Fig. 4

MiR-34a suppresses trophoblast invasion by targeting MYC mRNA. a Western blot was performed to assess the protein levels of MYC in non-transfected JEG-3 cells and in JEG-3 cells that were transfected with p-EGFP-miR-34a (miR-34a) or p-EGFP empty vector. β-actin was used as an internal control for grayscale analysis. Compared with the non-transfected cells, ***p < 0.001. b JEG-3 cells were co-transfected with pcDNA3.1-MYC (MYC) (or pcDNA3.1 vector) and p-EGFP-miR-34a (or p-EGFP vector), and the protein levels of MYC were examined by Western blot analysis 48 h after transfection. β-actin was used as an internal control, and grayscale analysis shows the mean ± standard deviation of three independent experiments (### p < 0.001). c MYC 3’-UTR was cloned into the firefly (FL) pmirGLO luciferase reporter vector. pmirGLO-MYC 3’-UTR (or pmirGLO vector) was co-transfected with p-EGFP vector, p-EGFP-miR-34a (miR-34a) or p-EGFP-miR-34a mutant (miR-34a_mut) into JEG-3 cells. Renilla (RL) luciferase reporter vector was also co-transfected as the internal reference. The luciferase activity (FL/RL) indicates the silencing effect resulting from the binding of miR-34a or miR-34a_mut to MYC 3’-UTR (# p < 0.05). d, e JEG-3 cells were co-transfected with MYC and miR-34a using the corresponding empty vectors as control, and the transfected cells were subjected to Matrigel-based invasion assay. The photographs of the invading cells were taken at 200× magnification, and the scales are 100 μm. The figure shows the representative images from three independent experiments and the values are expressed as the mean ± standard deviation. ## p < 0.01