Figure 3

MIR31 promotes the translocation of YAP1 into the nucleus and promotes the transcription of CCND1. (a) Representative cells of immunofluorescence for GFP and YAP1, x600. The nuclei are indicated by white arrows. *The translocation of YAP1 into the nucleus was not observed in the cells unsuccessfully transfected with pre-MIR31. The cells were cultured under standard conditions with 5% fetal bovine serum. (b) Ratio of nuclear YAP1 staining. *p < 0.05, unpaired two-tailed Student’s t-test. (c) The CCND1 levels were increased by MIR31 overexpression and the CCND1 levels were decreased by the MIR31-specific inhibitor on immunoblotting for CCND1 and α-tubulin. The ratio of CCND1/α-tubulin is shown in the bar graph. (d) The CCND1 levels were increased by YAP1 overexpression. Immunoblotting for YAP1, CCND1 and α-tubulin. (e) The luciferase activity after transfection of the reporter constructs containing the LATS2 promotor region normalized to the GAPDH promotor region (top). Immunoblotting for YAP and α-tubulin following siRNA transfection (bottom). Mock and MIR31 cells were transfected with non-targeting siRNA. *p < 0.05, unpaired two-tailed Student’s t-test. (f) Correlation between the MIR31 expression and results of the immunohistochemical analysis of LATS2 and CCND1 in vivo. Representative results are shown in micrographs, x100. All experiments were performed in triplicate.