Fig. 5

The 20-nt pyknon motif influences the functional role of N-BLR. a. miR-200c-3p levels following 48 h of co-transfection with empty pcDNA 3.1 vector, WT N-BLR vector, WT N-BLR del miR-200c-3p, WT N-BLR double del for both miR-200c-3p and miR-141-3p binding sites in HT-29 cell lines. The levels of miR-200c-3p were significantly reduced by the overexpression of the WT N-BLR compared to the empty vector, whereas they were restored by the overexpression of the mutant vector. b miR-200c-3p expression levels following 48 h of co-transfection with empty pcDNA 3.1 vector, pyk90-DEL N-BLR vector, pyk90-DEL N-BLR del miR-200c-3p, pyk90-DEL N-BLR double del for both miR-200c-3p and miR-141-3p binding sites. The lack of the pyk90 motif within N-BLR is likely to critically impair the binding between N-BLR and miR-200c-3p, thus the levels of the miRNA do not decrease significantly compared to the empty vector. c Comparison of miR-200c-3p expression levels between WT N-BLR and pyk90-DEL N-BLR cells: the binding of miR-200c-3p to N-BLR is partly dependent on the presence of the pyk90 motif. Y-axis values represent the ratio of miR-200c-3p and miR-141-3p to U6. Ratios were calculated with the 2^–ΔCt method using U6 levels for normalization. For each set of co-transfection experiments, the expression levels of miR-200c-3p were corrected by subtracting the values derived from the corresponding miRNA mimic negative control. Data are shown as mean ± SEM: n = 4. d Migration assays at 24 h show a significant increase of migrating cells with stable overexpression of WT N-BLR. Conversely, stable overexpression of pyk90-DEL N-BLR leads to a dramatic decrease of migratory capabilities even compared to the empty vector stable clone. e Similarly to migration, invasion assays at 36 h identified a significant increase of the invading population among the stable WT BLR overexpressing cells compared with the empty vector stable clone. While overexpression of pyk90-DEL N-BLR did not produce such gain of function, although not significant, it still conferred an edge of invasion over the empty clones. Data are shown as mean ± SEM: n = 3. f E-cadherin, ZEB1, and vimentin detection by immunofluorescence in HCT116 N-BLR overexpressing clones. The signal of E-cadherin (green color) was markedly decreased in WT N-BLR clone. The ZEB1 signal was absent in cells with empty vector (green color) but visible in WT N-BLR overexpressing clone. Blue color indicates nuclei. Single green, blue, and merged channel images of ZEB1 are reported in Additional file 3: Figure S9C. g Representative H&E images and immunohistochemical staining of Ki67 in liver metastases from nude mice after approximately four to six weeks of intrasplenic injection with empty vector, WT N-BLR, and pyk90-DEL N-BLR overexpressing HCT116 clones are shown. h Quantification of Ki-67 staining is reported. i WT N-BLR enhances liver metastases in the injected mice. Weekly imaging was performed using the Xenogen IVIS spectrum system within 12 min following injection of D-Luciferin (150 mg/mL). Living image 4.1 software was used to determine the regions of interest (ROI), and average photon radiance (p/s/cm2/sr) was measured for each mouse. Data were log-transformed before analysis. Data are shown as mean ± SEM: EMPTY n = 4, WT N-BLR n = 5, pyk90-DEL N-BLR n = 7. (Student’s t-test; *p < 0.05; **p < 0.01; ***p < 0.001; ****p < 0.0001)