Fig. 3: Circulating miR-9-5p promoted cholesterol to 25-HC conversion by targeting ATF3. | Nature Communications

Fig. 3: Circulating miR-9-5p promoted cholesterol to 25-HC conversion by targeting ATF3.

From: Tumor-derived miR-9-5p-loaded EVs regulate cholesterol homeostasis to promote breast cancer liver metastasis in mice

Fig. 3

a Livers collected from mice xenografted with MDA-MB-231cells and 231/miR-9 KO cells were subjected to RNA-seq and GSEA, showing enrichment of genes related to oxysterol derived from cholesterol pathway (Permutation test, n = 3 mice per group). b Diagram of cholesterol conversion to oxysterol. c LC-MS/MS analysis of oxysterol derived from cholesterol in liver in indicated xenografted NSG mice (one-way ANOVA, n = 5 mice per group). d LC-MS/MS analysis of oxysterol derived from cholesterol in liver in indicated mice receiving tail vein injections of EVs (one-way ANOVA, n = 5 mice per group). e Quantification of gene expression in liver in indicated xenografted NSG mice by qRT-PCR (one-way ANOVA, n = 6 mice per group). f Quantification of gene expression in liver in indicated NSG mice receiving tail vein injections of EVs by qRT-PCR (one-way ANOVA, n = 6 mice per group). g Predicted miR-9-5p binding sites in human and mouse ATF3 genes. The corresponding sequences in WT and mutated reporters are shown. h Responsiveness of wild-type and mutant ATF3 3’UTR reporters to miR-9-5p in MCF-10A or MCF-10A/miR-9 cells. Luciferase reporter assay showing responsiveness of the reporters to transfected wild-type and mutant ATF3 3’UTR in MCF-10A and MCF-10A/miR-9 cell (unpaired two-tailed t test, n = 3 independent batches). i Quantification of gene expression in MCF-10A cell transfected with miR-9-5p mimic or control by qRT-PCR (one-way ANOVA, n = 3 independent batches). j Immunoblot analysis of MCF-10A cell transfected with miR-9-5p mimic or control. k Quantification of gene expression of primary hepatocytes after EVs/PBS treatment for 48 h by qRT-PCR (one-way ANOVA, n = 3 independent batches). l Immunoblot analysis of primary hepatocytes after EVs/PBS treatment for 48 h. m Immunoblot analysis of liver in indicated xenografted mice. n IHC of ATF3 and CH25H in liver sections in indicated xenografted mice. Scale bars, 100 μm. o Immunoblot analysis of liver in indicated mice received tail vein injections of EVs. p IHC of ATF3 and CH25H in liver sections in indicated mice received tail vein injections of EVs. Scale bars, 100 μm. Data are presented as mean ± s.d. Source data and exact P value are provided as a Source data file.

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