Blockade of invasion and metastasis of breast cancer cells via targeting CXCR4 with an artificial microRNA Zhongxing Liang a, * , Hui Wu a , Santosh Reddy a , Aizhi Zhu a , Sijia Wang a , Dean Blevins a , Younghyoun Yoon a , Yawei Zhang a , Hyunsuk Shim a,b a Department of Radiology, Winship Cancer Institute, Emory University, Atlanta, GA 30322, USA b Department of Hematology/Oncology, Winship Cancer Institute, Emory University, Atlanta, GA 30322, USA Received 31 August 2007 Available online 14 September 2007 Abstract miRNAs have been shown to function as regulatory molecules and to play an important role in cancer progression. Very little is cur- rently known about the increasing invasion and metastasis of breast cancer due to the loss of expressive levels of certain miRNAs in breast tumor cells. In order to determine whether the CXCR4/SDF-1 pathway is regulated by expression of miRNAs, we designed and synthesized pre-miRNA against CXCR4. This double-stranded miRNA gene was ligated with a miR-155-based Block-iT Pol II miR RNAi Expression Vector (Invitrogen). Expression levels of CXCR4 in CXCR4–miRNA-transfected breast tumor cells had signif- icantly declined. These cells exhibited reduced migration and invasion in vitro. Furthermore, they formed fewer lung metastases in vivo compared to ctrl-miRNA-transfected cells. These data support the conclusion that miRNA against CXCR4 can serve as an alterative means of therapy to lower CXCR4 expression and to block the invasion and metastasis of breast cancer cells. Ó 2007 Elsevier Inc. All rights reserved. MicroRNAs (miRNAs) are a recently discovered family of small, noncoding, double-stranded RNA molecules in cells that can negatively regulate the expression of target genes with their complementary sequence [1,2]. In mam- mals, hundreds of miRNAs have now been identified, some of which are expressed in a tissue-specific and developmen- tal stage-specific manner. Since the recent discovery of this regulatory RNA phenomenon, much progress has been made towards understanding the mechanisms by which this occurs and in the identification of cellular machinery involved in RNA-mediated silencing [1,2]. More recently, miRNA down-regulation was suggested to play a role in cancer progression [3–6]. Currently, a few investigations have also successfully targeted oncogenes with artificial synthetic miRNAs [7–9]. In addition, down-regulation of miR-15a in pituitary adenomas was found to correlate with a greater tumor diameter [10]. In this study, we investigated the down-modulation of CXCR4 expression and function by artificial miRNA by measuring mRNA and protein levels in the breast cancer cell line MDA-MB-231, which has a high level of CXCR4 expression. The results suggested that miRNA targeting CXCR4 may be a potential therapeutic avenue to prevent breast cancer metastasis. Materials and methods Cell culture and reagents. Human breast carcinoma cell line, MDA- MB-231 was routinely maintained in RPMI-1640 medium (Sigma, St. Louis, MO) supplemented with 10% FBS, 100 U/ml of penicillin sodium and 100 lg/ml of streptomycin sulfate, at 37 °C in humidified air con- taining 5% carbon dioxide air atmosphere. Design of artificial miRNAs and transfection. We designed the CXCR4 targeting sequence (5 0 -TGTTGGCT(GC)CTTACTACATT, GenBank Accession No. NM_003467). These miRNA gene double-strands were 0006-291X/$ - see front matter Ó 2007 Elsevier Inc. All rights reserved. doi:10.1016/j.bbrc.2007.09.007 Abbreviations: miRNA, microRNA; CXCR4, CXC chemokine recep- tor-4; SDF-1, stromal-derived factor-1; RT-PCR, reverse transcription polymerase chain reaction. * Corresponding author. Fax: +1 404 778 5550. E-mail address: zliang@emory.edu (Z. Liang). www.elsevier.com/locate/ybbrc Biochemical and Biophysical Research Communications 363 (2007) 542–546