The TaqMan primers and probes for HMGA1 were previously described [19]. have also shown that overexpression of induces transformation both and in animal models (reviewed in [6, 7]). The causal role of in breast cancer development and metastasis is supported by studies in cell lines [14, 22, 23] as well as by the analysis of clinical specimens [15, 16]. For example, elevated HMGA1 protein expression has been reported in breast carcinomas and hyperplastic lesions with cellular atypia, in contrast with normal breast tissue where HMGA1 was not detected [15, 16]. Similarly, HMGA1 overexpression has been observed in human breast cancer cell lines, with the highest levels in known metastatic lines, such as Hs578T and MDA-MB-231 [14, 22, 23]. Moreover, exogenous overexpression of HMGA1a was shown to induce transformation of the human non-tumorigenic mammary myoepithelial cell line Hs578Bst [14] and to increase the metastatic ability of MCF7 breast cancer cells [22]. Conversely, decreasing expression in Hs578T breast cancer cells was shown to cause a reduction in anchorage-independent growth, which is a typical feature of cancer cells [14]. is considered an attractive target for therapeutic intervention because its expression is virtually absent in normal adult tissue and knockdown of has been shown to interfere with the tumorigenic growth of multiple cancer cell lines [6, 7]. We therefore sought to determine if silencing can affect breast cancer development and metastatic progression using a human xenograft mouse NF 279 model. MATERIALS AND NF 279 METHODS Cell lines, transfections and proliferation assays The MDA-MB-231 breast cancer cell line was obtained from American Type Culture Collection, and cultured in DMEM (Cellgro 10-013), supplemented with 10% FBS NF 279 and 5 g/ml gentamicin. Cells were propagated for two weeks, aliquoted in media supplemented with 5% DMSO and stored in LAIR2 liquid nitrogen. Each aliquot was used for less than six months. MDA-MB-231 cells were transfected using Lipofectamine 2000 (Invitrogen). Stable transfectants were selected by adding 50 g/ml of blasticidin to the media, and propagated in media without blasticidin. Anchorage-independent growth was assessed on soft agar as previously described [17]. Tumorsphere formation from single cell suspensions was assessed using MammoCult media (STEMCELL Technologies) in ultralow adherence plates (Corning). Spheres were counted using an NF 279 inverted microscope after seven days of growth. Secondary tumorspheres were generated from single cells suspensions obtained by enzymatic digestion of the primary tumorspheres using 0.05% trypsin (Invitrogen). All kits and reagents were used according to the manufacturers instructions. silencing construct The silencing construct pHMGA1-394-EmGFP-miR was generated using the BLOCK-iT Pol II miR RNAi with EmGFP system (Invitrogen), following the manufacturers instructions. Briefly, a silencing microRNA (miRNA) RNA interference (RNAi) oligonucleotide was designed based on the reference sequence “type”:”entrez-nucleotide”,”attrs”:”text”:”NM_145899.1″,”term_id”:”22208966″,”term_text”:”NM_145899.1″NM_145899.1, using Invitrogens RNAi designer. The target sequence with the best rank score (5-AGCGAAGTGCCAACACCTAAG) was incorporated into a pre-miRNA sequence and cloned into the pcDNA6.2-GW/EmGFP-miR vector using the reagents and competent supplied with the kit. Five clones were isolated and the constructs sequenced. One construct with verified miRNA sequence was selected for transfection. A vector harboring a non-targeting miRNA sequence provided with the kit was used as control. Gene expression Analysis RNA expression was assessed by quantitative Real-Time PCR (qRT-PCR) after reverse transcription. RNA was extracted using the RNeasy Mini Kit (Qiagen) applying the on-column DNase treatment. The amount and quality of the RNA were verified by measuring the absorbance NF 279 at 260 and 280 nm. Reverse transcription was performed with the High Capacity cDNA Reverse Transcription Kit, and duplex qPCR of the resulting cDNA was performed on a 7500 Real Time PCR System, using the TaqMan Gene Expression Master Mix, and the Human.