Fig. 2: Exosomal miR-9-5p induced cholesterol biosynthesis by targeting INSIG1/2. | Nature Communications

Fig. 2: Exosomal miR-9-5p induced cholesterol biosynthesis by targeting INSIG1/2.

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

Fig. 2

a The enriched miRNAs from MDA-MB-231 EVs versus MCF-10A EVs overlapping with miRNA pools targeting both Insig1 and Insig2 from Targetscan database in cholesterol pathway. b Left: qRT-PCR-determined miR-9-5p levels in an equal amount of EVs derived from serum of normal (n = 6) and breast cancer patients (n = 12) (unpaired two-tailed t test). Right: The RNA quality of serum EVs was demonstrated. c RT-qPCR-determined miR-9-5p levels in liver (n = 5) and serum (n = 5) in indicated tumor-bearing or tumor-free mice (one-way ANOVA). d RT-qPCR-determined miR-9-5p levels in liver from mice receiving tail vein injections of indicated EVs (one-way ANOVA, n = 6 mice per group). e In situ hybridization for miR-9-5p in liver of mice from tumor free, 231/Ctrl and 231/Rab27a KD. Scale bar, 50 μm. f Predicted miR-9-5p binding sites in human and mouse Insigs genes (Insig1 and Inisg2, respectively). The corresponding sequences in WT and mutated reporters are shown. g Luciferase reporter assay showing responsiveness of the reporters to transfected human and mouse Insig1 or Insig2 in MCF-10A and MCF-10A/miR-9 cells (unpaired two-tailed t test, n = 3 independent batches). h Immunoblot analysis of liver sample collected from indicated mice received EVs injection. i, j Total cholesterol (TC) and triglycerides (TG) level in liver and serum in indicated mice received EVs (unpaired two-tailed t test, n = 6 mice per group). k Quantification of gene expression of HL7702 cell after EVs/PBS treatment for 48 h using qRT-PCR (one-way ANOVA, n = 3 independent batches). l Immunoblot analysis of HL7702 cell after EVs/PBS treatment for 48 h. m Immunoblot analysis of liver sample collected from indicated tumor bearing mice. n According to the gates in phasor plot, LD region was segmented in livers of indicated groups. Scale bars, 20 μm. o Total cholesterol (TC) and triglycerides (TG) level in liver (n = 6) and serum (n = 5) in indicated tumor bearing mice (unpaired two-tailed t test). p Liver sections were stained with oil red O in indicated groups. Scale bars, 50 μm. Data are presented as mean ± s.d. Source data and exact P value are provided as a Source data file.

Back to article page