Fig. 5

Identification of miR-675-5p targets involved in apoptosis. A: The network diagram obtained using the mirWalk database [29] illustrates the presumed 3’UTR targets of miR-675-5p involved in apoptosis (KEGG Pathway hsa04210#Apoptosis). B: Expression level analysis (qRT-PCR) of miR-675-5p in HCT116 after overexpression of miR-675-5p under normoxic conditions. The miR-675-5p levels were normalized for RNU6 (U6 Small Nuclear 1), and the ΔΔCt was calculated with respect to the Scrambled Negative Control (Scr). C-D: Representative images and densitometric analysis of Western blots respectively for pro-caspase 9 and pro-caspase 3 on proteins lysates from HCT116 transfected with miR-675-5p mimic or Scrambled Negative Control (Scr) for 24 h in normoxia. The graphs ordinate shows the OD (Optical Density) of the indicated proteins normalized for the housekeeping’s OD (β-actin). Corresponding uncropped full-length blots are included in a Supplementary Materials. E: The Firefly Luciferase assay validates pro-caspase 3 as the target of miR-675-5p. Luminescence was normalized for RFP values end presented in the graph as relative Luciferase activity in cells treated with mimic-miR-675-5p (Luc/RFP + Mimic miR-675-5p) with respect to cells treated with the Negative Control (Luc/RFP + Scr). All Data are expressed as mean ± SD of three independent experiments and statistical significance was analyzed using Student’s t-test (* = p < 0.05; ** = p < 0.01)