Research article

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

Li Xu Yan^1,2†, Qi Nian Wu^1,2†, Yan Zhang^1,2†, Yang Yang Li^1,2, Ding Zhun Liao^1,2, Jing Hui Hou^1,2, Jia Fu^1,2, Mu Sheng Zeng^1,3, Jing Ping Yun^1,2, Qiu Liang Wu^1,2, Yi Xin Zeng^1,3 and Jian Yong Shao^1,2,3*

• * Corresponding author: Jian Yong Shao shaojy@sysucc.org.cn

• † Equal contributors

Author Affiliations

¹ State Key Laboratory of Oncology in Southern China, Sun Yat-Sen University Cancer Center, 651 Dong Feng Road East, Guangzhou, 510060, PR China

² Department of Pathology, Sun Yat-Sen University Cancer Center, 651 Dong Feng Road East, Guangzhou, 510060, PR China

³ Department of Experiment Research, Sun Yat-Sen University Cancer Center, 651 Dong Feng Road East, Guangzhou, 510060, PR China

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Breast Cancer Research 2011, 13:R2 doi:10.1186/bcr2803

Published: 10 January 2011

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 (r_s = -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.