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miR-155 promotes macroscopic tumor formation yet inhibits tumor dissemination from mammary fat pads to the lung by preventing EMT

miR-155, a micro-RNA, is over-expressed in many types of cancer cells, including breast cancer, and its role(s) in tumor metastasis has been studied on a very limited basis. Tumor metastasis is a multi-step process with the last step in the process being formation of macroscopic tumor in organs distant from the primary tumor site. This step is the least studied. Here, we report that stable expression of miR-155 in 4T1 breast tumor cells reduces significantly the aggressiveness of tumor cell dissemination as a result of preventing epithelial-to-mesenchymal transition (EMT) of tumor cells in vivo. Further, miR-155 directly suppresses the expression of the transcription factor TCF4, which is an important regulator of EMT. However, when tumor cells are injected directly into the bloodstream, miR-155 remarkably promotes macroscopic tumor formation in the lung. Analysis of gene expression profiling identified a group of genes that are associated with promoting macroscopic tumor formation in the lung. Importantly, most of these genes are over-expressed in epithelial cells. Our findings provide new insight into how miR-155 modulates the development of tumor metastasis. This study suggests that the location of tumor cells over-expressing miR-155 is a critical factor: in mammary fat pads miR-155 prevents tumor dissemination; whereas in the lung miR-155 apparently maintains the epithelial phenotype of tumor cells that is critical for macroscopic tumor formation.

to the cell cultures and the cultures incubated at 37°C for an additional 2 h. The cells were then trypsined and washed with Dubecco's phosphate buffered saline containing 1% bovine serum albumin. The cells were FACS analyzed according to the protocol provided by the manufacturer (Invitrogen)

Western blot assay
Total cell lysates were loaded in equal protein amounts (50 μg) determined by BCA (Pierce, Rockford, IL). Proteins were resolved by SDS-PAGE (Novex, Invitrogen), followed by transfer onto a nitrocellulose membrane using the iBlot Dry Blotting System (Invitrogen). A previously described method (Liu et al., 2009) was used to process the membranes for western blot analysis. Expression of proteins was determined by probing membranes with antibodies against primary antigens; β-actin antibody served as a 2 control (Santa cruz). Bound primary antibody was detected with Alex fluor 680 labeled second antibody (Invitrogen).

Reverse transcription-PCR
Total RNA was extracted using TRIzol Reagent (Invitrogen) following the manufacturer's protocol. RNA (500 ng) was reverse-transcribed by random hexamers and SuperScript III reverse transcriptase (Invitrogen) in a volume of 20 μL. The reverse transcription reaction (1 μL) was used as a template in a 20 μL PCR reaction mixture using a primer set specific for the genes of interest (all primers used from RT-PCR are listed in supplemental table 2). An equivalent amount of RNA without reverse transcription served as negative control. As a positive control for amplification from the cDNA, GAPDH and β-actin were used.

Luciferase reporter assay
The psiCHECK-2 vector (Promega, Madison, WI) was used to clone the 3'-UTR of mouse TCF4 or Sabt1 mRNA. The vector contains a multiple cloning region downstream of the stop codon of an SV40 promoter-driven Renilla luciferase gene. This allows expression of a Renilla transcript with the 3'-UTR sequence of interest. Renilla luciferase activity is then used to assess the effect of the 3'-UTR on transcript stability and translation efficiency. The psiCHECK-2 vector also contains a constitutively expressed firefly luciferase gene. Firefly luciferase is used to normalize transfections and eliminates the need to transfect a second vector control. TCF4 or Sabt1 mutants were made with the QuikChange® site-directed mutagenesis kit (Stratagene). Each of 3 the mutants was sequenced to confirm the mutated products. 293T cells were seeded in the wells of a 6-well plate 1 day pretransfection and then each well transfected with a mixture of 200 ng 3' -UTR luciferase reporter vectors and 1800 ng of MDH1-PGK-GFP-BIC or miR-223 as a negative control. Twenty-four hours posttransfection, cells were lysed and luciferase activity was measured using a luminometer (Applied Biosystems) and the Dual-Luciferase Reporter Assay System (Promega). The ratio of Renilla luciferase to firefly luciferase was calculated.

Realtime quantification of microRNA by Stem-looped RT-PCR
We followed the protocol developed by Tang et al. (2006) (Tang et al.) to measure microRNA expression. Briefly, Primers and Taqman probes were designed according to the sequence published (Tang et al., 2006). Reverse primers used to synthesize the cDNA were mixed to a concentration of 200 nM for each reverse primer. One μl of this premixed reverse primer mixture was used in reverse transcription reaction (working concentration for each reverse primer was 10 nM). SuperScript III First-Strand Synthesis System (Invitrogen) was used to reverse transcribe RNA into cDNA. We