Fig 1: LNP-miRs reduce the levels of VEGFA, TGFß1, CCL5 and CXCL2 and inhibit the proliferation of BRAFi resistant melanoma cells.A Schematic illustration of the biological assays carried out upon melanoma cells exposure to LNPs. B Quantification of miR-199b-5p and miR-204-5p by using qRT–PCR in A375 and M14 BRAFi-resistant cells upon 48 h of exposure to LNP-Scr or LNP-miRs. C Elisa assays measuring VEGFA, TGFß1, CCL5 and CXCL2 soluble levels in CM coming from A375 res (upper panels) and M14 res (lower panels) cells upon LNPs’ exposure (48 h). For these experiments cells have been serum starved for 24 h and then CM have been collected; results were determined by measuring absorbance at 450 nm into a microplate reader. D Luciferase reporter assays of the constructs containing CCL5 and CXCL2 3’UTRs co-transfected with the indicated miRNAs alone or in combination for 48 h have been performed in HEK293 cells. CCL5 and CXCL2 reporter plasmids were transfected at 500 ng; pLX313-Renilla plasmid has been used to normalize results at 50 ng. E Cell viability evaluation by measuring ATP content in A375 and M14 BRAFi-resistant cells left untreated, treated with the sole Dabrafenib (BRAFi, 500 nM) or in combination with the MEKi, i.e. Trametinib at 10 nM (MAPKi) in the presence of LNP-Scr or LNP-miR (30 µg each). *p < 0.05; **p < 0.01; and ***p < 0.001. qRT-PCR data are represented as mean (n = 3) ± SD; Elisa, luciferase and cell viability results are expressed as the mean of at least three independent experiments ±SEM.
Fig 2: Macrophage signatures and VEGFA, TGFß1, CCL5 and CXCL2 correlate with survival of melanoma patients based on SKCM data.Kaplan–Meier curves estimating the clinical relevance of M2 (left graphs) or M1 (right graphs) macrophage infiltrates in association with patient clinical outcome alone (A) or in combination (B) with Up-genes (i.e. VEGFA, TGFß1, CCL5 and CXCL2). The hazard ratio, p values for Cox models and the log-rank p values are shown on the Kaplan–Meier plots. The hazard ratio, Cox models and the log-rank p values were evaluated to plot KM curves.
Fig 3: Pro-angiogenic and pro-inflammatory factors controlled by oncosuppressor miRNAs are distinguishing features of MAPKi-resistant melanomas.A Venn Diagram showing the intersected data obtained by RNA-seq, GSEA and cytokinome analyses for the identification of VEGFA, TGFß1, CCL5 and CXCL2. B Quantification of miR-199b-5p and miR-204-5p by using qRT–PCR in A375 and M14 BRAFi-resistant cells vs. sensitive counterparts. C Elisa assays measuring VEGFA, TGFß1, CCL5 and CXCL2 soluble levels in cell media (CM) deriving from A375 (upper panels) and M14 (lower panels). For these experiments cells have been serum starved for 24 h and then CM have been collected; results were determined by measuring absorbance at 450 nm into a microplate reader. D Representative results of miR-204-5p and miR-199b-5p expression from matched formalin-fixed paraffin-embedded (FFPE) melanoma samples before initiation of targeted therapy (Pre) and after disease progression (PD). E VEGFA, TGFß1, CCL5 and CXCL2 mRNA expression levels (Up-genes) measured by qRT-PCR in PD vs. Pre-therapy samples (n = 14). F Spearman correlation calculated using qRT-PCR data of the two Down-miRs (miR-204-5p and miR-199b-5p) and the four Up-genes in PD (right panel) and in Pre-therapy (left panel) samples. *p < 0.05; **p < 0.01; and ***p < 0.001. qRT-PCR data are represented as the mean (n = 3) ±SD; Elisa results are expressed as mean of at least three independent experiments ± SEM.
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