Jacobsen A, Lindow M, Krogh A, Lund AH: Programmed cell death 4 (PDCD4) is an important functional target of the microRNA miR-21 in breast cancer cells. J Biol Chem. 2008, 283: 1026-1033. 10.1074/jbc.M707224200.CrossRefPubMed
9.
Si ML, Zhu S, Wu H, Lu Z, Wu F, Mo YY: miR-21-mediated tumor growth. Oncogene. 2007, 26: 2799-2803. 10.1038/sj.onc.1210083.CrossRefPubMed
10.
Zhu S, Wu H, Wu F, Nie D, Sheng S, Mo YY: MicroRNA-21 targets tumor suppressor genes in invasion and metastasis. Cell Res. 2008, 18: 350-359. 10.1038/cr.2008.24.CrossRefPubMed
11.
Oh SY, Ju Y, Park H: A highly effective and long-lasting inhibition of miRNAs with PNA-based antisense oligonucleotides. Mol Cells. 2009, 28: 341-345. 10.1007/s10059-009-0134-8.CrossRefPubMed
12.
Pathology and Genetics of Tumours of the Breast and Female Genital Organs. Edited by: Tavassoli F, Devilee P. 2003, Lyon: IARC Press
13.
Singletary SE, Allred C, Ashley P, Bassett LW, Berry D, Bland KI, Borgen PI, Clark GM, Edge SB, Hayes DF, Hughes LL, Hutter RV, Morrow M, Page DL, Recht A, Theriault RL, Thor A, Weaver DL, Wieand HS, Greene FL: Staging system for breast cancer: revisions for the 6th edition of the AJCC Cancer Staging Manual. The Surgical Clinics of North America. 2003, 83: 803-819. 10.1016/S0039-6109(03)00034-3.CrossRefPubMed
14.
Obernosterer G, Martinez J, Alenius M: Locked nucleic acid-based in situ detection of microRNAs in mouse tissue sections. Nature Protocols. 2007, 2: 1508-1514. 10.1038/nprot.2007.153.CrossRefPubMed
15.
Lee SH, Kunz J, Lin SH, Yu-Lee LY: 16-kDa prolactin inhibits endothelial cell migration by down-regulating the Ras-Tiam1-Rac1-Pak1 signaling pathway. Cancer Res. 2007, 67: 11045-11053. 10.1158/0008-5472.CAN-07-0986.CrossRefPubMed
16.
Wu L, Li Z, Zhang Y, Zhang P, Zhu X, Huang J, Ma T, Lu T, Song Q, Li Q, Guo Y, Tang J, Ma D, Chen KH, Qiu X: Adenovirus-expressed human hyperplasia suppressor gene induces apoptosis in cancer cells. Mol Cancer Ther. 2008, 7: 222-232. 10.1158/1535-7163.MCT-07-0382.CrossRefPubMed
17.
Melo SA, Ropero S, Moutinho C, Aaltonen LA, Yamamoto H, Calin GA, Rossi S, Fernandez AF, Carneiro F, Oliveira C, Ferreira B, Liu CG, Villanueva A, Capella G, Schwartz SJr, Shiekhattar R, Esteller M: A TARBP2 mutation in human cancer impairs microRNA processing and DICER1 function. Nat Genet. 2009, 41: 365-370. 10.1038/ng.317.CrossRefPubMedPubMedCentral
18.
Patterson TA, Lobenhofer EK, Fulmer-Smentek SB, Collins PJ, Chu TM, Bao W, Fang H, Kawasaki ES, Hager J, Tikhonova IR, Walker SJ, Zhang L, Hurban P, de Longueville F, Fuscoe JC, Tong W, Shi L, Wolfinger RD: Performance comparison of one-color and two-color platforms within the MicroArray Quality Control (MAQC) project. Nat Biotechnol. 2006, 24: 1140-1150. 10.1038/nbt1242.CrossRefPubMed
19.
20.
Yan LX, Huang XF, Shao Q, Huang MY, Deng L, Wu QL, Zeng YX, Shao JY: MicroRNA miR-21 overexpression in human breast cancer is associated with advanced clinical stage, lymph node metastasis and patient poor prognosis. RNA. 2008, 14: 2348-2360. 10.1261/rna.1034808.CrossRefPubMedPubMedCentral
21.
Gramantieri L, Ferracin M, Fornari F, Veronese A, Sabbioni S, Liu CG, Calin GA, Giovannini C, Ferrazzi E, Grazi GL, Croce CM, Bolondi L, Negrini M: Cyclin G1 is a target of miR-122a, a microRNA frequently down-regulated in human hepatocellular carcinoma. Cancer Res. 2007, 67: 6092-6099. 10.1158/0008-5472.CAN-06-4607.CrossRefPubMed
22.
Grimson A, Farh KK, Johnston WK, Garrett-Engele P, Lim LP, Bartel DP: MicroRNA targeting specificity in mammals: determinants beyond seed pairing. Mol Cell. 2007, 27: 91-105. 10.1016/j.molcel.2007.06.017.CrossRefPubMedPubMedCentral
23.
Griffiths-Jones S, Saini HK, van Dongen S, Enright AJ: miRBase: tools for microRNA genomics. Nucleic Acids Res. 2008, 36: D154-158. 10.1093/nar/gkm952.CrossRefPubMed
24.
Hsu SD, Chu CH, Tsou AP, Chen SJ, Chen HC, Hsu PW, Wong YH, Chen YH, Chen GH, Huang HD: miRNAMap 2.0: genomic maps of microRNAs in metazoan genomes. Nucleic Acids Res. 2008, 36: D165-169. 10.1093/nar/gkm1012.CrossRefPubMed
25.
Krek A, Grun D, Poy MN, Wolf R, Rosenberg L, Epstein EJ, MacMenamin P, da Piedade I, Gunsalus KC, Stoffel M, Rajewsky N: Combinatorial microRNA target predictions. Nat Genet. 2005, 37: 495-500. 10.1038/ng1536.CrossRefPubMed
26.
27.
Song CH, Park SY, Eom KY, Kim JH, Kim SW, Kim JS, Kim IA: Potential prognostic value of heat-shock protein 90 in the presence of phosphatidylinositol-3-kinase overexpression or loss of PTEN, in invasive breast cancers. Breast Cancer Res. 2010, 12: R20-10.1186/bcr2557.CrossRefPubMedPubMedCentral
28.
Gamallo C, Palacios J, Suarez A, Pizarro A, Navarro P, Quintanilla M, Cano A: Correlation of E-cadherin expression with differentiation grade and histological type in breast carcinoma. The American Journal of Pathology. 1993, 142: 987-993.PubMedPubMedCentral
29.
Tuominen VJ, Ruotoistenmaki S, Viitanen A, Jumppanen M, Isola J: ImmunoRatio: a publicly available web application for quantitative image analysis of estrogen receptor (ER), progesterone receptor (PR), and Ki-67. Breast Cancer Res. 2010, 12: R56-10.1186/bcr2615.CrossRefPubMedPubMedCentral
30.
Sempere LF, Christensen M, Silahtaroglu A, Bak M, Heath CV, Schwartz G, Wells W, Kauppinen S, Cole CN: Altered MicroRNA expression confined to specific epithelial cell subpopulations in breast cancer. Cancer Res. 2007, 67: 11612-11620. 10.1158/0008-5472.CAN-07-5019.CrossRefPubMed
31.
Matkovich SJ, Van Booven DJ, Youker KA, Torre-Amione G, Diwan A, Eschenbacher WH, Dorn LE, Watson MA, Margulies KB, Dorn GW: Reciprocal regulation of myocardial microRNAs and messenger RNA in human cardiomyopathy and reversal of the microRNA signature by biomechanical support. Circulation. 2009, 119: 1263-1271. 10.1161/CIRCULATIONAHA.108.813576.CrossRefPubMedPubMedCentral
32.
Wickramasinghe NS, Manavalan TT, Dougherty SM, Riggs KA, Li Y, Klinge CM: Estradiol downregulates miR-21 expression and increases miR-21 target gene expression in MCF-7 breast cancer cells. Nucleic Acids Res. 2009, 37: 2584-2595. 10.1093/nar/gkp117.CrossRefPubMedPubMedCentral
33.
Bhat-Nakshatri P, Wang G, Collins NR, Thomson MJ, Geistlinger TR, Carroll JS, Brown M, Hammond S, Srour EF, Liu Y, Nakshatri H: Estradiol-regulated microRNAs control estradiol response in breast cancer cells. Nucleic Acids Res. 2009, 37: 4850-4861. 10.1093/nar/gkp500.CrossRefPubMedPubMedCentral
34.
Eulalio A, Huntzinger E, Izaurralde E: Getting to the root of miRNA-mediated gene silencing. Cell. 2008, 132: 9-14. 10.1016/j.cell.2007.12.024.CrossRefPubMed
35.
Sudo K, Takahashi E, Nakamura Y: Isolation and mapping of the human EIF4A2 gene homologous to the murine protein synthesis initiation factor 4A-II gene Eif4a2. Cytogenet Cell Genet. 1995, 71: 385-388. 10.1159/000134145.CrossRefPubMed
36.
Williams-Hill DM, Duncan RF, Nielsen PJ, Tahara SM: Differential expression of the murine eukaryotic translation initiation factor isogenes eIF4A(I) and eIF4A(II) is dependent upon cellular growth status. Arch Biochem Biophys. 1997, 338: 111-120. 10.1006/abbi.1996.9804.CrossRefPubMed
Metadata
Title
Knockdown of miR-21 in human breast cancer cell lines inhibits proliferation, in vitro migration and in vivotumor growth
Authors
Li Xu Yan
Qi Nian Wu
Yan Zhang
Yang Yang Li
Ding Zhun Liao
Jing Hui Hou
Jia Fu
Mu Sheng Zeng
Jing Ping Yun
Qiu Liang Wu
Yi Xin Zeng
Jian Yong Shao
Publication date
01-02-2011
Publisher
BioMed Central
Published in
Breast Cancer Research / Issue 1/2011
Electronic ISSN: 1465-542X
DOI
https://doi.org/10.1186/bcr2803

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