Figure 3

TIMP3, a tumor suppressor gene, as a potential target of MiR-4435. (a) miR-4435 and TIMP3 seed sequences identified using miRmap database. (b,c) Protein expression levels of TIMP3 in mutant UQCRB-expressing cell lines and colon cancer cells. β-actin was used as an internal control. (d) Schematic representation of reporter plasmids carrying a miR-4435 target site [psiCHECK-2_TIMP3(WT)] or its mismatched target [psiCHECK-2_TIMP3(MT)] at their 3′-UTR (top). The miR-4435 core seed-matched sequence and its mutated one were shown in red. HEK293 cells were co-transfected with each indicated reporter plasmid and miR-10a-5p mimic or miR-4435 mimic. At 24 h post-transfection, normalized luciferase activity (Rluc/Fluc) compared with WT vector-transfected control was determined (bottom). miR-10a-5p, a miRNA mimic used as a control. All quantified data are presented as mean ± S.E.M compared to control. (***indicates p < 0.001). (e,f) Expression levels of miR-4435 after transfection of mutant UQCRB-expressing cell lines and colon cancer cells with miR-4435 inhibitor. (g,h) Expression levels of TIMP3 after transfection of mutant UQCRB-expressing cell lines and colon cancer cells with miR-4435 inhibitor. β-actin was used as an internal control. All quantified data are presented as mean ± S.E.M compared to control (*p < 0.05, **p < 0.01, ***p < 0.0001). These all images are representative of 3 repeated independently repeated experiments.