Fig. 3

miR-381 targets the 3’UTR and then decreases the mRNA stabilization of YAP. a Putative miR-381 binding sites in the 3′-UTR sequences of YAP. b Luciferase activity of A549 cells transfected with plasmids carrying a wild-type or mutant 3′UTR of YAP in response to miR-381 mimics or inhibitors. c Schematic images of a construction carrying a wild-type or mutant 3′UTR of YAP combined with MS2 binding sequence (up panel). MS2-RIP followed by miR-381 qPCR to measure miR-381 associated with 3′UTR of YAP in HBEC and 95-D cells (down panels). d AGO2-RIP followed by qPCR to evaluate 3′UTR of YAP level after miR-381 knockdown or overexpression and miR-381 WT or Mut, respectively. e The cell lysates from A549 cells with transfection with miR-381 mimics or inhibitors were incubated with biotin-labeled miR-381 probe. The RNA level of miR-381 was analyzed by qPCR to detect the effect of probe. f A549 cells were co-transfected YAP 3′UTR WT or Mut and then the cell lysates were incubated with biotin-labeled miR-381 WT or Mut, respectively. The relative enrichment of YAP 3′UTR was analyzed by qPCR. g A549 cells were co-transfected miR-381 WT or Mut and then the cell lysates were incubated with biotin-labeled YAP 3′UTR WT or Mut probe, respectively. The relative enrichment of miR-381 was analyzed by qPCR. h A549 cells were co-transfected with miR-381 and YAP 3′UTR WT for 24 h. The locations of YAP 3′UTR and miR-381 were analyzed by FISH (fluorescence in situ hybridization). (i, j) A549 cells were transfected with miR-381 mimics (i) or inhibitors (j), respectively. The mRNA and protein levels of YAP were analyzed by RT-PCR, western blot and qPCR assays. k A549 cells were transfected with miR-381 mimics or inhibitors, respectively. The protein level of YAP was analyzed by immunofluorescent staining. l, m RT-PCR, western blot and qPCR results indicated that miR-381 dose- and time- dependently decreased YAP expression. n, o qPCR results indicated that miR-381 inhibitor dose- and time- dependently increased YAP, Cyr61 and CTGF expressions in A549 and 95-D cells. Results were presented as mean ± SD, and the error bars represent the SD of three independent experiments. *P < 0.05 or **P < 0.01 indicates a significant difference between the indicated groups (two-tailed, unpaired Student’s t-test or one-way analysis of variance). ns, not significant