Additional file 3 of miR-146a-5p impairs melanoma resistance to kinase inhibitors by targeting COX2 and regulating NFkB-mediated inflammatory mediators
Creators: name: Vergani, Elisabetta
name: Dugo, Matteo
name: Cossa, Mara
name: Frigerio, Simona
name: Di Guardo, Lorenza
name: Gallino, Gianfrancesco
name: Mattavelli, Ilaria
name: Vergani, Barbara
name: Lalli, Luca
name: Tamborini, Elena
name: Valeri, Barbara
name: Gargiuli, Chiara
name: Shahaj, Eriomina
name: Ferrarini, Marina
name: Ferrero, Elisabetta
name: Gomez Lira, Macarena
name: Huber, Veronica
name: Vecchio, Michele Del
name: Sensi, Marialuisa
name: Leone, Biagio Eugenio
name: Santinami, Mario
name: Rivoltini, Licia
name: Rodolfo, Monica
name: Vallacchi, Viviana
Publisher: name: figshare
Year: 2020
Subjects: subject: Medicine
subject: Cell Biology
subject: Genetics
subject: FOS: Biological sciences
subject: Molecular Biology
subject: Pharmacology
subject: Biotechnology
subject: Developmental Biology
subject: Cancer
subject: 111714 Mental Health
subject: FOS: Health sciences
subject: Hematology
subject: 110309 Infectious Diseases
subject: 60506 Virology

description: Additional file 2: Supplementary Fig. S1. Altered miR-146a expression influences BRAF/MEKi sensitivity and apoptosis in melanoma. A) Boxplots representing IC50 values of 6 matched PLX4032-resistant (R) and sensitive (S) melanoma cell lines to PLX4032 (vemurafenib), BRAFi (dabrafenib), MEKi (trametinib), to the combined treatment BRAF/MEKi, and to sTRAIL-induced apoptosis. B) Inverse correlation between miR-146a expression levels and IC50 values of PLX4032, BRAFi and MEKi in melanoma cell lines (Spearman analysis). C) Forced expression of miR-146a (+m-miR-146a) in LM16R cell line and in LM69 and LM70 short term cultures increased the effects of PLX4032 treatment as shown by reduced cell growth and increased cell cytotoxicity and apoptosis, evaluated by CCK8, LDH and caspase 8 and 3/7 activity. D) Inhibition of miR-146a expression (i-miR-146a) in LM16 cells increased cell proliferation and decreased the release of LDH and the apoptosis rate as evaluated by CCK8, LDH and caspase 3/7 activity. E) Overexpression of miR-146a (m-miR-146a) upon PLX4032, BRAF/MEKi and sTRAIL treatments in LM47R cells increased cell cytotoxicity and apoptosis, as evaluated by LDH and caspase 8 and 3/7 activity. Data are plotted compared to scrambled control. *: p &lt; 0.05, **: p &lt; 0.01, ***: p &lt; 0.0001 by Student’s unpaired t test. Supplementary Fig. S2. COX2 inhibition increases sensitivity to PLX4032 and to BRAF/MEKi and reduces PGE2 release. A) Relative luciferase activity after co-transfection of PTGS2 3’UTR luciferase reporter vector or control vector with miR-146a mimic or mimic negative control. Experiment was performed in LM16R cells. B) Inverse correlation between miR-146a expression levels and IC50 values of PLX4032 in melanoma cell lines (Spearman analysis). C) Western blot analyses showing downregulation of COX2 after transfection with siRNA against COX2 (siCOX2) compared to scrambled control (S). COX2 protein levels were downregulated to 15%, as determined by quantification of the signal and expressed as the ratio of COX2/Actin intensity. Experiment was performed with LM47R cells. D) Silencing of COX2 by siRNA transfection enhanced cell growth inhibition and increased cell cytotoxicity and apoptosis upon treatment with PLX4032 compared to scrambled control. E) PGE2 release in culture supernatants following 24 h treatment with the COX2 inhibitors celecoxib and NS398, alone or in combination with BRAF/MEKi. F) Combined treatment with celecoxib increased cell growth inhibition and cytotoxicity by PLX4032. ●: interaction index = 1. G) Dose-dependent effect of celecoxib alone (5, 10, 20, 40, 50 and 80 μM) and combined with PLX4032 (3 μM) on cell growth. ●: interaction index = 1 at the dose of 50 μM of celecoxib combined with PLX4032. P values were calculated by Student’s unpaired t test in A, D and F, by one-way ANOVA followed by Bonferroni correction in E, and by two-way ANOVA followed by Bonferroni correction in G. *: p &lt; 0.05, **: p &lt; 0.01, ***: p &lt; 0.0001. Supplementary Fig. S3. miR-146a expression levels are inversely correlated with levels of target genes. Analysis of correlation between miR-146a expression and its target genes EGFR, CCL2 and BCL2L1 in metastatic melanoma specimens. The rp and p values resulting from Pearson analysis are shown. Supplementary Fig. S4. Dynamic culture in bioreactor preserves TME architecture of 3D tumor explants. Representative histological images of H&amp;E-stained 3D tumor sections after 3 days culture in bioreactor. The marked areas in the left panels are shown at higher magnification in the right panels to display the preserved cellularity and tumor tissue histo-architecture. Scale bar 80 μM. Supplementary Fig. S5. miR-146a overexpression increases drug sensitivity in 3D cultures of tumor explants. A) Modulation of direct and indirect miR-146a target genes upon transfection of miR-146a mimic (m-miR-146a) compared to scrambled-transfected control (S) in 3D cultures of BRAF-mutated melanoma tumors from naïve patients. AU: arbitrary units. B) Decreased staining for Ki67 and pERK proliferating cells and increased Caspase 3 positive cells in 3D cultures upon treatment with BRAF/MEKi and transfection with miR-146a (m-miR-146a) or scrambled control (S). Scale bar 10 um.