Fig. 5. Expression pattern and role of miR-130b-3p in MCF7 xenografts of immune-deficient mice.

A Mammary fat pad injection of MCF7 cells or PBS (negative control). The graph shows tumor growth in the MCF7-injected group. B Mouse plasma was collected at weeks 2, 3, and 4 (each group, n = 4) after tumor cell injection. miR-130b-3p in plasma was quantified through RT-qPCR assay and normalized by the average Ct levels of miR-23a-3p, miR-191-5p, miR-425-5p, and miR-451a. C Tumors from the MCF7 group and normal fat pads from the PBS group were isolated at weeks 2, 3, and 4. Tissues were homogenized in QIAzol lysis reagent and the miR-130b-3p level was examined. miRNAs from tissues were normalized by the expression of RNU6. D, Co-staining of miR-130b-3p (green) and SPIN90 (red) and the fibroblast markers α-SMA for CAFs and Vimentin for normal fibroblasts (magenta) in tumor and adjacent normal tissues (n = 4 per group) from the MCF7 group. White dotted lines and arrows, stromal fibroblasts. The merged images show samples co-stained for miR-130b-3p and SPIN90, and with DAPI. Scale bar, 25 μm. E Expression pattern of miR-130b-3p and SPIN90 in tumor and normal tissues. The fluorescent intensity in the stroma was analyzed by the ImageJ software. F Representative IHC images of xenografted tumors derived from MCF7 NC-i (MCF7 cells stably expressing negative control for the inhibitor) and MCF7 130b-i (MCF7 cells stably expressing the oligonucleotide inhibitor of miR-130b-3p). Relative expression levels of α-SMA and SPIN90 of randomly selected ten areas were calculated using the ImageScope (Leica) software. Scale bar, 100 μm. G MCF7 cancer cells and HBF cells stably expressing miR-130b-3p inhibitor or negative control oligonucleotide were co-injected into the mammary fat pads of mice (n = 5). Tumor tissues were harvested 6 weeks later and weighed. All data are presented as mean ± standard deviation. *p ≤ 0.05; **p ≤ 0.01; ***p ≤ 0.001 (Student’s t-test).