Fig. 5

Identification of IL-11 as a bona fide target of miR-124. a and b Real-time RT-PCR analysis of IL-11 mRNA expression (a) or western blot analysis of IL-11 protein expression (b) in MDA-MB-231 cells transfected with miR-124 mimic (miR-124) or negative control (NC) (top) and in MCF7 cells transfected with miR-124 inhibitor (in-miR-124) or negative control (NC) (bottom). c IHC staining analysis of IL-11 expression in the tibia from mice injected with MDA-MB-231 cells stably expressing miR-124 (miR-124) or NC. d IL-11 protein expression was determined by Western blot in normal human breast tissue (Normal) and eight kinds of human breast cancer cell lines. e Expression of IL-11, as determined by semi-quantitative analysis using BandScan, in normal human breast tissue and human breast cancer cell lines was negatively related to the level of miR-124 as detected by Real-time RT-PCR. r² = 0.5300, P = 0.0262 by Spearman correlation analysis. f Reporter assay identified IL-11 as a direct target of miR-124. Sketch of the construction of wild type (WT) or mutant psicheck2-IL-11 3’UTR vectors. The seed sequence and mutant binding sites are underlined (top). Luciferase reporter plasmid carrying IL-11 3’UTR (WT-IL-11) or IL-11 3’UTR with point mutation in miR-124 target sequence (MUT-IL-11) was cotransfected into HEK293T cells with miR-124 mimic or NC (bottom)