Fig. 4

RB1 was the direct target through which miR-661 regulated EMT and invasion of NSCLC. a. Schematic diagram of miR-661 binding sites in the RB1 3′UTR. Sequences were compared between the mature miR-661 and wild-type (wt) or mutant (Mut) putative target sites in the 3′UTR of RB1 mRNA. b. Relative luciferase activity of 3′UTR RB1 and 3′UTR mut RB1 in pre-miR-661 (miR-661) or negative control premiRNAs (Control) transfected A549 cell. Data are mean (n = 3) ± SEM (*, P < 0.01, # P > 0.05). c. Western blotting of RB1 expression in A549 and SPC-A1 cells after transient transfection with miR-661. d. Immunostaining of RB1 in subcutaneous tumor formed by A549 infected with LV-miR-661 or LV-NC. e. Correlation analysis between RB1 and miR-661 expression in 50 paired tumorous and adjacent tissues. f. Transwell assay of A549 and SPC-A1 with miR-661 or miR-661 + RB1 overexpression. Data are mean (n = 3) ± SEM. g. Western blotting of EMT markers in A549 and SPC-A1 with miR-661 or miR-661 + RB1 overexpression. h. Wound healing assay of A549 and SPC-A1 with miR-661 or miR-661 + RB1 overexpression at 48 h after transfection. Data are mean (n = 3) ± SEM