Eupatilin Inhibits Renal Cancer Growth by Downregulating MicroRNA-21 through the Activation of YAP1
Figure 5
YAP1 is a direct target of miR-21 in renal cancer cells. (a) Schematic representation of YAP1 mRNA with a putative 3′-UTR miR-21-binding site and sequences of wild-type (YAP1 WT) and mutant (YAP1 MUT) miR-21 target sites. The putative miR-21 binding site in the YAP1 3′-UTR is indicated in red. This site was identified using TargetScan. (b) Luciferase reporter assay of 786-O cells transfected with a vector with an inserted YAP1 3′-UTR sequence (YAP1 3′-UTR WT) or a vector with an inserted mutated 3′-UTR sequence (YAP1 3′-UTR MUT), together with negative control miRNA (miR-NC) or miR-21 mimics. Luciferase activity was normalized to Renilla luciferase activity. Data represent the mean ± SEM (n = 3) of three independent experiments. (P < 0.01 versus miR-NC). (c) 786-O cells infected with miR-21 mimics or control lentivirus were treated with Eupatilin (10 mM) for 24 h. The protein expression of YAP1 in 786-O cells was detected by western blotting. (d) Quantification of the results in (c). Data represent the mean ± SEM (n = 3) of three independent experiments. (P < 0.01 versus miR-NC; ## P < 0.01 versus miR-NC+Eupatilin). (e) Mice injected with 786-O cells infected with miR-21 mimics or control lentivirus were treated with Eupatilin. The protein expression of YAP1 in tumors was assessed by western blotting. (f) Quantification of the results in (e). Data represent the mean ± SEM (n = 3) of three independent experiments. (P < 0.01 versus miR-NC; ## P < 0.01 versus miR-NC+Eupatilin).