Identification of microRNAs associated with lung metastasis. (A) Flowchart outlining procedures used to establish sublines from MB231RN, a MDA-MB-231 variant expressing red fluorescent protein (RFP) and a neomycin resistance marker. The MB231RN-LM subline was isolated from spontaneous lung metastases, whereas the MB231RN-MFP subline was isolated from primary tumors grown in mammary fat pads (MFPs). (B) Representative hematoxylin and eosin–stained lung sections from mice killed 7 weeks after tumor cell inoculation (2.5 × 105 cells/injection/mouse, 5 mice/group) are shown. (C) Fold changes of microRNAs (miRNAs) differentially expressed in MB231RN-LM cells compared to MB231RN-MFP cells (gray bars) and parental MB231RN cells (black bars). The results are presented as mean ± SD (n = 3). (D) Expression of miR-18a and miR-17 in sublines MB231RN-CE-MFP and MB231RN-CE-LM, which were generated from primary tumors (CE-MFP) or lung metastases (CE-LM) harvested from mice inoculated with cells expanded from a single colony of the MB231RN line. Relative expression levels of miRNAs are presented as mean ± SD (n = 3). *P < 0.05 by Student’s t-test. n, the number of experimental repeats.