Fig. 4. miR-130b-3p, which is secreted mostly from luminal A breast cancer, acts as a SPIN90 repressor in fibroblasts.

A Dicer knockdown in HBF. Dicer and SPIN90 expression were detected by Western blotting. B Representative images showing expression of miR-130b-3p (green) and the epithelial cell marker E-cadherin (red) in tumor and paired normal tissues from a commercially available tumor microarray (BR804b, Luminal A (n = 16), Luminal B (n = 13), Her2-enriched (n = 6), TNBC (n = 5)). Graph indicates log ratio for the intensity of miR-130b-3p in tumor tissues relative to normal tissues. Scale bar, 50 μm. C RT-qPCR analysis quantified the expression of mature miR-130b-3p in cancer cell lines of various luminal types, compared to that in normal MCF10A cells (n = 3). The level of each miRNA was normalized to that of RNU6. D Relative expression of miR-130b-3p in CM enriched from luminal A cancer and normal cell lines, normalized by the average Ct level of miR-23a-3p, miR-191-5p, miR-425-5p, and miR-451a (n = 3). E Western blotting analysis for SPIN90 and α-SMA in HBFs incubated for 48 h in the indicated CM (n = 3). F Relative amounts of miR-130b-3p in MCF7 CM treated with RNase in the presence or absence of Triton X-100 (n = 3 each). MCF7-derived CM was incubated with 10 μg/mL RNase or 1% (v/v) Triton X-100 at 37 °C for 30 min. G Western blot analysis of HBF cells incubated with MCF7 CM in the presence or absence of RNase and/or Triton X-100. The expression of each protein was normalized relative to that of α-tub. Buffer in MCF7 CM was replaced using a centrifugal filter to minimize Triton X-100 cytotoxicity. H HBFs were treated with CM from MCF7 cells stably expressing an oligonucleotide inhibitor for miR-130b-3p (130b-i) or a negative control oligonucleotide (NC-i) (n = 3). All data are presented as mean ± standard deviation. *p ≤ 0.05; **p ≤ 0.01; ***p ≤ 0.001 (Student’s t-test).