Fig. 2: Cancer cell-secreted miR-204 induces hypoxia by targeting VHL.

a The eWAT collected from orthotopic tumour mice were subjected to RNA-seq and GSEA analysis, showing enrichment of genes related to hypoxia pathways (n = 3 mice for Control, n = 3 mice for bearing MDA-MB-231 tumour). NES: normalized enrichment score. Nominal P-values and false discovery rate (FDR) q-values are shown. b Validation by qRT-PCR analysis of mRNA abundance of Vhl and Hif1α in iWAT and eWAT from mice bearing indicated tumours (n = 5 mice per group; one-way ANOVA followed by Dunnett multiple comparison test). c Immunoblot analysis of VHL and HIF1Α in iWAT and eWAT from tumour group mice (tumour-free group, n = 4; 231/Ctrl group, n = 4; 231/Rab27a KD group, n = 5). Vinculin (VCL) was used as loading control. d Validation by qRT-PCR analysis of mRNA abundance of Vhl and Hif1α in iWAT and eWAT from mice in sEVs injected groups (n = 6 mice per group; one-way ANOVA followed by Dunnett multiple comparison test). e Immunoblot analysis of VHL and HIF1Α in iWAT and eWAT from sEVs treated mice (PBS group, n = 4; MCF-10A sEVs group, n = 4; MDA-MB-231 sEVs group, n = 5). f Target gene prediction of miR-204-5p with miRNA-seq analysis and related bioinformatics tools. g Predicted miR-204 binding sites in human and mouse VHL/Vhl genes (hVHL and mVhl, respectively). The corresponding sequences in WT and mutated reporters are shown. h Responsiveness of the reporters to miR-204 in transfected in MCF-10A (unpaired two-tailed t-test, n = 3 biological replicates). i Western blots showing levels of indicated proteins in differentiated primary adipocytes transfected with miR-204 mimic or control miRNA mimic. Repeated three times independently with similar results obtained. j Representative ISH images showing miR-204 level in adipose tissue of indicated groups of mice. Repeated three times independently with similar results obtained. Scale bar, 100 μm. Representative immunohistochemistry staining of HIF1Α in iWAT and eWAT from xenografted tumour mice k and sEVs injected mice l and their respective control groups. Repeated three times independently with similar results obtained. Scale bar, 200 μm. Data are presented as mean ± s.e.m; ^*P < 0.05, ^**P < 0.01, ^***P < 0.001, ^****P < 0.0001, ns: not significant. Source data and exact P value are provided as a Source data file.