Fig 2: MSD assay signal in “free” (a) vs “total” (b) assay setups with pre-incubation of therapeutic mAb:IL-11 to verify assay species measured.

Fig 3: The miR-124/IL-11 axis is perturbed in breast cancer patients with bone metastasis. a Kaplan-Meier analysis for bone metastasis-free survival of 79 patients who received breast cancer surgery and a follow-up of the development of bone metastasis. The median value of miR-124 expression in primary breast cancer tissues as detected by ISH from all 79 samples was chosen as the cut-off point. P = 0.033 by log rank test. b A correlation analysis was performed between miR-124 expression of bone metastasis tissues from 34 patients that received bone metastasis resection and the time from primary breast cancer surgery to bone metastasis development. r = 0.45, p = 0.0065 by Spearman correlation analysis. c-e Percentages of specimens with low or high miR-124 expression were relative to percentages of specimens with KPS = 80 or KPS < 80 (c), and specimens with single bone lesion or multiple bone lesions (d) as well as specimens with Ki67 < 14% or Ki67 = 14% (e). * P < 0.05 by Fisher’s exact test. f Kaplan-Meier analysis for the overall survival of 34 patients with bone metastasis. The median value of all 34 samples was chosen as the cut-off point. P = 0.035 by log rank test. g Percentages of specimens with low or high miR-124 expression relative to the levels of IL-11 protein. ** P < 0.01 by Fisher’s exact test. h Kaplan-Meier analysis was used to compare the overall survival of patients with higher expression of IL-11 and lower expression of IL-11. The median value of IL-11 in all 34 samples was chosen as the cut-off point. P = 0.007 by log rank test. i A schematic representation of the miR-124/IL-11 regulatory axis in the interaction between primary breast cancer and bone microenvironment

Fig 4: IL-11 partially mediates the inhibition breast cancer bone metastasis by miR-124 both in vitro and in vivo. a-c IL-11 neutralizing antibody treatment (5 µg/ml) efficiently reversed the promoting effects of conditioned medium from MDA-MB-231 cells transfected with miR-124 inhibitor (i-miR-124-CM) on the proliferation (a) and differentiation of BMMs as determined by TRAP staining (b and c). Representative images of TRAP-positive multinucleated osteoclasts are shown in b and statistical analysis is shown in c. d-f Recombinant human IL-11 treatment (10 ng/ml) partially reversed the suppressive effects of conditioned medium from MDA-MB-231 cells transfected with miR-124 (miR-124-CM) on the proliferation (d) and differentiation of BMMs as determined by TRAP staining (e and f). * P < 0.05, ** P < 0.01 and *** P < 0.001 by two-tailed Student’s t test. Experiments were performed in triplicate and data are shown as mean ± SD. g MCF7 cells stably expressing miR-124 inhibitor (i-miR-124) or NC (i-NC) were firstly inoculated into the left ventricle of nude mice, and then 5 µg IL-11 neutralizing antibody (IL-11 Ab) or the control IgG (IgG) were injected into the tail veins of the mice. Luciferase signals were measured with an ex vivo imaging system. h Statistical analysis showed IL-11 neutralizing antibody partially reverses the promoting effect of miR-124 inhibitor on the metastasis of MCF7 cells. Horizontal line indicates median value. * P < 0.05 by Mann–Whitney U test

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)