Fig. 6
miR-1 targets CXCR4/FOXM1/RRM2, alters FOXM1 accessibility to RRM2 promoter, and modulates downstream signaling. A Schematic for the cloning of 3'-UTR sequences for dual luciferase assay. The upper panel of the inset shows the alignment of mature miR-1 sequences with 3'-UTR of CXCR4, and the lower panel shows the CXCR4 UTR residues mutated to abrogate the miR-1 binding. Primer sequences used to amplify 3'-UTR of CXCR4 were presented next to the inset. B Dual-luciferase assay validating CXCR4 as a target of miR-1. SBC5 cells were co-transfected with the luciferase-3'-UTR construct of CXCR4 (wild type/mutant), and miR-1 mimic, or scramble (SCR) and luciferase activity was measured. Chromatin immunoprecipitation of FOXM1 and RRM2 qPCR confirming the binding of FOXM1 with RRM2 promoter, (C) miR-1 decreased the interaction of FOXM1 with RRM2 promoter, (D) miR-1 sponging (miR-1Zip) enhances the accessibility of RRM2 promoter to FOXM1. E FOXM1 binding to RRM2 promoter in the presence of CXCL12 (100 ng/ml for 48-72 h), higher binding in miR-1Zip cells is due to high CXCR4 expression in these cells. F miR-1 sponging increased the expression of FOXM1 and RRM2 in SBC3 cells. G Immunoblotting studies showed that overexpression of miR-1 decreased the expression of FOXM1 and RRM2 in SBC5 and NCI-H69 cells. H Schematic to understand the viability of the CXCR4-FOXM1-RRM2 axis in SCLC cell lines. I-J SBC3-miR-1Zip and SBC5 cells were treated with AMD3100 (CXCR4 inhibitor) and FDI-6 (FOXM1 inhibitor) and analyzed for the expression of FOXM1 and RRM2 by Western blot. AMD3100 or FDI-6 treatment decreased the expression of FOXM1 and RRM2, like miR-1. Immunoblotting analysis demonstrated that; (K) miR-1 sponging increased the activation of AKT and ERK, and expression of epithelial to mesenchymal markers snail and Zeb-1 in SBC3 cells, (L) miR-1 overexpression decreased AKT and ERK activation in SBC5 and NCI-H69 cells. miR-1 also decreased the expression of snail in SBC5 cells. Statistical significance was calculated using two-tailed Student’s t-test for C-D, and one-way ANOVA for E. *, p < 0.05; **, p < 0.01; ***, p < 0.001; ns, non-significant