Fig. 7: miR-147b overexpression leads to increased tumor burden in vivo.

A Subcutaneous injection of A549-miR-SCR and A549-miR-147b cells into the right flank of immunodeficient NSG mice. Tumors were harvested after 40 days. A Representative macroscopic pictures of subcutaneous tumors, scale bar 5 mm. B Measurement of tumor size during tumor progression (n = 5) and C tumor mass after 40 days. Validation of D miR-147b and E DUSP8 mRNA expression in mice tumor samples (n = 5). F DUSP8 level measured by ELISA (n = 5). G Western blot analysis of DUSP8 in miR-147b OE tumors compared to miR-SCR tumors (n = 3). H Intravenous injection of A549-miR-SCR and A549-miR-147b cells into the tail vein of immunodeficient NSG mice. Representative images of micro-CT scans, extracted lung images, H&E-stained sections, and immunofluorescence staining for Ki67 and vWF (green) and DAPI (blue) in tumor sections. Scale bars, 50 μm. I Quantification of average lung intensity (n = 5). Quantification of proliferating J Ki67⁺cells (green) (n = 5, 5 images per animal) and K vWF (n = 5, 5 images per animal) counterstained with DAPI (blue) within the tumor counted per high power field (HPF) using Fiji Software. Validation of L miR-147b and M DUSP8 mRNA expression in mice lung tumor samples (n = 5). N DUSP8 level measured by ELISA (n = 5). O Western blot analysis of DUSP8 expression (n = 3). P Schematic representation of miR-147b-mediated tumor progression via suppression of DUSP8. Suppression of DUSP8 by miR-147b leads to inhibition of JNK de-phosphorylation resulting in activation of JNK signaling. Overexpression of miR-147b alters the phosphorylation of MAPKs and leads to increased proliferation and migration. The opposite effects were observed by silencing miR-147b expression and thereby restoring DUSP8 function with cancer-suppressive properties, leading to increased apoptosis. Data are shown as mean ± standard error of the mean using two-way ANOVA with Bonferroni’s multiple comparisons (B) and two-tailed unpaired t-test with Welch’s correction (C–O). P-values ≤ 0.05 were considered statistically significant for all analyses, *p ≤ 0.05, **p ≤ 0.01, ***p ≤ 0.001 and ****p ≤ 0.0001.